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erbB4 expression and activation in later life
European Neuropsychopharmacology (2012) 22, 356–363
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Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life Teresa Marie du Bois ⁎, Kelly Anne Newell, Xu-Feng Huang Centre for Translational Neuroscience, School of Health Sciences, Illawarra Health and Medical Research Institute, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia Schizophrenia Research Institute (SRI), Sydney, Australia
Received 25 January 2011; received in revised form 16 August 2011; accepted 1 September 2011
KEYWORDS Neuregulin 1; erbB4; NMDA receptor; Schizophrenia; Rat; Brain
Abstract Schizophrenia is a complex and devastating mental disorder of unknown etiology. Hypofunction of N-methyl-D-aspartate (NMDA) receptors are implicated in the disorder, since phencyclidine (PCP) and other NMDA receptor antagonists mimic schizophrenia-like symptoms in humans and animals so well. Moreover, genetic linkage and post mortem studies strongly suggest a role for altered neuregulin 1 (Nrg1)/erbB4 signaling in schizophrenia pathology. This study investigated the relationship between the NMDA receptor and Nrg1 signaling pathways using the perinatal PCP animal model. Rats (n = 5/group) were treated with PCP (10 mg/kg) or saline on postnatal days (PN) 7, 9 and 11 and were sacrificed on PN12, 5 weeks and 20 weeks for biochemical analyses. Western blotting was used to determine total and phosphorylated levels of proteins involved in NMDA receptor/Nrg1 signaling in the prefrontal cortex and hippocampus. In the cortex, PCP treatment altered Nrg1/erbB4 expression levels throughout development, including decreased Nrg1 and erbB4 at PN12 (−25–30%; p b 0.05); increased erbB4 and p-erbB4 (+18–27%; p b 0.01) at 5 weeks; and decreased erbB4 and p-erbB4 (−16–18%; p b 0.05) along with increased Nrg1 (+33%; p b 0.01) at 20 weeks. In the hippocampus, levels of Nrg1/erbB4 were largely unaffected apart from a significant decrease in p-erbB4 at 20 weeks (−13%; p b 0.001); however NMDA receptor subunits and PSD-95 showed increases at PN12 and 5 weeks (+20–32%; p b 0.05), and decreases at 20 weeks (−22–29%; p b 0.05). This study shows that NMDA receptor antagonism early in development can have long term effects on Nrg1/erbB4 expression which could be important in understanding pathological processes which might be involved in schizophrenia. © 2011 Elsevier B.V. and ECNP. All rights reserved.
⁎ Corresponding author at: University of Wollongong, School of Health Sciences, Northfields Avenue, NSW 2522, Australia. Tel.: +61 2 42215294; fax: +61 2 42215945. E-mail address: [email protected] (T.M. du Bois). 0924-977X/$ - see front matter © 2011 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2011.09.002
Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life
1. Introduction Glutamatergic N-methyl-D-aspartate (NMDA) receptors play a key role in brain development, controlling processes such as neuronal migration, synaptogenesis and synaptic plasticity (McDonald and Johnston, 1990; Pearce et al., 1987). The NMDA receptor is comprised of a heteromeric assembly of subunits, including an obligatory NR1 subunit plus distinct combinations of NR2A–D subunits, and rarely NR3 subunits (Madry et al., 2007). The immature form of the NMDA receptor channel complex, present at postnatal day (PN) 1–14 in rats, is highly sensitive to glutamate, with less sensitivity to magnesium block (Sircar, 2000). The longest and largest NMDA receptor currents are also observed at this time (Colwell et al., 1998; Hestrin, 1992). During this period, the brain is vulnerable to the toxic effects of modulation of the NMDA receptor (Haberny et al., 2002). The physiological properties of NMDA receptors correlate with developmental variation in receptor subunits. In rats, the only forms present at birth are the NR1, NR2B and NR2D subunits; during postnatal weeks 1–3, NR2B and NR2D decline whereas NR2A becomes abundant, then all subtypes decrease to adult levels (Wenzel et al., 1996, 1997). NR2C emerges at day 5, peaks at 10 days and then remains unchanged (Wenzel et al., 1997). In rodents, treatment with NMDA receptor antagonists such as phencyclidine (PCP) and MK-801 during the perinatal period induces large increases in neuronal apoptosis (Hansen et al., 2004; Harris et al., 2003; Ikonomidou et al., 1999; Wang et al., 2001) and produces schizophrenia-relevant behavior such as impaired cognitive function (Andersen and Pouzet, 2004; Sircar, 2003; Stefani and Moghaddam, 2005; Wang et al., 2001; Wiley et al., 2003), sensorimotor gating deficits (Harris et al. 2003; Wang et al. 2001) and hyperlocomotor activity (du Bois et al., 2008a; Facchinetti et al., 1993; Wang et al., 2001). Further, we have shown that perinatal PCP treatment alters the expression of neurotransmitter receptors implicated in schizophrenia including dopamine D2, NMDA, GABAA and muscarinic M1/4 receptors (du Bois et al., 2008b, 2009a, 2009b). Hypofunction of NMDA receptors has been implicated in schizophrenia, since NMDA receptor antagonists such as PCP and ketamine mimic schizophrenia symptoms so closely in healthy individuals and strongly exacerbate symptoms in schizophrenia patients (Javitt and Zukin, 1991). Studies have also demonstrated changes in NMDA receptor binding, transcription and subunit expression in different brain regions of schizophrenia subjects; and many candidate susceptibility genes for schizophrenia such as D-amino acid oxidase (DAAO), dybindin-1, Disrupted in schizophrenia 1 (DISC-1) and neuregulin 1 (Nrg1) can directly or indirectly affect NMDA receptor signaling (see Banerjee et al., 2010 for review). Like the NMDAR, Nrg1 also plays a key role in brain development, regulating many important processes in both the developing and the mature brain (Harrison and Law, 2006). Consistent with its important developmental role, Nrg1 mRNA expression is highest early in development and declines with age (Corfas et al., 1995). The Nrg1 gene gives rise to at least 30 different isoforms, the most well characterized of which have been grouped into 3 classes, types I, II and III (Harrison and Law, 2006). In rodents, these isoforms are known to be expressed in a region and cortical layer specific
