Excitotoxic stimulation activates distinct pathogenic and protective expression signatures in the hippocampus

Abstract Excitotoxic events underlying ischaemic and traumatic brain injuries activate degenerative and protective pathways, particularly in the hippocampus. To understand opposing pathways that determine the brain's response to excitotoxicity, we used hippocampal explants, thereby eliminating systemic variables during a precise protocol of excitatory stimulation. N‐methyl‐d‐aspartate (NMDA) was applied for 20 min and total RNA isolated one and 24 h later for neurobiology‐specific microarrays. Distinct groups of genes exhibited early vs. delayed induction, with 63 genes exclusively reduced 24‐h post‐insult. Egr‐1 and NOR‐1 displayed biphasic transcriptional modulation: early induction followed by delayed suppression. Opposing events of NMDA‐induced genes linked to pathogenesis and cell survival constituted the early expression signature. Delayed degenerative indicators (up‐regulated pathogenic genes, down‐regulated pro‐survival genes) and opposing compensatory responses (down‐regulated pathogenic genes, up‐regulated pro‐survival genes) generated networks with temporal gene profiles mirroring coexpression network clustering. We then used the expression profiles to test whether NF‐κB, a potent transcription factor implicated in both degenerative and protective pathways, is involved in the opposing responses. The NF‐κB inhibitor MG‐132 indeed altered NMDA‐mediated transcriptional changes, revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi‐target treatment strategies and implications for predictive medicine.


| INTRODUC TI ON
Excitotoxicity, related to ischaemic stroke, traumatic brain injury and seizures, occurs through over-activation of excitatory glutamate receptors. The glutamatergic receptors involved are those selectively activated by either N-methyl-D-aspartate (NMDA) or α-amino-3hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and their excessive activation causes neuropathology. 1 Interestingly, glutamatergic activity can induce repair signalling. For instance, excitotoxic stimulation of NMDA receptors induces neuronal impairment and death, but in the developing brain these receptors play a role in neuronal survival. 2 In addition, related signalling avenues have been suggested to promote cell survival in different in vitro and in vivo models. [3][4][5] Thus, identifying competing genetic programmes is important to fully understand the influence of opposing pathways underlying the brain's response to injury. The brain's response to an excitotoxic injury involves pathogenic and protective pathways, and clinical outcome is likely determined by the temporal relationships between such counteracting gene responses. Excitatory over-activation, in particular, activates such opposing pathways, with excitotoxicity causing transcriptional induction of both cell death-and cell survival-related genes. 6,7 Examples of differential gene expression during cerebral ischaemia and models of excitotoxic events were demonstrated by measuring brain RNA expression profiles. [7][8][9][10] Notably, temporal expression changes were identified in the transient middle cerebral artery occlusion (MCAO) stroke model by employing neurobiology-focussed arrays. 7 The MCAO study provides strong evidence of distinct acute versus delayed gene expression profiles comprised of diverse functional categories.
The present study investigates the excitotoxic injury response in the hippocampus using the cultured hippocampal slice, a physiologically relevant three-dimensional tissue model with native pathogenic responsiveness with regard to neurodegenerative insults. 3,4,[11][12][13] Different excitotoxic insults in the explants indeed resulted in the expected delayed neuronal damage along with associated compensatory signals found involved in governing cellular damage. 3,4,6 The hippocampus is particularly important to study as it is a higher brain centre involved in memory encoding and behavioural responses, as well as being distinctly vulnerable to excitotoxicity. Here, excitotox- with media composed of 50% basal medium Eagle, 25% Earle's balanced salts, 25% horse serum and defined supplements. 12,13 Slices were maintained at 37°C, and media changes occurred every 2-3 days. Slices were allowed to mature for 16-20 days in culture before being used in experiments. For immunostaining, subsets of cultured slices were quickly harvested for homogenate preparation and immunoblotting with specific antibodies, or they were cold fixed in 4% paraformaldehyde for 4 h, placed in 20% sucrose overnight and sectioned at 20µm thickness for immunohistochemistry protocols. revealing components of opposing expression signatures that converge on the single response element. Overall, this study identified counteracting avenues among the distinct responses to excitotoxicity, thereby suggesting multi-target treatment strategies and implications for predictive medicine.

