A translational EEG-based approach to assess modulation of long-lasting NMDAR-dependent synaptic plasticity.

BACKGROUND: NYX-2925 is a novel N-methyl-D-aspartate receptor (NMDAR) modulator that has been shown to facilitate both NMDAR-dependent long-term potentiation (LTP) in vitro and learning and memory in vivo. OBJECTIVE: The present studies examine the effects of NYX-2925 on NMDAR-dependent auditory LTP (aLTP) in vivo. METHODS: NMDAR-dependent aLTP and NMDAR-dependent auditory mismatch negativity (MMN) was measured, as well as changes in resting-state qEEG power. RESULTS: NYX-2925 (1, 10 mg/kg PO) increased aLTP 1 h after auditory tetanus measured by the post- minus pre-tetanus difference waveform 140-180 ms post tone onset. NYX-2925 (0.1, 1 mg/kg PO) facilitated MMN measured by the difference waveform (i.e., deviant minus standard tones). NYX-2925 (0.1, 1, 10 mg/kg PO) also enhanced resting-state alpha qEEG power. Conversely, the NMDAR glutamate site antagonist CPP (10 mg/kg IP) reduces alpha power and MMN and produces an opposite effect as NYX-2925 on aLTP. CONCLUSIONS: Together, these data suggest that the activation of the NMDAR by NYX-2925 enhances synaptic plasticity in vivo, which may both reduce symptoms of neurological disorders and serve as a biomarker for drug effects. This is the first demonstration of a long-lasting (1-h post-tetanus) effect of NMDAR modulation on synaptic plasticity processes in vivo using a noninvasive technique in freely behaving animals.

The long-lasting antidepressant effects of rapastinel (GLYX-13) are associated with a metaplasticity process in the medial prefrontal cortex and hippocampus.

Rapastinel (GLYX-13) is an N-methyl-d-aspartate receptor (NMDAR) modulator that has characteristics of a glycine site partial agonist. Rapastinel is a robust cognitive enhancer and facilitates hippocampal long-term potentiation (LTP) of synaptic transmission in slices. In human clinical trials, rapastinel has been shown to produce marked antidepressant properties that last for at least one week following a single dose. The long-lasting antidepressant effect of a single dose of rapastinel (3mg/kg IV) was assessed in rats using the Porsolt, open field and ultrasonic vocalization assays. Cognitive enhancement was examined using the Morris water maze, positive emotional learning, and contextual fear extinction tests. LTP was assessed in hippocampal slices. Dendritic spine morphology was measured in the dentate gyrus and the medial prefrontal cortex. Significant antidepressant-like or cognitive enhancing effects were observed that lasted for at least one week in each model. Rapastinel facilitated LTP 1day-2weeks but not 4weeks post-dosing. Biweekly dosing with rapastinel sustained this effect for at least 8weeks. A single dose of rapastinel increased the proportion of whole-cell NMDAR current contributed by NR2B-containing NMDARs in the hippocampus 1week post-dosing, that returned to baseline by 4weeks post-dosing. The NMDAR antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) blocked the antidepressant-like effect of rapastinel 1week post dosing. A single injection of rapastinel also increased mature spine density in both brain regions 24h post-dosing. These data demonstrate that rapastinel produces its long-lasting antidepressant effects via triggering NMDAR-dependent processes that lead to increased sensitivity to LTP that persist for up to two weeks. These data also suggest that these processes led to the alterations in dendritic spine morphologies associated with the maintenance of long-term changes in synaptic plasticity associated with learning and memory.

Positive emotional learning is regulated in the medial prefrontal cortex by GluN2B-containing NMDA receptors.