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manner (Kerber et al., 2003), each with independent functions in different brain regions (Meyer et al., 1997). Nrg1 functions are largely mediated by the receptor tyrosine kinases erbB2, 3 and 4. ErbB4 has been indicated as the predominant receptor for Nrg1 (Stefansson et al., 2004). It has been shown that erbB4 receptor protein expression does not vary with age (Thompson et al., 2007). Four structurally different erbB4 receptor isoforms have been identified in neural tissue: JM-a, JM-b, CYT-1 and CYT-2 (Junttila et al., 2000). ErbB4 receptor expression has been shown to be confined to inhibitory (GABAergic) interneurons (especially parvalbumincontaining interneurons), specifically at the postsynaptic density of dendrites receiving glutamatergic synapses (Fazzari et al., 2010). Linkage studies have identified both the Nrg1 and the erbB4 receptor as susceptibility genes for schizophrenia and there is evidence of altered mRNA and protein expression of Nrg1 and erbB4 receptors in post mortem human schizophrenia brain tissue in an isoform-specific manner (see Banerjee et al., 2010 for review). Given that Nrg1/erbB4 genetic mutations likely contribute to only a small proportion of schizophrenia cases, it has been suggested that changes in expression levels of Nrg1 and erbB4 in the schizophrenia brain are secondary to other molecular changes related to the disease such as NMDA receptor hypofunction (Li et al., 2007). ErbB4 receptors are anchored at PSD-95 along with NMDA receptor 2A–D subunits (Fazzari et al., 2010; Garcia et al., 2000; Huang et al., 2000), which places the glutamatergic NMDA receptor and Nrg1 signaling pathways together at the synapse, allowing for crosstalk between the systems. While studies have shown that Nrg1 can influence NMDA receptor function (Bjarnadottir et al., 2007; Gu et al., 2005; Li et al., 2007), it is unclear how NMDA receptor activity can modulate Nrg1/erbB4. This information is important in order to understand how NMDA receptor, Nrg1 and GABAergic systems interact together to control synaptic activity and brain function. The present study investigated whether perinatal PCP treatment could lead to long term changes in Nrg1 and erbB4 expression in the rat brain. This study examined total and activated (phosphorylated) forms of key proteins linking NMDA receptor and Nrg1/erbB4 systems including selected NMDA receptor subunits, Nrg1, erbB4 and the anchoring protein PSD-95. We were also interested in Akt expression because erbB4 receptor signaling activates the phosphotidylinositol-3 kinase/Akt pathway (Junttila et al., 2000). We hypothesized that Nrg1/erbB4, PSD-95 and Akt expression would be highly influenced by glutamatergic/ NMDA receptor activity, and follow the same expression pattern as the NMDA receptor.
2. Experimental procedures 2.1. Animals Timed, pregnant female Sprague–Dawley rats were obtained from the Animal Resource Centre (Perth, Australia) at day 14 of gestation. The dams were housed individually with a regular 12 h light–dark cycle (lights on 07:00 h, off at 19:00 h) with food and water ad libitum. Day of birth was considered PN0. Pups were sexed on PN7 and litters were randomly assigned to PCP or saline groups. Each litter consisted of ten to twelve pups. Females were kept in litters, but only male pups were used for these experiments. This study was approved by the
358 Animal Ethics Committee of the University of Wollongong, and procedures complied with the Australian Code of Practice for the Care and Use of Animal for Scientific Purposes, which conforms to International Guiding Principles for Biomedical Research Involving Animals. All efforts were made to minimize numbers of animals used and their suffering.
2.2. Experimental design Male rat pups were a given subcutaneous injection on PN 7, 9 and 11 with 10 mg/kg PCP (Sigma, Castle Hill, NSW, Australia) or saline at a volume of 1 ml/kg once per day between 09:00 h and 10:00 h. The dose and treatment regime is based on previous experiments showing that 10 mg/kg PCP at PN7, 9 and 11 induces apoptotic neurodegeneration and long-term behavioral and neurochemical changes (Wang et al., 2001; du Bois et al., 2008a, 2009a). Pups were sacrificed by decapitation on PN12, or by CO2 asphyxiation at 5 weeks and 20 weeks. These time points were chosen to represent perinatal, adolescent and adult ages which are important and distinct brain developmental periods (Andersen, 2003) and because NMDA receptor and Nrg1 expression levels vary across these developmental time-points (Colwell et al., 1998; Corfas et al., 1995). The prefrontal cortex and hippocampus were dissected on ice with the aid of a standard rat brain atlas (Paxinos and Watson, 1997), snap frozen in liquid nitrogen and stored at − 80 °C. The prefrontal cortex (equivalent to the PrL, prelimbic cortex) was dissected at the level of Bregma 3.7 mm to 2.7 mm. At the level of Bregma − 1.6 mm, the cortex was peeled away and the hippocampus was collected. These regions were specifically examined for this study as both areas are highly implicated in schizophrenia pathology and our previous studies show altered neurotransmitter receptor binding in these areas in animal models (du Bois et al., 2008b, 2009a, 2009b). Tissue was homogenized in NP-40 lysis buffer (Invitrogen Australia Pty Ltd, Mulgrave, Australia); containing protease inhibitor cocktail, beta-glycerophoshate and phenylmethanesulfonylfluoride (PMSF; Sigma). Homogenates were spun at 20,000 ×g for 10 min at 4 °C. The supernatants were collected and equal amounts of protein were separated on 4–12% Bis–Tris gels (Bio-Rad Laboratories, Gladesville, Australia) using SDS-PAGE. Protein concentration was determined using the DC assay (Bio-Rad), according to the manufacturer's instructions. Following electrophoresis (100 V for 50 min), proteins were transferred to polyvinylidene difluoride membranes (200 V for 60 min). Membranes were blocked in 5% bovine serum albumin (BSA), followed by incubation with the primary antibody in 1% BSA overnight at 4 °C. Following washes (3× 5 min) in Tris Buffered Saline +0.1% Tween 20 (TBST), membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at 25 °C. Blots were visualized using enhanced chemiluminescence (ECL) detection reagents (Ge Healthcare, Rydalmere, Australia). The bands corresponding to the various proteins of interest were scanned and densitometrically analyzed using an automatic imaging analysis system, Quantity One (BioRad). All quantitative analyses were normalized to β-actin, based on previous studies in the literature (Chong et al., 2008).