K E Y W O R D S
compensatory signalling, excitotoxic response, gene array, neuroprotection, repair pathway, transcription factor

| Induction of excitotoxicity
The hippocampal slice model of excitotoxicity involves the excitotoxic insult achieved by infusing the long-term cultured slices with serum-free media containing 200 μM NMDA (Tocris; Ellisville, Missouri), a sufficient concentration to ensure rapid activation of NMDA receptors throughout the three-dimensional tissue cultures.
For a consistent 20-min insult period, the excitatory activation was quickly stopped using multiple media washes containing 40 μM each of MK801 and CNQX (Tocris), two selective antagonists to quench any further activity of NMDA and AMPA receptors, respectively.
In separate experiments, cultured hippocampal slices were treated with 60 μM MG-132, a proteasome inhibitor, for 60 min before as well as during and after the NMDA infusion and quenching steps.
The slice cultures were then placed in fresh media for the post-insult period of 1-24 h. Start times were adjusted such that all treated and control slices were collected on the same day, harvesting with a gentle brush to remove them from the culture inserts. To verify the gene array results, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out on selected genes in NMDA-treated and control samples. Extracted total RNA was assessed for concentration as determined from optical density measurement at 260 and 280 nm. Routine two-step qRT-PCR analyses were then conducted in duplicate with an Applied Biosystems detection system, utilizing TaqMan assays specific to the rodent genes being assessed.

| Gene array preparation and hybridization
A subset of hippocampal slices harvested 0.5-to 40-h post-insult was also subjected to nuclear extraction after rapid homogenization in ice-cold nuclear extract buffer. For measures of MG-132-sensitive NF-κB activation, equal aliquots of the nuclear extracts (7.5 μg protein) were subjected to the electrophoretic mobility shift assay (EMSA) described previously, 6 using a labelled NF-κB consensus oligonucleotide probe.

| Annotation and analysis of gene array data
Over 1300 sequences relevant to neurobiology research are included in the Affymetrix microarray design. Statistically significant alterations to RNA transcript levels relative to control slices were determined using the Affymetrix Genechip software MAS 5.0.
Greater than 50% induction or repression in expression was applied as a filtering threshold among those genes identified by the software to have a statistically significant change. For each treatment, three gene chips were used and compared with corresponding sham samples. Differentially expressed gene transcripts were sorted through complete linkage clustering using a Euclidean distance measurement transformed into heat maps using R. 14 Filtered gene sets were also subjected to gene-by-gene literature searches and divided into functional classes. Genes linked to predominately pro-survival pathways versus predominately pathogenic pathways were categorized to assemble gene profiles of potential opposing pathway responses. Co-expressed genes were determined using the GeneMania database. 15 Gene ontological (GO) terms were determined using the DAVID database 16  PKCβ, 34 PKCγ 35 and ILK. 36 The list of genes linked to pathogenesis includes the following: cJun, 37 IL-1β, 38 NGFI-B, 39 caspase 1 (previously known as interleukin-1β-converting enzyme or ICE), 40