In rats, hedonic ultrasonic vocalizations (USVs) is a validated model of positive affect and is best elicited by rough-and-tumble play. Here we report that modulation of GluN2B-containing NMDA receptors (NMDAR) in the medial prefrontal cortex (MPFC) is involved in positive emotional learning. Rough and tumble play increased both GluN1 and GluN2B NMDAR subunit mRNA and protein levels in the frontal cortex. GLYX-13, a GluN2B-preferring, NMDAR glycine-site partial agonist (1 mg/kg, i.v.) significantly increased positive emotional learning whereas the GluN2B receptor-specific antagonist, ifenprodil (10 mg/kg, i.p.), inhibited positive emotional learning. Animals selectively bred for low rates of hedonic USVs were returned to wild-type levels of positive emotional learning following GLYX-13 treatment. MPFC microinjections of GLYX-13 (0.1-10 µg/side) significantly increased rates of positive emotional learning. Thus GluN2B-containing NMDARs may be involved in positive emotional learning in the MPFC by similar mechanisms as spatial/temporal learning in the hippocampus.

Regional changes in gene expression after limbic kindling.

Repeated electrical stimulation results in development of seizures and a permanent increase in seizure susceptibility (kindling). The permanence of kindling suggests that chronic changes in gene expression are involved. Kindling at different sites produces specific effects on interictal behaviors such as spatial cognition and anxiety, suggesting that causal changes in gene expression might be restricted to the stimulated site. We employed focused microarray analysis to characterize changes in gene expression associated with amygdaloid and hippocampal kindling. Male Long-Evans rats received 1 s trains of electrical stimulation to either the amygdala or hippocampus once daily until five generalized seizures had been kindled. Yoked control rats carried electrodes but were not stimulated. Rats were euthanized 14 days after the last seizures, both amygdala and hippocampus dissected, and transcriptome profiles compared. Of the 1,200 rat brain-associated genes evaluated, 39 genes exhibited statistically significant expression differences between the kindled and non-kindled amygdala and 106 genes exhibited statistically significant differences between the kindled and non-kindled hippocampus. In the amygdala, subsequent ontological analyses using the GOMiner algorithm demonstrated significant enrichment in categories related to cytoskeletal reorganization and cation transport, as well as in gene families related to synaptic transmission and neurogenesis. In the hippocampus, significant enrichment in gene expression within categories related to cytoskeletal reorganization and cation transport was similarly observed. Furthermore, unique to the hippocampus, enrichment in transcription factor activity and GTPase-mediated signal transduction was identified. Overall, these data identify specific and unique neurochemical pathways chronically altered following kindling in the two sites, and provide a platform for defining the molecular basis for the differential behaviors observed in the interictal period.

Uncovering the molecular basis of positive affect using rough-and-tumble play in rats: a role for insulin-like growth factor I.

Positive emotional states have been shown to confer resilience to depression and anxiety in humans, but the molecular mechanisms underlying these effects have not yet been elucidated. In laboratory rats, positive emotional states can be measured by 50-kHz ultrasonic vocalizations (hedonic USVs), which are maximally elicited by juvenile rough-and-tumble play behavior. Using a focused microarray platform, insulin-like growth factor I (IGFI) extracellular signaling genes were found to be upregulated by hedonic rough-and-tumble play but not depressogenic social defeat. Administration of IGFI into the lateral ventricle increased rates of hedonic USVs in an IGFI receptor (IGFIR)-dependent manner. Lateral ventricle infusions of an siRNA specific to the IGFIR decreased rates of hedonic 50-kHz USVs. These results show that IGFI plays a functional role in the generation of positive affective states and that IGFI-dependent signaling is a potential therapeutic target for the treatment of depression and anxiety.

Rats selectively bred for low levels of 50 kHz ultrasonic vocalizations exhibit alterations in early social motivation.

In rats, the rates of 50 kHz ultrasonic vocalizations (USVs) can be used as a selective breeding phenotype and variations in this phenotype can be an indicator of affective states. The 50 kHz USV is elicited by rewarding stimuli (e.g., food, sexual behavior) and therefore can express a positive affective state. Conversely, the 22 kHz USV is elicited by aversive stimuli (e.g., presence of a predator, social defeat) indicating a negative affective state. In the present study, we tested the effect of selectively breeding for 50 kHz USVs on a variety of maternal social/emotional behaviors in young rat pups (PND 10-12). These measures consisted of an assessment of isolation calls and conditioned odor preference paradigm. Results indicate that animals selected for low levels of 50 kHz USVs show the greatest alterations in social behaviors compared to the control animals. The low line animals had an increase in isolation calls tested during place preference conditioning and a decrease in 50 kHz ultrasonic calls in all conditions. These same low line animals failed to show a typical preference for a maternally-associated odor during the place preference test. The different social behaviors of the high line animals did not consistently vary from those of the control group. These results have important implications for the study of genetic and epigenetic mechanisms underlying emotional states, and possibly contribute to the research underlying the emotional changes in developmental disorders such as autistic spectrum disorder by providing a novel animal model that displays communication deficits that are interdependent with significant social behavioral impairments.