2.3. Antibodies Polyclonal antibodies for the NR1 subunit of the NMDA receptor (sc-1467), phosphorylated NR1 (p-NR1; ser896/897; sc-12890), NR2A subunit of the NMDA receptor (sc-1468), NR2B subunit of the NMDA receptor (sc-1469), Akt1,2,3 (sc-8312), p-Akt1,2,3 (ser473; sc-7985-R) Nrg1 (sc-348), erbB4 (sc-283) and p-erbB4 (Tyr1056; sc-33040) were purchased from Santa Cruz Biotechnology Inc (Scoresby, Victoria, Australia). Polyclonal antibodies for PSD-95 (ab18258), p-PSD-95 (ser295; ab16495), p-NR2A (Y1325; ab16646) and p-NR2B (ser1480; ab18533) were purchased from Abcam (Waterloo, Australia). Primary antibody dilution ranged from 1:100 to 1:3,000. Secondary antibodies
T.M. du Bois et al. were purchased from Millipore (AP307P, AP308P; North Ryde, Australia) and were used at a concentration of 1:3,000. β-Actin was purchased from Millipore (MAB1501) and used at a concentration of 1:100,000. Antibody specificity was determined by incubating antibodies with 100–200 fold molar excess of their corresponding epitope peptides; or from the documented literature (Cobbs et al., 2007; Marvizon et al., 2002) and antibody datasheets.
2.4. Statistical analyses The relative density for each protein of interest is based on 5 biological replicates per treatment and time-point, with experiments performed in 2–4 technical replicates. Differences between treatment and control groups were analyzed at individual time-points and brain regions using student's t-tests. Statistical significance was set at an alpha level of p b 0.05.
3. Results Single specific bands were identified at approximately corresponding molecular weights for NR1, p-NR1, NR2A, p-NR2A, NR2B, p-NR2B, Akt, p-Akt (60 kDa band), PSD-95, p-PSD-95. Nrg1 and erbB4 displayed multiple specific bands. For this study, the 85 kDa band of Nrg1 and the 185 kDa band of erbB4 were quantified, which are suggested to represent the Nrg1 type III isoform (Cabedo et al., 2004) and erbB4 full length protein (Chong et al., 2008; Plowman et al., 1993). The Nrg1 type III band was specifically chosen as it is highly implicated in schizophrenia and animal models. For example, Nrg1 type III knockout mice are found to exhibit more pronounced schizophrenia-like behavioral deficits, in particular cognitive deficits, which other Nrg1 knockouts do not (Chen et al., 2008). These bands were eliminated following preabsorbtion of Nrg1 or erbB4 antibodies with their epitope peptides, indicating that the bands were specific for Nrg1 and erbB4.
3.1. Prefrontal cortex 3.1.1. PN12 In the prefrontal cortex at PN12, PCP treatment caused a significant reduction in expression of Nrg1 (−30%, p b 0.001) and erbB4 (−25%, p b 0.05) (Fig. 1A). Levels of p-Akt were also significantly reduced (−19%, p b 0.05). Changes in NMDA receptor subunits did not reach statistical significance.
3.1.2. 5 weeks At 5 weeks in the prefrontal cortex, Nrg1 expression remained decreased (− 13%, p b 0.01), while significant increases were seen in total erbB4 (+ 27%, p b 0.01), and phosphorylated levels of erbB4 (+ 18%, p b 0.001; Fig. 1B). When looking at the effects on the NMDA receptor system, there was a significant increase in phosphorylated levels of NR2A (+ 32%, p b 0.01). Total levels of the anchoring molecule, PSD-95, were also significantly increased (+ 11%, p b 0.05).
3.1.3. 20 weeks Conversely, in the prefrontal cortex at 20 weeks, Nrg1 expression levels were significantly increased in the PCP group (+33%, p b 0.01), while erbB4 (−18%, p b 0.01) and phosphorylated erbB4 (−16%, p b 0.05) levels were significantly decreased. Expression levels of NMDA receptor subunits and PSD-95 returned to control level (Fig. 1C).
Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life
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Figure 1 Protein expression in the prefrontal cortex at A) PN12, B) 5 week and C) 20 week time-points. ^Statistical trend; *p b 0.05; **p b 0.01; ***p b 0.001 vs. controls.
3.2. Hippocampus 3.2.1. PN12 In the hippocampus at PN12, PCP treatment did not alter Nrg1 or erbB4 expression levels (Fig. 2A). However, in terms of the NMDA receptor system, treatment significantly increased phosphorylated levels of NR1 (+ 20%, p b 0.05; Fig. 2A). PSD-95 also showed a significant increase in expression (+ 24%, p b 0.05).
3.2.2. 5 weeks At 5 weeks in the hippocampus, PCP treatment did not alter Nrg1 or erbB4 expression levels (Fig. 2B). When looking at the NMDA receptor system, treatment significantly increased NR2A (+32%, p b 0.05) and NR1 (+24%, p b 0.05) expression levels (Fig. 2B). Additionally, levels of PSD-95 (+27%, p b 0.05) and phosphorylated Akt (+22%, p b 0.01) were significantly increased (Fig. 2B).
3.2.3. 20 weeks In the hippocampus at 20 weeks, there was a slight but significant reduction in expression of phosphorylated erbB4 levels (− 13%, p b 0.001; Fig. 2C). In terms of treatment effects on the NMDA receptor system, PCP-treated rats showed a significant reduction in total NR2A (−22%, p b 0.001) and phosphorylated NR2A (− 26%, p b 0.05), as well as NR2B (−29%, p b 0.05) levels (Fig. 2C). Total levels of NR1 and phosphorylated levels of
NR2B also tended to be decreased though not significantly (p's N 0.05). In addition, PSD-95 expression was significantly decreased at this time-point (−23%, p b 0.05; Fig. 2C).
4. Discussion This study for the first time has examined the effects of perinatal PCP treatment on expression of Nrg1/erbB4 during development in the rat brain in an effort to see whether altering NMDA function has long term effects on Nrg1/erbB4 expression and activation. In the cortex, PCP altered Nrg1 and total and phosphorylated levels of erbB4 throughout development while in the hippocampus, PCP did not alter total Nrg1/erbB4 levels, although levels of phosphorylated erbB4 were reduced at adulthood. Results are discussed in the context of parallel changes to NMDA receptor subunits, PSD-95 and Akt.
4.1. PCP affects cortical expression of Nrg1/erbB4 throughout development In the present study, PCP treatment reduced Nrg1 and erbB4 expressions during development in the cortex, as well as phosphorylated Akt, which is a downstream target of
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Figure 2 Protein expression in the hippocampus at A) PN12, B) 5 week and C) 20 week timepoints. ^Statistical trend; *p b 0.05; **p b 0.01; ***p b 0.001 vs. controls.
erbB4. This result is not surprising given that NMDA antagonist treatment early during development block excitation of neurons, triggering decreased activation of cell survival pathways (i.e. Akt) and ultimately widespread apoptosis (Lei et al., 2008). Nrg1/erbB4 signaling plays a crucial role in the control of cortical GABA circuitry development (Fazzari et al., 2010). It has recently been confirmed by Fazzari et al. (2010) that the erbB4 receptor is specifically expressed on inhibitory interneurons, particularly parvalbumin-containing chandelier and basket cells, where it localized to axon terminals and postsynaptic densities receiving glutamatergic input. Here, erbB4 promotes formation of axo-axonic inhibitory synapses over pyramidal neurons and regulates formation of excitatory synapses to interneurons. ErbB4 has also shown to be expressed exclusively on interneurons in the hippocampus (Buonanno, 2010). Given the important role of Nrg1/erbB4 signaling on GABAergic system development, reduced cortical Nrg1/erbB4 protein expression early in brain development seen shortly after perinatal PCP treatment could affect the development of GABAergic interneurons and wiring of GABAergic circuits. In this regard, Wang et al. (2008) have shown that perinatal PCP treatment causes a loss of parvalbumin positive interneurons in the cortex.
Similar losses are seen after treatment with another NMDA receptor antagonist, MK-801 (Braun et al., 2007). Moreover, we have previously shown increased GABAA receptor binding in several brain regions in adulthood following perinatal PCP treatment, which may represent a compensatory upregulation of receptors in response to deficits in GABAergic system function (du Bois et al., 2009a). When looking at Nrg1/erbB4 expression at adolescence, Nrg1 remained slightly lower in PCP-treated rats, while both erbB4 and phosphorylated levels of erbB4 were upregulated. This may represent a compensatory upregulation in response to reduced signaling earlier in development. Also occurring at this time-point was increased PSD-95 and phophorylated levels of the NR2A subunit, suggesting increased activation of NMDA receptors. PSD-95 may be facilitating enhanced erbB4 and NMDA receptor signaling at the synapse at adolescence in PCP-treated rats. At the 20 week time-point, Nrg1 was increased, while total and phosphorylated erbB4 levels were decreased. NMDA receptor subunit and PSD-95 levels returned to control. The latter finding is consistent with our previous autoradiography data showing increased NMDA receptor binding at adolescence in cortical areas after perinatal PCP treatment, then a return to control level at adulthood (du Bois
Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life et al., 2009a). The mechanisms underlying the switch in direction of change of Nrg1 are not completely understood at present. However it can be speculated that an increase in NMDA receptor activity (increased p-2A) at adolescence in the cortex drives the increase in Nrg1 at later stages as a feedback mechanism, as Nrg1 signaling attenuates NMDA receptor activity (Bjarnadottir et al., 2007; Gu et al., 2005; Hahn et al., 2006). This is supported by the finding that soluble Nrg1 is released from presynaptic nerve terminals in response to neuronal activity (Fernandez et al., 2000; Ozaki et al., 2004).
4.2. Hippocampal Nrg1/erbB4 expression is largely unaffected altered by perinatal PCP treatment PCP treatment affected the NMDA receptor system throughout development in the hippocampus, while changes to the Nrg1 system were limited to a small but significant decrease in phosphorylated erbB4 levels at adulthood. At PN12, activation of NR1 was increased, along with increased PSD-95 expression. By the adolescent time-point, more changes were evident in the NMDA receptor system, including increases in NR2A and NR1, as well as increases in phosphorylated Akt and PSD-95. Other studies also report an upregulation of NMDA receptor levels after PCP treatment (Sircar, 2003; Wang et al., 2001). The upregulation of NMDA receptors at adolescence may have allowed for the accumulation of toxic levels of intracellular free calcium, even more so if endogenous levels of glutamate are increased in this model through a loss of GABAergic inhibition of pyramidal cell output. On its own, calcium overload acts to suppress NR1 transcription (Gascon et al., 2005). This may explain why at 20 weeks, there was a switch in the direction of change with decreases in NMDA receptor subunits 2A, phosphorylated 2A and 2B. Phosphorylated levels of PSD-95 were also significantly decreased. A decrease in p-PSD-95 at ser295 could affect the ability of PSD-95 to accumulate and recruit receptors and decrease synaptic potentiation (Kim et al., 2007). Instability of the PSD-95/NR1 + NR2A complex can lead to receptor internalization and ultimately downregulation of NMDA receptors (Dong et al., 2004) and as a consequence may impact on NMDA receptor-Nrg1 interactions by affecting NMDA/ErbB4 receptor cross-talk. This may be why a decrease in erbB4 activity appears without a change in Nrg1 or erbB4 levels. These deficits in hippocampal NMDA receptor function may contribute to the deficits in cognitive function in rodents in the long term after treatment with PCP (see du Bois and Huang, 2007). It is interesting that the prefrontal cortex and hippocampus did not show the same pattern of alterations to protein expression following perinatal PCP treatment in this study. In the hippocampus, more changes were evident with the NMDA receptor; while in the prefrontal cortex, Nrg1/erbB4 were more affected compared to the hippocampus. It is common to see varying patterns of change within the brain due to its heterogeneity in cyto- and chemo-architecture among brain regions. Perinatal PCP treatment may have affected the developmental trajectories and connections of these proteins/systems to varying degrees early on in development.
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5. Summary and conclusion This study has demonstrated that Nrg1/erbB4 expression and activation levels are altered throughout development as a consequence of NMDA receptor antagonism during early brain development. This demonstrates that changes in Nrg1/erbB4 can be secondary to that of the NMDA receptor, which is relevant to both the NMDA receptor hypofunction and neurodevelopmental hypotheses of schizophrenia. These findings are relevant to pathological changes which may be occurring in the schizophrenia brain, though it is difficult to directly compare specific changes in expression across species. However in future studies it will still be useful to examine the effects of specific NMDA receptor blockers on Nrg1/erbB4 signaling and how this relates to GABAergic system function as these three systems are closely linked and have a disrupted balance in schizophrenia.
Role of the funding source Funding for this study was provided by National Health and Medical Research Council (NHMRC) Project Grant ID573426, as well as the Schizophrenia Research Institute (SRI) Australia, utilizing infrastructure from New South Wales Health. The funding sources had no further role in study design, in the collection, analysis and interpretation of data; in the writing of the report and in the decision to submit the paper for publication.
Contributors Xu-Feng Huang, Kelly Newell and Teresa du Bois designed the study. Teresa du Bois and Kelly Newell performed the animal experiments. Teresa du Bois wrote the protocol for and performed the Western blot experiments, quantified the results, performed the statistical analyses and wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest The authors declare that, except for income received from primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.
Acknowledgments None.
