Gregers Wegener

Reference Genes for Normalization: A Study of Rat Brain Tissue

Quantitative real‐time polymerase chain reaction (qPCR) has become a widely used tool in the search for disease genes. When examining gene expression with qPCR in psychiatric diseases, endogenous reference gene(s) must be used for normalization. Traditionally, genes such as β‐actin (ActB), Gapd, and 18s rRNA, assumed to be stably expressed, have been used for normalization. However, it has become clear that expression of these genes is influenced by different experimental paradigms. Since stable gene expression of houskeeping genes (HKGs), therefore, cannot be expected, alternative strategies are warranted. With the overall aim to inspect the gene expression of three target genes, NMDAR1, SORT, and CREB, in rat hippocampus, we tested a panel of eight HKGs, 18s rRNA, ActB, CycA, Gapd, Hmbs, Hprt1, Rpl13A, and Ywhaz in order to select the most stably expressed gene, using the NormFinder and geNorm software applets. Additionally, we have applied four different normalization approaches for normalization of the three target genes. We found using the NormFinder software that Ywhaz is the most stably expressed gene among the eight tested HKGs. However, the results of the analysis of the target genes are highly dependent on the choice of normalization approach. Moreover, the number of HKGs, used for selection of the most stable HKG, also influences on the result of the gene expression analysis of target genes. These results underline the importance of choosing a proper normalization strategy when analyzing gene expression with qPCR. The strategy should be unbiased and carried out in every specific experimental setting. Synapse 62:302–309, 2008.

Antidepressant treatment is associated with epigenetic alterations in the promoter of P11 in a genetic model of depression

P11 (S100A10) has been associated with the pathophysiology of depression both in human and rodent models. Different types of antidepressants have been shown to increase P11 levels in distinct brain regions and P11 gene therapy was recently proven effective in reversing depressive-like behaviours in mice. However, the molecular mechanisms that govern P11 gene expression in response to antidepressants still remain elusive. In this study we report decreased levels of P11, associated with higher DNA methylation in the promoter region, in the prefrontal cortex of the Flinders Sensitive Line (FSL) genetic rodent model of depression. This hypermethylated pattern was reversed to normal, as indicated by the control line, after chronic administration of escitalopram (a selective serotonin reuptake inhibitor; SSRI). The escitalopram-induced hypomethylation was associated with both an increase in P11 gene expression and a reduction in mRNA levels of two DNA methyltransferases that have been shown to maintain DNA methylation in adult forebrain neurons (Dnmt1 and Dnmt3a). In conclusion, our data further support a role for P11 in depression-like states and suggest that this gene is controlled by epigenetic mechanisms that can be affected by antidepressant treatment.

Repeated electroconvulsive seizures increase the total number of synapses in adult male rat hippocampus

The underlying mechanism of the therapeutic effect of electroconvulsive therapy (ECT) is still unclear. Here we investigated whether repeated electroconvulsive seizures (ECS), an animal model of ECT, in rats induce neuroplastic changes in the subregions of the hippocampus. ECS or sham treatment was given daily for 10 days to adult male rats. Stereological principles were employed to quantify volumes and the number of neurons and synapses. Volumes of granule cell layer (GCL) and Hilus in Dentate Gyrus of the hippocampus were significantly larger in the ECS treatment group. The neuron numbers in GCL, synapse numbers (including total synapses, spine synapses, and both perforated and nonperforated spine synapse subtypes) and synapse height in CA1 were significantly increased in the ECS treatment group. Our results indicated that repeated ECS induces neurogenesis, synaptogenesis and remodelling of synapses in rat hippocampus. This could provide a potential mechanism to explain the therapeutic effect of ECS.

Inverse correlation of brain and blood BDNF levels in a genetic rat model of depression

There is accumulating evidence that brain-derived neurotrophic factor (BDNF) plays a critical role in the pathophysiology of depression. Decreased serum levels have been reported in major depression, and a correlation between BDNF reduction and the severity of the disease was found. Moreover, in post-mortem hippocampal tissue, increased levels of BDNF immunoreactivity have been reported in subjects treated with antidepressants compared to untreated subjects. These findings indicate parallel changes in brain and serum BDNF levels during depression. BDNF has been measured in selected brain areas in several animal models. In investigations between Flinders Sensitive Line (FSL) and Flinders Resistant Line (FRL) rats, a genetic rat model of depression, no differences were found in BDNF levels in the frontal cortex and hippocampus, areas believed to be core brain regions in depression. However, to our knowledge brain and serum BDNF levels have never been reported in parallel for any psychiatric disease model. Therefore, we examined the levels of BDNF in whole blood, serum, cerebrospinal fluid (CSF), hippocampus, and frontal cortex in male FSL and FRL rats. BDNF levels in serum and whole blood of FSL rats were significantly increased compared to FRL rats. In contrast, in the hippocampus the BDNF level was significantly decreased in FSL compared to FRL rats while no differences were found in the frontal cortex and CSF. The differential regulation of the BDNF levels in hippocampus, serum, and whole blood in FSL/FRL rats adds to the hypothesis that neurotrophic factors are related to the pathophysiology of depression.