Modeling depression: social dominance-submission gene expression patterns in rat neocortex.

Gene expression profiles in the cortex of adult Long-Evans rats as a function of a stressful social loss and victory in inter-male fighting encounters were examined. This social dominance and subordination model has been postulated to simulate early changes in the onset of depression in the losers. Microarrays were fabricated containing 45mer oligonucleotides spotted in quadruplicate and representing 1178 brain-associated genes. Dynamic range, discrimination power, accuracy and reproducibility were determined with standard mRNA "spiking" studies. Gene expression profiles in dominant and subordinate animals were compared using a "universal" reference design [Churchill GA (2002) Fundamentals of experimental design for cDNA microarrays. Nat Genet 32 (Suppl):490-495]. Data were analyzed by significance analysis of microarrays using rank scores [Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116-5121; van de Wiel MA (2004) Significance analysis of microarrays using rank scores. Kwantitatieve Methoden 71:25-37]. Ontological analyses were then performed using the GOMiner algorithm [Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein JN (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 4(4):R28]. And finally, genes of special interest were further studied using quantitative reverse transcriptase polymerase chain reaction. Twenty-two transcripts were statistically significantly differentially expressed in the neocortex between dominant and subordinate animals. Ontological analyses revealed that significant gene changes were clustered primarily into functional neurochemical pathways associated with protein biosynthesis and cytoskeletal dynamics. The most robust of these were the increased expression of interleukin-18, heat shock protein 27, beta3-tubulin, ribosome-associated membrane protein 4 in subordinate animals. Interleukin-18 has been found to be over-expressed in human depression and panic disorder as well as other physiological stress paradigms [Takeuchi M, Okura T, Mori T, Akita K, Ohta T, Ikeda M, Ikegami H, Kurimoto M (1999) Intracellular production of interleukin-18 in human epithelial-like cell lines is enhanced by hyperosmotic stress in vitro. Cell Tissue Res 297(3):467-473] and heat shock proteins have been shown to be involved in the pathogenesis of many neurodegenerative and psychiatric disorders [Iwamoto K, Kakiuchi C, Bundo M, Ikeda K, Kato T (2004) Molecular characterization of bipolar disorder by comparing gene expression profiles of postmortem brains of major mental disorders. Mol Psychiatry 9(4):406-416; Pongrac JL, Middleton FA, Peng L, Lewis DA, Levitt P, Mirnics K (2004) Heat shock protein 12A shows reduced expression in the prefrontal cortex of subjects with schizophrenia. Biol Psychiatry 56(12):943-950]. Thus, the gene expression changes that we have observed here are consistent with and extend the observations found in the clinical literature and link them to the animal model used here thereby reinforcing its use to better understand the genesis of depression and identify novel therapeutic targets for its treatment.

The identification of novel therapeutic targets for the treatment of malignant brain tumors.

A two-step strategy was developed consisting of differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) with cultured normal human fetal astrocytes and U-373MG glioma cells followed by reverse Northern analysis of normal brain and primary tumor tissues. hu-dek, alpha-NAC, ribosomal proteins L7a and L35a, and five novel genes were identified. Since none of these genes has been previously shown to be associated with malignant brain tumor formation, this approach may be useful to identify novel targets for the diagnosis and treatment of brain tumors.

The role of mRNA stability and transcription in O6-methylguanine DNA methyltransferase (MGMT) expression in Mer+ human tumor cells.