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Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life Teresa Marie du Bois ⁎, Kelly Anne Newell, Xu-Feng Huang Centre for Translational Neuroscience, School of Health Sciences, Illawarra Health and Medical Research Institute, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia Schizophrenia Research Institute (SRI), Sydney, Australia
Received 25 January 2011; received in revised form 16 August 2011; accepted 1 September 2011
KEYWORDS Neuregulin 1; erbB4; NMDA receptor; Schizophrenia; Rat; Brain
Abstract Schizophrenia is a complex and devastating mental disorder of unknown etiology. Hypofunction of N-methyl-D-aspartate (NMDA) receptors are implicated in the disorder, since phencyclidine (PCP) and other NMDA receptor antagonists mimic schizophrenia-like symptoms in humans and animals so well. Moreover, genetic linkage and post mortem studies strongly suggest a role for altered neuregulin 1 (Nrg1)/erbB4 signaling in schizophrenia pathology. This study investigated the relationship between the NMDA receptor and Nrg1 signaling pathways using the perinatal PCP animal model. Rats (n = 5/group) were treated with PCP (10 mg/kg) or saline on postnatal days (PN) 7, 9 and 11 and were sacrificed on PN12, 5 weeks and 20 weeks for biochemical analyses. Western blotting was used to determine total and phosphorylated levels of proteins involved in NMDA receptor/Nrg1 signaling in the prefrontal cortex and hippocampus. In the cortex, PCP treatment altered Nrg1/erbB4 expression levels throughout development, including decreased Nrg1 and erbB4 at PN12 (−25–30%; p b 0.05); increased erbB4 and p-erbB4 (+18–27%; p b 0.01) at 5 weeks; and decreased erbB4 and p-erbB4 (−16–18%; p b 0.05) along with increased Nrg1 (+33%; p b 0.01) at 20 weeks. In the hippocampus, levels of Nrg1/erbB4 were largely unaffected apart from a significant decrease in p-erbB4 at 20 weeks (−13%; p b 0.001); however NMDA receptor subunits and PSD-95 showed increases at PN12 and 5 weeks (+20–32%; p b 0.05), and decreases at 20 weeks (−22–29%; p b 0.05). This study shows that NMDA receptor antagonism early in development can have long term effects on Nrg1/erbB4 expression which could be important in understanding pathological processes which might be involved in schizophrenia. © 2011 Elsevier B.V. and ECNP. All rights reserved.
⁎ Corresponding author at: University of Wollongong, School of Health Sciences, Northfields Avenue, NSW 2522, Australia. Tel.: +61 2 42215294; fax: +61 2 42215945. E-mail address: [email protected] (T.M. du Bois). 0924-977X/$ - see front matter © 2011 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2011.09.002
Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life
1. Introduction Glutamatergic N-methyl-D-aspartate (NMDA) receptors play a key role in brain development, controlling processes such as neuronal migration, synaptogenesis and synaptic plasticity (McDonald and Johnston, 1990; Pearce et al., 1987). The NMDA receptor is comprised of a heteromeric assembly of subunits, including an obligatory NR1 subunit plus distinct combinations of NR2A–D subunits, and rarely NR3 subunits (Madry et al., 2007). The immature form of the NMDA receptor channel complex, present at postnatal day (PN) 1–14 in rats, is highly sensitive to glutamate, with less sensitivity to magnesium block (Sircar, 2000). The longest and largest NMDA receptor currents are also observed at this time (Colwell et al., 1998; Hestrin, 1992). During this period, the brain is vulnerable to the toxic effects of modulation of the NMDA receptor (Haberny et al., 2002). The physiological properties of NMDA receptors correlate with developmental variation in receptor subunits. In rats, the only forms present at birth are the NR1, NR2B and NR2D subunits; during postnatal weeks 1–3, NR2B and NR2D decline whereas NR2A becomes abundant, then all subtypes decrease to adult levels (Wenzel et al., 1996, 1997). NR2C emerges at day 5, peaks at 10 days and then remains unchanged (Wenzel et al., 1997). In rodents, treatment with NMDA receptor antagonists such as phencyclidine (PCP) and MK-801 during the perinatal period induces large increases in neuronal apoptosis (Hansen et al., 2004; Harris et al., 2003; Ikonomidou et al., 1999; Wang et al., 2001) and produces schizophrenia-relevant behavior such as impaired cognitive function (Andersen and Pouzet, 2004; Sircar, 2003; Stefani and Moghaddam, 2005; Wang et al., 2001; Wiley et al., 2003), sensorimotor gating deficits (Harris et al. 2003; Wang et al. 2001) and hyperlocomotor activity (du Bois et al., 2008a; Facchinetti et al., 1993; Wang et al., 2001). Further, we have shown that perinatal PCP treatment alters the expression of neurotransmitter receptors implicated in schizophrenia including dopamine D2, NMDA, GABAA and muscarinic M1/4 receptors (du Bois et al., 2008b, 2009a, 2009b). Hypofunction of NMDA receptors has been implicated in schizophrenia, since NMDA receptor antagonists such as PCP and ketamine mimic schizophrenia symptoms so closely in healthy individuals and strongly exacerbate symptoms in schizophrenia patients (Javitt and Zukin, 1991). Studies have also demonstrated changes in NMDA receptor binding, transcription and subunit expression in different brain regions of schizophrenia subjects; and many candidate susceptibility genes for schizophrenia such as D-amino acid oxidase (DAAO), dybindin-1, Disrupted in schizophrenia 1 (DISC-1) and neuregulin 1 (Nrg1) can directly or indirectly affect NMDA receptor signaling (see Banerjee et al., 2010 for review). Like the NMDAR, Nrg1 also plays a key role in brain development, regulating many important processes in both the developing and the mature brain (Harrison and Law, 2006). Consistent with its important developmental role, Nrg1 mRNA expression is highest early in development and declines with age (Corfas et al., 1995). The Nrg1 gene gives rise to at least 30 different isoforms, the most well characterized of which have been grouped into 3 classes, types I, II and III (Harrison and Law, 2006). In rodents, these isoforms are known to be expressed in a region and cortical layer specific