| RE SULTS
For the study of transcriptional events elicited specifically in the hippocampus, we prepared rat hippocampal slices and maintained them in culture on Millipore inserts ( Figure 1A). The explants displayed long-term maintenance of the major neuronal subfields of the hippocampus ( Figure 1B) and their dense processes populated with evident staining of synaptic terminals ( Figure 1C-E). Former studies reported that the slice cultures exhibit connectivity, plasticity and pathogenic responsiveness as found in vivo. 11,13,[50][51][52] Here, we used an NMDA infusion period that led to pathogenic changes as previously described. 6,51 The protocol for the excitotoxic stimulation and subsequent measuring of gene expression profiles is shown in Figure 1F, in which the explants were infused with NMDA for 20 min followed by rapid washout in the presence of glutamatergic antagonists for precise insult duration due to quenching any further excitatory activation.
To test for early versus delayed expression profiles after the defined excitotoxic insult, RNA was isolated from groups of slice cultures, cDNA was generated and used to synthesize biotinylated cRNA, which was followed by the chip hybridization protocol employing Affymetrix Rat Neurobiology U34 arrays. Across the approximately 1300 neurobiologically relevant sequences in the arrays, the scatter plot in Figure 1F indicates a close correlation of hybridization signal intensities between non-treated control samples.
The linear distribution of expression intensities confirms reproducible measures of transcription levels.
Compared with the expression levels measured in vehicletreated control slices, the NMDA insult led to 106 differentially expressed genes across the two post-insult times. The small set F I G U R E 1 NMDA-mediated excitotoxicity in stable hippocampal explants to measure transcriptional responses. Organotypic cultures of rat hippocampal slices were maintained on the Biopore membrane of Millicell inserts (A). The Nissl-stained slice cultures have preserved organization of the major neuronal subfields (B), and the neurons were stained for βIII-tubulin (C) and found to be densely populated with synapses positive for the markers synaptophysin (D) and GluR-1 (E). Size bars: B, 280 μm; C-E, 24 μm. The excitotoxic insult protocol and gene expression profiling consisted of a 20-min infusion of 200 μM NMDA, rapid antagonist quenching of the cultures, followed by RNA isolation and synthesis of biotinylated cRNA (F). Using chip hybridization with Affymetrix Neurobiology arrays, expressed genes were found to have a close linear distribution of hybridization signal intensities between like treatment groups (R 2 = 0.959 for the lower scatter plot). For explants harvested 1 h and 24 h after the NMDA-induced excitotoxic stimulation and compared with vehicle treatment, expression profiles were visualized using a heat map for the most significant gene clusters (G). Differential expression between the 1-and 24-h postinsult groups was found, with green, black and red indicating low, intermediate and high gene expression, respectively. Dendrograms were formulated through complete linkage clustering using a Euclidean distance measurement. Pie charts for each cluster depict percentages of those genes in the listed functional categories from the 1300 neurobiologically relevant genes met the criteria of at least a 50% increase or decrease in expression and represented a wide range of functionality. In Table 1   Interestingly, it should be pointed out that the excitotoxic hippocampal slice model was without any gene expression changes that F I G U R E 2 Cellular responses to excitotoxicity are defined by distinct gene expression profiles found early vs. delayed after the insult. RNA from control and NMDA exposed hippocampal slices was subjected to microarray analysis using Affymetrix GeneChips. (A) Genes that are up-regulated early and their 24-h counterparts. Genes that are (B) upregulated or (C) down-regulated late (24 h) after the insult and their respective early counterparts. Note that green identifies those genes that were up-regulated above 50% with a significant increase call. Red bars represent those genes that were down-regulated 50% with a significant decrease call, while those genes that did not change or fit the 50% criteria, are shown as grey bars. Only those genes that were consistently altered in three independent repetitions are listed met the minimal criteria of ≥50% decline at 1-h post-insult. Among the delayed down-regulation events, 9 of the 63 genes were markedly reduced in expression by 89%-95%. The nine genes represent mostly synaptic proteins or proteins linked to synaptic mechanisms.
Among the synaptic proteins, GluR-K3 expression was reduced by 94% and synapsin 2 was reduced by 89% (see Table 1 Omnibus via GeneMania. In this way, we informed our neurospecific gene expression network with prior gene expression studies but the network was not biased by cell or tissue type. As shown in Figure 3A, the generated network clustered into two distinct sets based on the gene coexpression data from the two post-insult assessments. We then overlaid our early and late gene expression data onto our coexpression network. Interestingly, we found that The categorical filtering also found evidence for opposing pathways. Early responses to the NMDA insult consisted of degenerative increases in the 15 primarily pathogenic genes listed above as well as protective increases in eight primarily pro-survival genes (see Table 2). The 24-h post-insult findings were more complicated but The degenerative and protective gene differences were subsequently used to assess networks involved in the distinct pathways. Starting from the gene coexpression networks generated in Figure 3A, all nodes not categorized as pathogenic and/or protective based on literature searches were removed. Next, the remaining nodes were then annotated based on four functional categories (from Table 1 To further understand how the temporal changes at a gene expression level might impact system behaviour, functional networks involving the early versus delayed differentially expressed genes were generated by mapping the gene name to biological function using Gene Ontology (GO). Networks were subsequently created using Cytoscape, where each node represents a GO biological function category (Figure 4). Node sizes represent the number of genes within a given GO category while edge sizes denote the number of genes shared between the category nodes.
To identify a candidate signalling avenue activated by NMDA and involved in opposing gene responses, we tested the role of NF-κB which also served to test the value of the signalling profiles F I G U R E 3 Gene networks associated with NMDA-mediated excitotoxicity. Differentially expressed gene networks at 1-h (left) and 24-h post-insult (right) mirror coexpression network clustering (A). Green nodes represent underexpressed genes, red denotes overexpressed genes, and grey denotes genes that have no measured change relative to control. Edges denote co-expressed genes found via GeneMania. Node shapes denote the following categories: transcription factors (downward arrow), cytokines (triangle), receptors and channels (parallelogram), kinases and transduction (diamond), and all others (squares). Next, collecting genes by pathogenic and protective classification demonstrated that early and delayed pathogenic and delayed protective networks also resemble coexpression networks (B). As in panel A, green nodes represent underexpressed genes, red denotes overexpressed genes, and grey denotes no significant change relative to control explants Note: Categorical filtering was applied to the differentially expressed genes influenced by NMDA exposure. Many of the genes have been primarily linked to either pathogenic events or cell repair/survival. From that list, we tabulated early degenerative changes consisting of up-regulated pathogenic gene expression, as well as delayed degenerative changes consisting of increased pathogenic genes as well as down-regulated expression of a putative set of survival genes. Highlighted in bold, MCP and C/EBP exhibited enhancement of expression at early and delayed post-insult times. Also tabulated are early protective increases in primarily pro-survival genes, as well as protective down-regulation of three pathogenic genes. In bold among pro-survival elements, only HO-1 exhibited sustained enhancement. Biphasic responding genes are noted with asterisks. established in the present study. NF-κB was selected for testing due to being activated by synaptic stimulation, 6,53 and this transcription factor has been implicated in both cell survival and cell death. 6,54 A set of 22 opposing genes were first assessed for whether they possess an NF-κB-binding site in their promoter region. In the second tier of assessment, the excitotoxic NMDA treatment was applied to hippocampal slices that were also treated with an inhibitor of NF-κB activity (MG-132) before, during, and after the NMDA infusion to identify those genes linked to NF-κB. MG-132 was confirmed to block the distinct early and delayed phases of NMDA-induced NF-κB activation (see Figure 5A). The resulting data listed in Table 3 suggest that NF-κB is responsible for a subset of opposing genes involved in the brain's response to an excitotoxic episode. The early inductions of IL-1β, IRF-I and TNFα expression were blocked by the NF-κB inhibitor, and these pathogenic genes indeed possess a regulatory site for the transcription factor (see rows in bold in Table 3A).
Besides the degenerative role of NF-κB regulating the three aforementioned pathogenic genes, NF-κB also has an apparent regulatory role with an opposing pro-survival gene, HES-1, early after the NMDA insult (Table 3B). Note that three other survival genes possess an NF-κB binding site (Egr-1, IL-6 and SOCS-3), however, the MG-132 inhibitor had no effect on their NMDA-induced expression.
Of the pathogenic genes induced at 24 h post-insult, MCP, caspase 1 and cJun have an NF-κB-binding site but blocking NF-κB activity only disrupted the enhanced expression of caspase 1 (Table 3C). Also at 24 h, two NMDA-induced survival genes with NF-κB-binding sites were blocked by the NF-κB inhibitor (JAK2 and IGFII), whereas three others with binding sites (HSP27, HSP10 and IGFII BP3) did not have their regulated expression blocked by MG-132 (Table 3D). These data provide an example of determining induced transcriptional responses in order to build an understanding of how a single regulator can influence opposing pathways. Illustrated in Figure 5B