Imipramine treatment increases the number of hippocampal synapses and neurons in a genetic animal model of depression.

The aim was to investigate treatment effects of the antidepressant imipramine on the markers of neuronal plasticity. We investigated changes in neuron and synapse numbers in a rat strain that displays a genetic susceptibility to depressive behavior, the Flinders Sensitive and Resistant Lines (FSL/FRL). All rats were treated with imipramine (15 mg/kg) or saline (i.p) once daily for 25 days. The volume, neuron and synapse numbers in the hippocampus were estimated using design‐based stereological methods. Under untreated conditions, the volume and the number of neurons and synapses were significantly smaller in the FSL saline group (untreated “depressed” rats) compared with the FRL saline group (normal rats), showing correlation to the observed decreased immobility in the forced swim test. Imipramine treatment significantly increased the number of neurons in the granule cell layer (GCL) and spine synapses in the CA1 in the FSL imipramine group (treated “depressed” rats) compared with the FSL saline group. The neuron numbers in the GCL and Hilus showed no differences in the FSL imipramine group compared to the FRL saline group. In conclusion, baseline levels of the volume and the number of neurons and spine synapses in hippocampus were significantly smaller in the untreated FSL rats. Our findings indicate that chronic imipramine treatment reverses the suppression of neurogenesis and synaptogenesis in the hippocampus of the “depressed” FSL rats, and this occurs in correlation with behavioral effects. Our results support the neuronal plasticity hypothesis that depressive disorders may be related to impairments of structural plasticity and neuronal viability in hippocampus, furthermore, antidepressant treatment counteracts the structural impairments.

Azure B, a metabolite of methylene blue, is a high-potency, reversible inhibitor of monoamine oxidase

Methylene blue (MB) has been shown to act at multiple cellular and molecular targets and as a result possesses diverse medical applications. Among these is a high potency reversible inhibition of monoamine oxidase A (MAO-A) that may, at least in part, underlie its adverse effects but also its psycho- and neuromodulatory actions. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl species, is the major metabolite. Similar to MB, azure B also displays a variety of biological activities and may therefore contribute to the pharmacological profile of MB. Based on these observations, the present study examines the interactions of azure B with recombinant human MAO-A and -B. The results show that azure B is a potent MAO-A inhibitor (IC₅₀=11 nM), approximately 6-fold more potent than is MB (IC₅₀=70 nM) under identical conditions. Measurements of the time-dependency of inhibition suggest that the interaction of azure B with MAO-A is reversible. Azure B also reversibly inhibits the MAO-B isozyme with an IC₅₀ value of 968 nM. These results suggest that azure B may be a hitherto under recognized contributor to the pharmacology and toxicology of MB by blocking central and peripheral MAO-A activity and as such needs to be considered during its use in humans and animals.

A high-fat diet exacerbates depressive-like behavior in the Flinders Sensitive Line (FSL) rat, a genetic model of depression

Major depressive disorder (MDD) and diabetes mellitus type II (T2DM) are two of the major health challenges of our time. It has been shown that MDD and T2DM are highly co-morbid, and recent work has proposed a bi-directional connection between the diseases. The aim of the current study was to investigate the effect of a high-fat diet (HFD) on behavior and metabolism in a genetic rat model of depression, the Flinders Sensitive and Resistant Line (FSL/FRL) rats. Age and weight matched rats were fed a HFD or control diet for 10 weeks and subjected to behavioral testing and metabolic assessment. We found that HFD exacerbated the depressive-like behavior of the FSL rat in the Forced Swim Test (FST), a depression screening tool, although it did not affect the non-depressed FRL rat despite a higher caloric intake. Moreover, the depressive-like phenotype was associated with reduced anxiety and impairment in novel object recognition memory, while HFD consumption led to diminished object recognition memory as well. In both strains HFD increased insulin levels during an oral glucose tolerance test, although fasting blood glucose levels were only significantly increased by HFD in the FSL rat, suggesting a greater metabolic susceptibility in this rat strain. We conclude that compared with the FRL rat, the FSL rat is more susceptible to developing aberrant behaviors related to depression following metabolic stress induced by HFD. Further studies with a mechanistic focus could potentially lead to a better understanding of a possible pathophysiological link between T2DM and MDD.