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manner (Kerber et al., 2003), each with independent functions in different brain regions (Meyer et al., 1997). Nrg1 functions are largely mediated by the receptor tyrosine kinases erbB2, 3 and 4. ErbB4 has been indicated as the predominant receptor for Nrg1 (Stefansson et al., 2004). It has been shown that erbB4 receptor protein expression does not vary with age (Thompson et al., 2007). Four structurally different erbB4 receptor isoforms have been identified in neural tissue: JM-a, JM-b, CYT-1 and CYT-2 (Junttila et al., 2000). ErbB4 receptor expression has been shown to be confined to inhibitory (GABAergic) interneurons (especially parvalbumincontaining interneurons), specifically at the postsynaptic density of dendrites receiving glutamatergic synapses (Fazzari et al., 2010). Linkage studies have identified both the Nrg1 and the erbB4 receptor as susceptibility genes for schizophrenia and there is evidence of altered mRNA and protein expression of Nrg1 and erbB4 receptors in post mortem human schizophrenia brain tissue in an isoform-specific manner (see Banerjee et al., 2010 for review). Given that Nrg1/erbB4 genetic mutations likely contribute to only a small proportion of schizophrenia cases, it has been suggested that changes in expression levels of Nrg1 and erbB4 in the schizophrenia brain are secondary to other molecular changes related to the disease such as NMDA receptor hypofunction (Li et al., 2007). ErbB4 receptors are anchored at PSD-95 along with NMDA receptor 2A–D subunits (Fazzari et al., 2010; Garcia et al., 2000; Huang et al., 2000), which places the glutamatergic NMDA receptor and Nrg1 signaling pathways together at the synapse, allowing for crosstalk between the systems. While studies have shown that Nrg1 can influence NMDA receptor function (Bjarnadottir et al., 2007; Gu et al., 2005; Li et al., 2007), it is unclear how NMDA receptor activity can modulate Nrg1/erbB4. This information is important in order to understand how NMDA receptor, Nrg1 and GABAergic systems interact together to control synaptic activity and brain function. The present study investigated whether perinatal PCP treatment could lead to long term changes in Nrg1 and erbB4 expression in the rat brain. This study examined total and activated (phosphorylated) forms of key proteins linking NMDA receptor and Nrg1/erbB4 systems including selected NMDA receptor subunits, Nrg1, erbB4 and the anchoring protein PSD-95. We were also interested in Akt expression because erbB4 receptor signaling activates the phosphotidylinositol-3 kinase/Akt pathway (Junttila et al., 2000). We hypothesized that Nrg1/erbB4, PSD-95 and Akt expression would be highly influenced by glutamatergic/ NMDA receptor activity, and follow the same expression pattern as the NMDA receptor.
2. Experimental procedures 2.1. Animals Timed, pregnant female Sprague–Dawley rats were obtained from the Animal Resource Centre (Perth, Australia) at day 14 of gestation. The dams were housed individually with a regular 12 h light–dark cycle (lights on 07:00 h, off at 19:00 h) with food and water ad libitum. Day of birth was considered PN0. Pups were sexed on PN7 and litters were randomly assigned to PCP or saline groups. Each litter consisted of ten to twelve pups. Females were kept in litters, but only male pups were used for these experiments. This study was approved by the
358 Animal Ethics Committee of the University of Wollongong, and procedures complied with the Australian Code of Practice for the Care and Use of Animal for Scientific Purposes, which conforms to International Guiding Principles for Biomedical Research Involving Animals. All efforts were made to minimize numbers of animals used and their suffering.
2.2. Experimental design Male rat pups were a given subcutaneous injection on PN 7, 9 and 11 with 10 mg/kg PCP (Sigma, Castle Hill, NSW, Australia) or saline at a volume of 1 ml/kg once per day between 09:00 h and 10:00 h. The dose and treatment regime is based on previous experiments showing that 10 mg/kg PCP at PN7, 9 and 11 induces apoptotic neurodegeneration and long-term behavioral and neurochemical changes (Wang et al., 2001; du Bois et al., 2008a, 2009a). Pups were sacrificed by decapitation on PN12, or by CO2 asphyxiation at 5 weeks and 20 weeks. These time points were chosen to represent perinatal, adolescent and adult ages which are important and distinct brain developmental periods (Andersen, 2003) and because NMDA receptor and Nrg1 expression levels vary across these developmental time-points (Colwell et al., 1998; Corfas et al., 1995). The prefrontal cortex and hippocampus were dissected on ice with the aid of a standard rat brain atlas (Paxinos and Watson, 1997), snap frozen in liquid nitrogen and stored at − 80 °C. The prefrontal cortex (equivalent to the PrL, prelimbic cortex) was dissected at the level of Bregma 3.7 mm to 2.7 mm. At the level of Bregma − 1.6 mm, the cortex was peeled away and the hippocampus was collected. These regions were specifically examined for this study as both areas are highly implicated in schizophrenia pathology and our previous studies show altered neurotransmitter receptor binding in these areas in animal models (du Bois et al., 2008b, 2009a, 2009b). Tissue was homogenized in NP-40 lysis buffer (Invitrogen Australia Pty Ltd, Mulgrave, Australia); containing protease inhibitor cocktail, beta-glycerophoshate and phenylmethanesulfonylfluoride (PMSF; Sigma). Homogenates were spun at 20,000 ×g for 10 min at 4 °C. The supernatants were collected and equal amounts of protein were separated on 4–12% Bis–Tris gels (Bio-Rad Laboratories, Gladesville, Australia) using SDS-PAGE. Protein concentration was determined using the DC assay (Bio-Rad), according to the manufacturer's instructions. Following electrophoresis (100 V for 50 min), proteins were transferred to polyvinylidene difluoride membranes (200 V for 60 min). Membranes were blocked in 5% bovine serum albumin (BSA), followed by incubation with the primary antibody in 1% BSA overnight at 4 °C. Following washes (3× 5 min) in Tris Buffered Saline +0.1% Tween 20 (TBST), membranes were incubated with horseradish peroxidase conjugated secondary antibodies for 1 h at 25 °C. Blots were visualized using enhanced chemiluminescence (ECL) detection reagents (Ge Healthcare, Rydalmere, Australia). The bands corresponding to the various proteins of interest were scanned and densitometrically analyzed using an automatic imaging analysis system, Quantity One (BioRad). All quantitative analyses were normalized to β-actin, based on previous studies in the literature (Chong et al., 2008).
2.3. Antibodies Polyclonal antibodies for the NR1 subunit of the NMDA receptor (sc-1467), phosphorylated NR1 (p-NR1; ser896/897; sc-12890), NR2A subunit of the NMDA receptor (sc-1468), NR2B subunit of the NMDA receptor (sc-1469), Akt1,2,3 (sc-8312), p-Akt1,2,3 (ser473; sc-7985-R) Nrg1 (sc-348), erbB4 (sc-283) and p-erbB4 (Tyr1056; sc-33040) were purchased from Santa Cruz Biotechnology Inc (Scoresby, Victoria, Australia). Polyclonal antibodies for PSD-95 (ab18258), p-PSD-95 (ser295; ab16495), p-NR2A (Y1325; ab16646) and p-NR2B (ser1480; ab18533) were purchased from Abcam (Waterloo, Australia). Primary antibody dilution ranged from 1:100 to 1:3,000. Secondary antibodies
T.M. du Bois et al. were purchased from Millipore (AP307P, AP308P; North Ryde, Australia) and were used at a concentration of 1:3,000. β-Actin was purchased from Millipore (MAB1501) and used at a concentration of 1:100,000. Antibody specificity was determined by incubating antibodies with 100–200 fold molar excess of their corresponding epitope peptides; or from the documented literature (Cobbs et al., 2007; Marvizon et al., 2002) and antibody datasheets.