We have recently reported that following depletion of O6-methylguanine DNA methyltransferase (MGMT) activity by acute streptozotocin (STZ) treatment to sensitize innately chloroethylnitrosourea (CENU)-resistant HT-29 cells, the eventual repletion of activity occurs with no concommitant alterations in steady-state MGMT mRNA levels. This suggestion of a potentially stable transcript prompted studies to define the relative contributions of MGMT mRNA stability and transcription to cellular MGMT expression. Northern analysis of MGMT mRNA in actinomycin D-treated HT-29, MR-1 and A2182 cells, ranging in relative MGMT expression from high to low respectively, demonstrates relatively long MGMT mRNA half-lives of > 10-12 h. Cell lines with low and moderate levels of MGMT mRNA appear to have longer mRNA half-lives than those with high levels. Run-on transcription in nuclei isolated from cells with low to moderate MGMT mRNA levels demonstrates undetectable basal MGMT transcription rates. Collectively these data suggest that a very low transcription rate, coupled with a stable mRNA molecule, might result in the translation of pre-existing mRNA molecules. This translation may be responsible for the gradual recovery of MGMT and CENU resistance over 24 h following MGMT depletion.

Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.

Prolonged depletion of O6-methylguanine DNA methyltransferase activity following exposure to O6-benzylguanine with or without streptozotocin enhances 1,3-bis(2-chloroethyl)-1-nitrosourea sensitivity in vitro.

This study was undertaken to ascertain the importance of prolonged depletion of O6-methylguanine DNA methyltransferase (MGMT) activity, following O6-benzylguanine (BG) and streptozotocin (STZ) exposure, in reversing 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance in vitro. We evaluated BCNU-induced cytotoxicity and measured the temporal recovery of MGMT activity in human colon carcinoma HT-29 cells following treatment with BG, STZ, or the combination of BG and STZ. The pretreatment regimens which provided the greatest potentiation of BCNU cytotoxicity were those exhibiting the greatest temporal inhibition of MGMT activity. The combination of BG (10 microM) and STZ (1.0 mM) produced sustained inhibition of MGMT activity through 24 h and potentiated BCNU cytotoxicity by at least one log greater than either agent alone. Similarly, BG (10-100 microM) produced marked reductions in MGMT activity and increased BCNU cytotoxicity in a dose-dependent fashion. A 100-microM dose of BG inhibited MGMT activity for 48 h and potentiated BCNU induced cell kill by 3 logs greater than BCNU alone. In addition, we observed that during the period of sustained inhibition of MGMT activity, no changes in the steady-state MGMT mRNA levels occurred. We conclude that prolonged inhibition of MGMT activity is an important determinant in reversing BCNU resistance and that chemotherapeutic regimens targeting the inactivation of MGMT activity should be optimized such that MGMT activity is depleted for at least 24 h following BCNU administration.

Effect of sodium salicylate on the human heat shock response.

Sodium salicylate, an anti-inflammatory agent, was examined for its effects on the heat shock response in cultured human cells. Salicylate activation of DNA binding by the heat shock transcription factor (HSF) was comparable to activation attained during heat shock. However, sodium salicylate did not induce heat shock gene transcription even though the HSF was bound in vivo to the heat shock elements upstream of the heat shock protein 70 (Hsp 70) gene. These results reveal that activation of the heat shock transcriptional response is a multistep process. Modulation of extracellular pH augments sensitivity to salicylate-induced activation of HSF.

Direct correlation between methylation status and expression of the human O-6-methylguanine DNA methyltransferase gene.

To assess the role of DNA cytosine methylation in the expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, the methylation status of selected CpG-containing dinucleotides in and surrounding the coding regions of the gene were examined and correlated with steady state expression of MGMT mRNA in 13 human cell lines. Additionally, tumor cells which exhibited very high levels of MGMT expression were chronically exposed to 5-azacytidine to assess the effects of changes in gene methylation on MGMT expression. Results of these studies demonstrate that the degree of methylation of multiple MGMT gene regions correlates with gene expression, but in a direct rather than an inverse fashion, and that 5-azacytidine-induced demethylation of the MGMT gene correlates with a significant reduction, rather than induction, of MGMT steady-state mRNA expression. These results suggest a unique, potentially alterable methylation-related regulatory mechanism for the MGMT gene.

Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes.