The inbred mouse strains, C57BL/6 and BALB/c have been used widely in preclinical psychiatric research. The differences in stress susceptibility of available strains has provided a useful platform to test pharmacological agents and behavioral responses. Previous brain gene profiling efforts have indicated that the inflammation and immune response gene pathway is the predominant gene network in the differential stress response of BALB/c and C57BL/6 mice. The implication is that a composite stress paradigm that includes a sequence of extended, varied and unpredictable stressors induces inflammation-related genes in the hippocampus. We hypothesized that the regulation of inflammation genes in the brain could constitute a primary stress response and tested this by employing a simple stress protocol, repeated exposure to the same stressor for 10 days, 2 h of restraint per day. We examined stress-induced regulation of 13 proinflammatory cytokine genes in male BALB/c and C57BL/6 mice using quantitative PCR. Elevated cytokine genes included tumor necrosis factor alpha (TNFα), interleukin 6 (IL6), interleukin 10 (IL10), tumor necrosis factor (TNF) super family members and interleukin 1 receptor 1 (IL1R1). In addition, we examined restraint stress-induced regulation of 12 glutamate receptor genes in both strains. Our results show that restraint stress is sufficient to elevate the expression of inflammation-related genes in the hippocampus of both BABLB/c and C57BL/6 mice, but they differ in the genes that are induced and the magnitude of change. Cell types that are involved in this response include endothelial cells and astrocytes.

Erythropoietin (EPO), a cytokine molecule, is best-known for its role in erythropoiesis. Preclinical studies have demonstrated that EPO has robust neuroprotective effects that appear to be independent of erythropoiesis. It is also being clinically tested for the treatment of neuropsychiatric illnesses due to its behavioral actions. A major limitation of EPO is that long-term administration results in excessive red blood cell production and increased blood viscosity. A chemical modification of EPO, carbamoylated erythropoietin (CEPO), reproduces the behavioral response of EPO in animal models but does not stimulate erythropoiesis. The molecular mechanisms involved in the behavioral effects of CEPO are not known. To obtain molecular insight we examined CEPO induced gene expression in neuronal cells. PC-12 cells were treated with CEPO followed by genome-wide microarray analysis. We investigated the functional significance of the gene profile by unbiased bioinformatics analysis. The Ingenuity pathway analysis (IPA) software was employed. The results revealed activation of functions such as neuronal number and long-term potentiation. Regulated signaling cascades included categories such as neurotrophin, CREB, NGF and synaptic long-term potentiation signaling. Some of the regulated genes from these pathways are CAMKII, EGR1, FOS, GRIN1, KIF1B, NOTCH1. We also comparatively examined EPO and CEPO-induced gene expression for a subset of genes in the rat dentate gyrus. The CEPO gene profile shows the induction of genes and signaling cascades that have roles in neurogenesis and memory formation, mechanisms that can produce antidepressant and cognitive function enhancing activity.

The two strains of inbred mice, BALB/c and C57BL/6, are widely used in pre-clinical psychiatry research due to their differences in stress susceptibility. Gene profiling studies in these strains have implicated the inflammation pathway as the main contributor to these differences. We focused our attention on female mice and tested their response to 5- or 10-day exposure to restraint stress. We examined the stress induced changes in the regulation of 11 inflammatory cytokine genes and 12 glutamate receptor genes in the hippocampus of female BALB/c and C57BL/6 mice using quantitative PCR. Elevated proinflammatory cytokine genes include Tumor Necrosis Factor alpha (TNFa), nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB), Interleukin 1 alpha (IL1a), Interleukin 1 receptor (IL1R), Interleukin 10 receptor alpha subunit (IL10Ra), Interleukin 10 receptor beta subunit (IL10Rb), and tumor necrosis factor (TNF) super family members. Our results show that BALB/c and C57BL/6 mice differ in the genes induced in response to stress exposure and the level of gene regulation change. Our results show that the gene regulation in female BALB/c and C57BL/6 mice differs between strains in the genes regulated and the magnitude of the changes.

Major depressive disorder and related illnesses are globally prevalent, with a significant risk for suicidality if untreated. Antidepressant drugs that are currently prescribed do not benefit 30% of treated individuals. Furthermore, there is a delay of 3 or more weeks before a reduction in symptoms. Results from preclinical studies have indicated an important role for trophic factors in regulating behavior. Erythropoietin (Epo), which is widely prescribed for anemia, has been shown to produce robust neurotrophic actions in the CNS. Although Epo's antidepressant activity has been successfully demonstrated in multiple clinical trials, the inherent ability to elevate RBC counts and other hematological parameters preclude its development as a mainstream CNS drug. A chemically engineered derivative, carbamoylated Epo (Cepo) has no hematological activity, but retains the neurotrophic actions of Epo. Cepo is therefore an attractive candidate to be tested as an antidepressant. Objective: To evaluate the antidepressant properties of Cepo in established antidepressant-responsive rodent behavioral assays. Methods: Adult male and female BALB/c mice were used for this study. Cepo (30 mu grams/kg BWT) or vehicle (PBS) was administered intraperitoneally for 4 days before the test of novelty induced hypophagia and subsequently at five hours before testing in forced swim test (FST), tail suspension test (TST) and open field test (OFT). To obtain mechanistic insight we examined the phosphorylation of the transcription factor cAMP response element binding protein (CREB). Results: Administration of Cepo at 30 mu grams/kg BWT, for 4 days produced significant reduction in latency to consume a palatable drink in a novel environment in male and female mice. Male BALB/c mice had a significant reduction in immobility in both tail suspension and forced swim tests, and female mice exhibited lower immobility in the forced swim test.

In recent years, erythropoietin (EPO) has emerged as a useful neuroprotective and neurotrophic molecule that produces antidepressant and cognitive-enhancing effects in psychiatric disorders. However, EPO robustly induces erythropoiesis and elevates red blood cell counts. Chronic administration is therefore likely to increase blood viscosity and produce adverse effects in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered modification of EPO, is non-erythropoietic but retains the neurotrophic and neurotrophic activity of EPO. Blood profile analysis after EPO and CEPO administration showed that CEPO has no effect on red blood cell or platelet counts. We conducted an unbiased, quantitative, mass spectrometry-based proteomics study to comparatively investigate EPO and CEPO-induced protein profiles in neuronal phenotype PC12 cells. Bioinformatics enrichment analysis of the protein expression profiles revealed the upregulation of protein functions related to memory formation such as synaptic plasticity, long term potentiation (LTP), neurotransmitter transport, synaptic vesicle priming, and dendritic spine development. The regulated proteins, with roles in LTP and synaptic plasticity, include calcium/calmodulin-dependent protein kinase type 1 (Camk1), Synaptosomal-Associated Protein, 25 kDa (SNAP-25), Sectretogranin-1 (Chgb), Cortactin (Cttn), Elongation initiation factor 3a (Eif3a) and 60S acidic ribosomal protein P2 (Rplp2). We examined the expression of a subset of regulated proteins, Cortactin, Grb2 and Pleiotrophin, by immunofluorescence analysis in the rat brain. Grb2 was increased in the dentate gyrus by EPO and CEPO. Cortactin was induced by CEPO in the molecular layer, and pleiotrophin was increased in the vasculature by EPO. The results of our study shed light on potential mechanisms whereby EPO and CEPO produce cognitive-enhancing effects in clinical and preclinical studies.