Genome-wide gene expression profiling in the hippocampus revealed that GluR1 ablation was associated with a range of gene expression changes, although only some of these changes were further confirmed by other methods. In addition, we found that a cluster of these changes occurred in the calcium handling and signaling group (Supporting Table S3
, Supporting Figure S3
). Intriguingly, the “on” reactions of calcium signaling appear to have been activated through up-regulation of cell membrane receptors and calcium channels; conversely, calcium is pumped back into endoplasmic/sarcoplasmic reticulum (ER/SR) through up-regulation of intracellular sarco/endoplasmic reticulum calcium-ATPase (SERCA), making it less effective. We also found that several effect molecules were down-regulated, thus suggesting that the “off” reactions of calcium signaling were also likely to be activated (). This seemingly contradictory regulation may shed new light on the delicate manner through which AMPARs regulate cellular calcium signaling cascades.
One of the key features of the calcium signaling pathway is that the calcium transient has two functions. In addition to activating cellular responses, it also functions as part of the feedback mechanism that regulates the transcriptional events responsible for maintaining signal stability. This calcium-dependant self-stability regulation might play a central role in the compensatory mechanisms that enable cells to adapt to modifications of their calcium signaling system (Berridge et al., 2003
). In light of this major feature of the calcium signaling pathway, we propose the following hypothesis: in GluR1−/− mice, cellular calcium signals are low, and membrane calcium permeable receptors and calcium channels increase to compensate for this state through feedback mechanisms. In addition, many downstream effectors of GluR1 are regulated through the calcium cascade, and a significant part of those downstream effectors are involved in neuroplasticity regulation. Thus, it appears that the calcium signaling pathway may play a central role in GluR1-induced neuroplasticity.
The complexity of the AMPA system and its wide range of regulatory roles complicate our understanding of its underlying molecular mechanisms and their role in regulating neuroplasticity. This study of GluR1 deficient rodents provides an important clue that the calcium signaling pathway may play a key role in the way that AMPA regulates neuroplasticity. The original study by Zamanillo and colleagues (Zamanillo et al., 1999
) showed that LTP was absent in the CA3 to CA1 synapses of GluR1-/- mice, suggesting the importance of AMPARs for hippocampal synaptic plasticity. Furthermore, it appears that postsynaptic elevations in calcium and calcium-dependent protein kinases are required to establish LTP, and that AMPARs are a likely target of these kinases (Barria et al., 1997
Evaluation at the protein level provided further evidence highlighting the crucial importance of GluR1 in regulating synaptic plasticity. For those calcium inflow enhancing proteins on the cell membrane, NMDAR1 was up-regulated in GluR1 KO mice. NMDAR1 itself is a key regulator of synaptic plasticity, especially in the hippocampal CA1 region (Tonegawa et al., 1996
). CaMKII consists of four distinct but highly homologous chains (α, β, γ, and δ) (Si et al., 2007
). The α chain of CaMKII, also known as CAMK2A, plays a very important role in controlling neuronal excitability, either by regulating excitatory neuronal transmission (Liu & Jones, 1997
), or by enhancing gamma-aminobutyric acid (GABA) synaptic response (Wang et al., 1995
). Notably, CAMK2A, like NMDAR1, also regulates synaptic plasticity in the hippocampal CA1 region (Hinds et al., 1998
); for instance, a deletion in the CAMK2A gene resulted in impaired LTP and LTD in the hippocampal CA1 region as well as spatial learning deficits (Silva et al., 1992a
; Silva et al., 1992b
). Interestingly, studies have shown that there are corresponding relationships between GluR1, NMDAR1, and CAMKII in regulating neuroplasticity (Zaitseva et al., 2003
; Zhao et al., 2008
). In this study, we found that NMDAR1 levels were elevated, but that CAMK2A levels were decreased. This result reflected the changes that occurred when one member of a group of key regulators was absent. At the cell membrane level, NMDAR1 was up-regulated, probably to compensate for the lack of GluR1, but with the increasing calcium levels in cell plasma, CAMK2A levels decreased in order to balance the increased cell excitability. In this situation, calcium was the key factor linking upstream regulators (like NMDAR1) to downstream regulators (such as CAMK2A). With the knockout of GluR1—which is a key regulator of synaptic plasticity as well as calcium (Barria et al., 1997
; Derkach et al., 1999
; Ye et al., 2006
)—calcium-linked balance was damaged, suggesting that calcium may be the major molecular mechanism at play in GluR1-mediated neuroplasticity.