Detection of brain-derived neurotrophic factor (BDNF) in rat blood and brain preparations using ELISA: Pitfalls and solutions

Quantification of endogenous brain-derived neurotrophic factor (BDNF) can be performed with an enzyme-linked immunosorbent assay (ELISA). Although BDNF has been determined in blood and brain preparations in numerous studies with ELISA kits, the methodological issues regarding measurements of BDNF concentrations in the blood and particularly in the brain have only been superficially investigated. We aimed at validating and optimizing the BDNF ELISA kit with respect to measurements in rat blood and brain samples. We found that the pre-analytical conditions were critical for plasma samples, but not serum or whole blood samples. The intra- and inter-assay variation and the accuracy and yield of the BDNF ELISA kit in rat serum and brain tissue were conducted with the optimal dilutions of frontal cortex and hippocampus extract. The optimal dilutions of frontal cortex and hippocampus extracts were determined to be 20 and 120 times, and we established that the intra-assay coefficient of variation (CV%) was 8 in hippocampus and 2 in frontal cortex and serum. The inter-assay variation was also low with a CV% of 11 or less in hippocampus, frontal cortex, and serum. Finally, we found that the accuracy and yield of the BDNF measurements were high in serum and low in hippocampus and frontal cortex. We conclude that the BDNF ELISA kit determines serum BDNF accurately and with high reproducibility. Furthermore it can be used for measurement of BDNF in rat brain preparations when particular precautions are taken and in particular with care regarding the dilution of the brain tissue samples.

Increased stress-evoked nitric oxide signalling in the Flinders sensitive line (FSL) rat: a genetic animal model of depression

Stress engenders the precipitation and progression of affective disorders, while stress-related release of excitatory mediators is implicated in the degenerative pathology observed especially in the hippocampus of patients with severe depression. Nitric oxide (NO) release following stress-evoked N-methyl-d-aspartate (NMDA) receptor activation modulates neurotransmission, cellular memory and neuronal toxicity. We have investigated the Flinders rat (FSL/FRL), a genetic animal model of depression, regarding the response of the hippocampal nitrergic system following exposure to an escapable stress/inescapable stress (ES-IS) paradigm. Hippocampal tissue from naive FSL/FRL rats and those exposed to ES-IS were studied with respect to constitutive nitric oxide synthase (cNOS) activity and neuronal nitric oxide synthase (nNOS) protein levels, as well as transcript expression of upstream regulatory proteins in the NMDA-NO signalling pathway, including NMDAR1, nNOS, CAPON, PIN and PSD95. Within stress-naive animals, no differences in hippocampal cNOS activity and nNOS expression or PIN were evident in FSL and FRL rats, although transcripts for NMDAR1 and CAPON were increased in FSL rats. Within the group of ES-IS animals, we found an increase in total hippocampal cNOS activity, nNOS protein levels and mRNA expression in FSL vs. FRL rats, together with an increase in PSD95 transcripts, and a reduction in PIN. In conclusion, ES-IS enhanced hippocampal cNOS activity in FSL rats, but not FRL rats, confirming the NMDA-NO cascade as an important vulnerability factor in the depressive phenotype of the FSL rat.

Differential expression of synaptic vesicle proteins after repeated electroconvulsive seizures in rat frontal cortex and hippocampus.

Electroconvulsive therapy (ECT) remains the treatment of choice for patients with severe or drug‐resistant depressive disorders, yet the mechanism behind its efficacy and the effect on neurotransmission is essentially unknown. As synaptic vesicle proteins (SVPs) are required for vesicle fusion and neurotransmitter release, we have examined the effect of single and repeated electroconvulsive seizures (ECS), an animal model of ECT, on the expression of 14 SVPs in the rat frontal cortex and the hippocampus using quantitative real‐time polymerase chain reaction (real‐time qPCR). Only in the frontal cortex, the mRNA level of synapsin II was significantly upregulated after repeated ECS. In contrast, the mRNA levels of 6 of the 14 SVPs were significantly regulated in the hippocampus after ECS. We found that SNAP29 was upregulated and synaptotagmin III was downregulated after one single ECS in the hippocampus. Furthermore, SNAP29, synapsin I, synapsin III, VAMP2, and VAMP5 were significantly upregulated, whereas synaptotagmin III was significantly downregulated after repeated ECS in the hippocampus. We suggest that these genes are highly important in the long‐term therapeutic effect of ECS, and thus it can behypothesized that the SVPs are involved in the pathophysiology of depression. Synapse 62:662–670, 2008.