2.4. Statistical analyses The relative density for each protein of interest is based on 5 biological replicates per treatment and time-point, with experiments performed in 2–4 technical replicates. Differences between treatment and control groups were analyzed at individual time-points and brain regions using student's t-tests. Statistical significance was set at an alpha level of p b 0.05.
3. Results Single specific bands were identified at approximately corresponding molecular weights for NR1, p-NR1, NR2A, p-NR2A, NR2B, p-NR2B, Akt, p-Akt (60 kDa band), PSD-95, p-PSD-95. Nrg1 and erbB4 displayed multiple specific bands. For this study, the 85 kDa band of Nrg1 and the 185 kDa band of erbB4 were quantified, which are suggested to represent the Nrg1 type III isoform (Cabedo et al., 2004) and erbB4 full length protein (Chong et al., 2008; Plowman et al., 1993). The Nrg1 type III band was specifically chosen as it is highly implicated in schizophrenia and animal models. For example, Nrg1 type III knockout mice are found to exhibit more pronounced schizophrenia-like behavioral deficits, in particular cognitive deficits, which other Nrg1 knockouts do not (Chen et al., 2008). These bands were eliminated following preabsorbtion of Nrg1 or erbB4 antibodies with their epitope peptides, indicating that the bands were specific for Nrg1 and erbB4.
3.1. Prefrontal cortex 3.1.1. PN12 In the prefrontal cortex at PN12, PCP treatment caused a significant reduction in expression of Nrg1 (−30%, p b 0.001) and erbB4 (−25%, p b 0.05) (Fig. 1A). Levels of p-Akt were also significantly reduced (−19%, p b 0.05). Changes in NMDA receptor subunits did not reach statistical significance.
3.1.2. 5 weeks At 5 weeks in the prefrontal cortex, Nrg1 expression remained decreased (− 13%, p b 0.01), while significant increases were seen in total erbB4 (+ 27%, p b 0.01), and phosphorylated levels of erbB4 (+ 18%, p b 0.001; Fig. 1B). When looking at the effects on the NMDA receptor system, there was a significant increase in phosphorylated levels of NR2A (+ 32%, p b 0.01). Total levels of the anchoring molecule, PSD-95, were also significantly increased (+ 11%, p b 0.05).
3.1.3. 20 weeks Conversely, in the prefrontal cortex at 20 weeks, Nrg1 expression levels were significantly increased in the PCP group (+33%, p b 0.01), while erbB4 (−18%, p b 0.01) and phosphorylated erbB4 (−16%, p b 0.05) levels were significantly decreased. Expression levels of NMDA receptor subunits and PSD-95 returned to control level (Fig. 1C).
Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life
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Figure 1 Protein expression in the prefrontal cortex at A) PN12, B) 5 week and C) 20 week time-points. ^Statistical trend; *p b 0.05; **p b 0.01; ***p b 0.001 vs. controls.
3.2. Hippocampus 3.2.1. PN12 In the hippocampus at PN12, PCP treatment did not alter Nrg1 or erbB4 expression levels (Fig. 2A). However, in terms of the NMDA receptor system, treatment significantly increased phosphorylated levels of NR1 (+ 20%, p b 0.05; Fig. 2A). PSD-95 also showed a significant increase in expression (+ 24%, p b 0.05).
3.2.2. 5 weeks At 5 weeks in the hippocampus, PCP treatment did not alter Nrg1 or erbB4 expression levels (Fig. 2B). When looking at the NMDA receptor system, treatment significantly increased NR2A (+32%, p b 0.05) and NR1 (+24%, p b 0.05) expression levels (Fig. 2B). Additionally, levels of PSD-95 (+27%, p b 0.05) and phosphorylated Akt (+22%, p b 0.01) were significantly increased (Fig. 2B).
3.2.3. 20 weeks In the hippocampus at 20 weeks, there was a slight but significant reduction in expression of phosphorylated erbB4 levels (− 13%, p b 0.001; Fig. 2C). In terms of treatment effects on the NMDA receptor system, PCP-treated rats showed a significant reduction in total NR2A (−22%, p b 0.001) and phosphorylated NR2A (− 26%, p b 0.05), as well as NR2B (−29%, p b 0.05) levels (Fig. 2C). Total levels of NR1 and phosphorylated levels of
NR2B also tended to be decreased though not significantly (p's N 0.05). In addition, PSD-95 expression was significantly decreased at this time-point (−23%, p b 0.05; Fig. 2C).
4. Discussion This study for the first time has examined the effects of perinatal PCP treatment on expression of Nrg1/erbB4 during development in the rat brain in an effort to see whether altering NMDA function has long term effects on Nrg1/erbB4 expression and activation. In the cortex, PCP altered Nrg1 and total and phosphorylated levels of erbB4 throughout development while in the hippocampus, PCP did not alter total Nrg1/erbB4 levels, although levels of phosphorylated erbB4 were reduced at adulthood. Results are discussed in the context of parallel changes to NMDA receptor subunits, PSD-95 and Akt.
4.1. PCP affects cortical expression of Nrg1/erbB4 throughout development In the present study, PCP treatment reduced Nrg1 and erbB4 expressions during development in the cortex, as well as phosphorylated Akt, which is a downstream target of
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Figure 2 Protein expression in the hippocampus at A) PN12, B) 5 week and C) 20 week timepoints. ^Statistical trend; *p b 0.05; **p b 0.01; ***p b 0.001 vs. controls.
erbB4. This result is not surprising given that NMDA antagonist treatment early during development block excitation of neurons, triggering decreased activation of cell survival pathways (i.e. Akt) and ultimately widespread apoptosis (Lei et al., 2008). Nrg1/erbB4 signaling plays a crucial role in the control of cortical GABA circuitry development (Fazzari et al., 2010). It has recently been confirmed by Fazzari et al. (2010) that the erbB4 receptor is specifically expressed on inhibitory interneurons, particularly parvalbumin-containing chandelier and basket cells, where it localized to axon terminals and postsynaptic densities receiving glutamatergic input. Here, erbB4 promotes formation of axo-axonic inhibitory synapses over pyramidal neurons and regulates formation of excitatory synapses to interneurons. ErbB4 has also shown to be expressed exclusively on interneurons in the hippocampus (Buonanno, 2010). Given the important role of Nrg1/erbB4 signaling on GABAergic system development, reduced cortical Nrg1/erbB4 protein expression early in brain development seen shortly after perinatal PCP treatment could affect the development of GABAergic interneurons and wiring of GABAergic circuits. In this regard, Wang et al. (2008) have shown that perinatal PCP treatment causes a loss of parvalbumin positive interneurons in the cortex.