Taken together, these studies support the notion that GluR1 regulates neuroplasticity by regulating the calcium signaling pathway. While AMPARs were previously considered almost impermeable to calcium (Mayer & Westbrook, 1987
), this view has since been challenged by the observation that non-NMDA glutamate receptors in a subset of cultured hippocampal neurons appear to be highly permeable to calcium (Iino et al., 1990
). These non-NMDARs were subsequently identified as AMPARs on the basis of their pharmacological profile (Ozawa & Iino, 1993
), and several additional studies have since supported this key finding (Bochet et al., 1994
; Jonas & Burnashev, 1995
). At excitatory synapses, the calcium influx through AMPARs is comparable to that through NMDARs at the resting membrane potential (Burnashev et al., 1995
; Koh et al., 1995
). The GluR1-containing complexes include GluR1/2, GluR1 homomers, and GluR1/3. GluR1/2 and GluR2/3 are the dominant forms of AMPAR channels and are calcium-impermeable; however, approximately 15-20% of GluR1 homo tetramers and a small percentage of the GluR1/3 expressed in the hippocampus are calcium permeable (Wenthold et al., 1996
). We would expect all the calcium-impermeable GluR1/2, calcium-permeable GluR1 homomers, and GluR1/3 to be reduced in GluR1 KO mice. In addition, because depolarization caused by activation of AMPARs is required to open NMDAR channels and other voltage dependent calcium channels, the reduction in calcium impermeable GluR1/2 receptors would eventually be expected to affect calcium influx in the neurons in response to various stimuli.
Another point of interest concerns voltage-dependent calcium channels (VDCCs). In the developing and mature CNS, VDCCs regulate the coupling of electrical excitation to gene expression, and modulate a wide variety of intracellular signaling pathways that, in turn, lead to neurite outgrowth, synaptogenesis, transmitter and hormone release, plasticity, and muscle contraction. Mammalian VDCCs are multimeric complexes of α1, β, α2δ, and γ subunits; α1 is the only subunit that forms a calcium-conducting pore. The synaptic proteins syntaxin-1A, synaptotagmin, SNAP-25, and synaptobrevin interact with the calcium channel complex of the α1 subunit, coupling excitation to neurotransmitter release at nerve terminals (Burgess & Noebels, 1999
). Our study showed that four calcium channel subunits were up-regulated in GluR1−/− mice, although we could not show altered expression at the protein level. Notably, three of these four are α1 subunits. At least eight identified mammalian genes encode α1 subunits. These subunits are noticeable because, of the four kinds of VDCC subunits, they are the only ones that form a calcium conducting pore.
The current study is also associated with certain limitations. First, only some of the positive microarray findings in this study were validated. Second, the current data apply to gene expression, which has only been partially correlated with changes at the protein or functional level. Finally, although cluster analysis revealed that gene expression in the calcium signaling group was the most frequently affected, the altered expression of other functional groups cannot be fully ruled out.
Nevertheless, GluR1 receptor malfunction has recently been highlighted in studies of CNS disorders (Burgess & Noebels, 1999
; Kwak & Weiss, 2006
). To our knowledge, our study is the first to systematically evaluate genome-wide gene expression changes in GluR1 KO animals. Our findings have focused on calcium signaling pathways and the role they play in AMPAR-regulated hippocampal plasticity, especially the GluR1 receptors. Of those targets identified here, the roles of NMDAR1 and CAMK2A in synaptic and behavioral plasticity have been well-documented, while the roles of other calcium signaling/handling genes whose expression levels are altered in GluR1 -/- mice are less clear. Further investigation of the molecules encoded by these genes may ultimately provide novel treatment targets for behavioral disorders.