| DISCUSS ION
Over-activation of glutamatergic synapses recapitulates many aspects of brain disorders linked to excitotoxicity. The present study utilized a defined over-activity period for NMDA-type glutamatergic receptors and identified early and delayed expression signatures in a vulnerable brain region, the hippocampus. The distinct signatures act together to make up the brain's response to cellular responses that occur early after an excitotoxic insult or Many of the induced signalling components indicate that competing pathways take part in the brain tissue's response to injury.
Adding to the many comprehensive lists of regulated genes from models of excitotoxic insults, this report will help advance understanding of the brain's response to hypoxic/ischaemic episodes that are linked to excitotoxicity. The identified responses to excitotoxic stimulation involved genes with a wide range of functionality. The present study supports the idea that the brain is not a passive recipient of pathogenic insults and the induction of degenerative processes, but rather, the brain can trigger pathways of cellular repair that counter pathological responses to injury.
Note that opposing proapoptotic and antiapoptotic genes were reported to be induced in the hippocampus of rats subjected to global cerebral ischaemia 56 as well as in hippocampal explants exposed to a defined NMDA exposure 6 as used in the present study.
Excitotoxicity through the activation of NMDA receptors shares many of the cellular cascades associated with a variety of brain injuries. The utilization of categorical filtering was an additional informatics step used to identify transcriptional regulation events with a high propensity of being involved in cellular degeneration vs. cellular protection. Further analyses in the current study found that the potent transcription factor NF-κB appears to play a role, at least in part, in both of the pathogenic and protective expression signatures identified in the excitotoxic hippocampus. Such also supports the assertion of improved profile effectiveness by including a categorical informatics step to identify candidate signalling elements, in this case one potentially responsible for opposing gene responses.
NF-κB has often been linked to neuronal survival, but with many studies also linking it to excitotoxic pathology. 1 In addition to regulating responses for a wide array of events including cellular proliferation, inflammation and tissue remodelling, NF-κB signalling facilitates both protection avenues in surviving cells and cell death pathways for the clearance of dying cells. Such a dynamic and dual role is likely important, due to the complexity of cellular decisions, to allow crosstalk between divergent pathways for cells to adapt to stress, repair injuries and foster tissue health. NF-κB is activated by synaptic over-stimulation and it represents signalling reported to underlie complex and often opposing responses, 6,54,57,58 with links to the MAPK pathway during biphasic gene regulation in surviving tissue. 8 The transcription factor also frequently responds to excitotoxic insults that have an early phase of cytoskeletal and synaptic compromise. 50,51,59 Interestingly, NMDA exposure in the current study activated pathogenic as well as protective genes in TA B L E 3 NF-κB activation blocker MG-132 identifies opposing genes regulated by NF-κB in the excitotoxic hippocampus Writing-review & editing (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.