Similar losses are seen after treatment with another NMDA receptor antagonist, MK-801 (Braun et al., 2007). Moreover, we have previously shown increased GABAA receptor binding in several brain regions in adulthood following perinatal PCP treatment, which may represent a compensatory upregulation of receptors in response to deficits in GABAergic system function (du Bois et al., 2009a). When looking at Nrg1/erbB4 expression at adolescence, Nrg1 remained slightly lower in PCP-treated rats, while both erbB4 and phosphorylated levels of erbB4 were upregulated. This may represent a compensatory upregulation in response to reduced signaling earlier in development. Also occurring at this time-point was increased PSD-95 and phophorylated levels of the NR2A subunit, suggesting increased activation of NMDA receptors. PSD-95 may be facilitating enhanced erbB4 and NMDA receptor signaling at the synapse at adolescence in PCP-treated rats. At the 20 week time-point, Nrg1 was increased, while total and phosphorylated erbB4 levels were decreased. NMDA receptor subunit and PSD-95 levels returned to control. The latter finding is consistent with our previous autoradiography data showing increased NMDA receptor binding at adolescence in cortical areas after perinatal PCP treatment, then a return to control level at adulthood (du Bois
Perinatal phencyclidine treatment alters neuregulin 1/erbB4 expression and activation in later life et al., 2009a). The mechanisms underlying the switch in direction of change of Nrg1 are not completely understood at present. However it can be speculated that an increase in NMDA receptor activity (increased p-2A) at adolescence in the cortex drives the increase in Nrg1 at later stages as a feedback mechanism, as Nrg1 signaling attenuates NMDA receptor activity (Bjarnadottir et al., 2007; Gu et al., 2005; Hahn et al., 2006). This is supported by the finding that soluble Nrg1 is released from presynaptic nerve terminals in response to neuronal activity (Fernandez et al., 2000; Ozaki et al., 2004).
4.2. Hippocampal Nrg1/erbB4 expression is largely unaffected altered by perinatal PCP treatment PCP treatment affected the NMDA receptor system throughout development in the hippocampus, while changes to the Nrg1 system were limited to a small but significant decrease in phosphorylated erbB4 levels at adulthood. At PN12, activation of NR1 was increased, along with increased PSD-95 expression. By the adolescent time-point, more changes were evident in the NMDA receptor system, including increases in NR2A and NR1, as well as increases in phosphorylated Akt and PSD-95. Other studies also report an upregulation of NMDA receptor levels after PCP treatment (Sircar, 2003; Wang et al., 2001). The upregulation of NMDA receptors at adolescence may have allowed for the accumulation of toxic levels of intracellular free calcium, even more so if endogenous levels of glutamate are increased in this model through a loss of GABAergic inhibition of pyramidal cell output. On its own, calcium overload acts to suppress NR1 transcription (Gascon et al., 2005). This may explain why at 20 weeks, there was a switch in the direction of change with decreases in NMDA receptor subunits 2A, phosphorylated 2A and 2B. Phosphorylated levels of PSD-95 were also significantly decreased. A decrease in p-PSD-95 at ser295 could affect the ability of PSD-95 to accumulate and recruit receptors and decrease synaptic potentiation (Kim et al., 2007). Instability of the PSD-95/NR1 + NR2A complex can lead to receptor internalization and ultimately downregulation of NMDA receptors (Dong et al., 2004) and as a consequence may impact on NMDA receptor-Nrg1 interactions by affecting NMDA/ErbB4 receptor cross-talk. This may be why a decrease in erbB4 activity appears without a change in Nrg1 or erbB4 levels. These deficits in hippocampal NMDA receptor function may contribute to the deficits in cognitive function in rodents in the long term after treatment with PCP (see du Bois and Huang, 2007). It is interesting that the prefrontal cortex and hippocampus did not show the same pattern of alterations to protein expression following perinatal PCP treatment in this study. In the hippocampus, more changes were evident with the NMDA receptor; while in the prefrontal cortex, Nrg1/erbB4 were more affected compared to the hippocampus. It is common to see varying patterns of change within the brain due to its heterogeneity in cyto- and chemo-architecture among brain regions. Perinatal PCP treatment may have affected the developmental trajectories and connections of these proteins/systems to varying degrees early on in development.
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5. Summary and conclusion This study has demonstrated that Nrg1/erbB4 expression and activation levels are altered throughout development as a consequence of NMDA receptor antagonism during early brain development. This demonstrates that changes in Nrg1/erbB4 can be secondary to that of the NMDA receptor, which is relevant to both the NMDA receptor hypofunction and neurodevelopmental hypotheses of schizophrenia. These findings are relevant to pathological changes which may be occurring in the schizophrenia brain, though it is difficult to directly compare specific changes in expression across species. However in future studies it will still be useful to examine the effects of specific NMDA receptor blockers on Nrg1/erbB4 signaling and how this relates to GABAergic system function as these three systems are closely linked and have a disrupted balance in schizophrenia.
Role of the funding source Funding for this study was provided by National Health and Medical Research Council (NHMRC) Project Grant ID573426, as well as the Schizophrenia Research Institute (SRI) Australia, utilizing infrastructure from New South Wales Health. The funding sources had no further role in study design, in the collection, analysis and interpretation of data; in the writing of the report and in the decision to submit the paper for publication.
Contributors Xu-Feng Huang, Kelly Newell and Teresa du Bois designed the study. Teresa du Bois and Kelly Newell performed the animal experiments. Teresa du Bois wrote the protocol for and performed the Western blot experiments, quantified the results, performed the statistical analyses and wrote the first draft of the manuscript. All authors contributed to and have approved the final manuscript.
Conflict of interest The authors declare that, except for income received from primary employer, no financial support or compensation has been received from any individual or corporate entity over the past three years for research or professional service and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.
Acknowledgments None.
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