K Rafa-Zablocka

α1-Adrenergic receptor subtypes in the central nervous system: insights from genetically engineered mouse models

α1-Adrenergic receptors (α1-ARs) are important players in peripheral and central nervous system (CNS) regulation and function and in mediating various behavioral responses. The α1-AR family consists of three subtypes, α1A, α1B and α1D, which differ in their subcellular distribution, efficacy in evoking intracellular signals and transcriptional profiles. All three α1-AR subtypes are present at relatively high densities throughout the CNS, but the contributions of the individual subtypes to various central functions are currently unclear. Because of the lack of specific ligands, functionally characterizing the α1-ARs and discriminating between the three subtypes are difficult. To date, studies using genetically engineered mice have provided some information on subtype-related functions of the CNS α1-ARs. In this mini-review, we discuss several CNS processes where the α1-ARs role has been delineated with pharmacological tools and by studies using mutated mice strains that infer specific α1-AR subtype functions through evaluation of behavioral phenotypes.

Selective Depletion of CREB in Serotonergic Neurons Affects the Upregulation of Brain-Derived Neurotrophic Factor Evoked by Chronic Fluoxetine Treatment

Neurotrophic factors are regarded as crucial regulatory components in neuronal plasticity and are postulated to play an important role in depression pathology. The abundant expression of brain-derived neurotrophic factor (BDNF) in various brain structures seems to be of particular interest in this context, as downregulation of BDNF is postulated to be correlated with depression and its upregulation is often observed after chronic treatment with common antidepressants. It is well-known that BDNF expression is regulated by cyclic AMP response element-binding protein (CREB). In our previous study using mice lacking CREB in serotonergic neurons (Creb1TPH2CreERT2 mice), we showed that selective CREB ablation in these particular neuronal populations is crucial for drug-resistant phenotypes in the tail suspension test observed after fluoxetine administration in Creb1TPH2CreERT2 mice. The aim of this study was to investigate the molecular changes in the expression of neurotrophins in Creb1TPH2CreERT2 mice after chronic fluoxetine treatment, restricted to the brain structures implicated in depression pathology with profound serotonergic innervation including the prefrontal cortex (PFC) and hippocampus. Here, we show for the first time that BDNF upregulation observed after fluoxetine in the hippocampus or PFC might be dependent on the transcription factor CREB residing, not within these particular structures targeted by serotonergic projections, but exclusively in serotonergic neurons. This observation may shed new light on the neurotrophic hypothesis of depression, where the effects of BDNF observed after antidepressants in the hippocampus and other brain structures were rather thought to be regulated by CREB residing within the same brain structures. Overall, these results provide further evidence for the pivotal role of CREB in serotonergic neurons in maintaining mechanisms of antidepressant drug action by regulation of BDNF levels.

The impact of selective CREB deletion in serotonergic neurons on the effects of fluoxetine administration and antidepressive behaviour

The majority of current antidepressant therapies is based on the enhancement of monoaminergic transmission observed directly after drug administration, yet alleviation of depressive symptoms occurs weeks later. Therefore, the mechanism of molecular changes arising during chronic antidepressant treatment is intensively researched. Many studies have pointed to an important role of cAMP response element binding (CREB) protein in the mechanism of antidepressant drug action, however the data are inconclusive. Generally, an increase of CREB level or activity after chronic antidepressant treatment was postulated, but in other studies opposite effects were observed [1]. Moreover, CREB-deficient animals show antidepressant-like phenotype. However, the feedback from knock-out studies could have been disturbed by cAMP response element modulator (CREM) upregulation occurring upon CREB loss [2], which was not taken into consideration. In our studies we investigated the role of CREB in the mechanism of antidepressant treatment using novel, inducible transgenic mouse model lacking CREB selectively in serotonergic neurons [3], maintained in CREM-deficient background (Creb1TPH2CreERT2Crem-/- mice). The animals have been phenotypically characterized showing no impairments in their basal behavior. However, single Creb1TPH2CreERT2 mutants of both sexes presented drug-resistant phenotype in tail suspension test after fluoxetine treatment, whereas male but not female double mutants (Creb1TPH2CreERT2Crem-/-) reacted to the drug similar to control animals [4]. The aim of current studies was to assess the molecular changes induced by chronic fluoxetine (10 mg/kg i.p., 21 days, 1x daily) treatment in hippocampus and prefrontal cortex (PFC). Animals were sacrificed 24h after last injection and the brain structures were collected. Assessment of mRNA expression for Creb1 and Bdnf genes was measured by RT-PCR using TaqMan probes. The levels of phosphorylated CREB, total CREB and BDNF proteins were studied using Western Blot. The results were analyzed using two-way ANOVA, followed by Fisher’s LSD test. We observed no effect in mRNA expression of Creb1 and Bdnf genes in control animals after fluoxetine treatment, nor in any of studied mutants. Moreover, no changes in phosphorylation of CREB or total CREB protein were visible in any of studied group. On the other hand, BDNF protein was strongly increased in response to drug in hippocampus of control males and females (by 212% and 120%, respectively), and in PFC of control females only (by 47%). Both treated and non-treated Creb1TPH2CreERT2 single mutant mice did not show any changes in BDNF in both studied brain structures; the result was significantly different from the effect observed in control animals after fluoxetine administration. Creb1TPH2CreERT2Crem-/- double mutants presented only non-significant tendency of increased BDNF protein level in hippocampus (both males and females) and PFC (females only). Despite the absence of any effect in CREB mRNA or protein levels observed in both mutant lines, the BDNF upregulation in fluoxetine-treated control mice suggests increased activity of CREB in hippocampus after antidepressant treatment as observed by other investigators [5]. Lack of molecular or behavioral changes after fluoxetine treatment in Creb1TPH2CreERT2 single mutant animals suggests that compensatory role of CREM upregulation in the inducible line may have different effects from that observed previously in constitutive CREB knockouts.

Lack of riluzole efficacy in the progression of the neurodegenerative phenotype in a new conditional mouse model of striatal degeneration

Background Huntington’s disease (HD) is a rare familial autosomal dominant neurodegenerative disorder characterized by progressive degeneration of medium spiny neurons (MSNs) located in the striatum. Currently available treatments of HD are only limited to alleviating symptoms; therefore, high expectations for an effective therapy are associated with potential replacement of lost neurons through stimulation of postnatal neurogenesis. One of the drugs of potential interest for the treatment of HD is riluzole, which may act as a positive modulator of adult neurogenesis, promoting replacement of damaged MSNs. The aim of this study was to evaluate the effects of chronic riluzole treatment on a novel HD-like transgenic mouse model, based on the genetic ablation of the transcription factor TIF-IA. This model is characterized by selective and progressive degeneration of MSNs. Methods Selective ablation of TIF-IA in MSNs (TIF-IAD1RCre mice) was achieved by Cre-based recombination driven by the dopamine 1 receptor (D1R) promoter in the C57Bl/6N mouse strain. Riluzole was administered for 14 consecutive days (5 mg/kg, i.p.; 1× daily) starting at six weeks of age. Behavioral analysis included a motor coordination test performed on 13-week-old animals on an accelerated rotarod (4–40 r.p.m.; 5 min). To visualize the potential effects of riluzole treatment, the striata of the animals were stained by immunohistochemistry (IHC) and/or immunofluorescence (IF) with Ki67 (marker of proliferating cells), neuronal markers (NeuN, MAP2, DCX), and markers associated with neurodegeneration (GFAP, 8OHdG, FluoroJade C). Additionally, the morphology of dendritic spines of neurons was assessed by a commercially available FD Rapid Golgi Stain™ Kit. Results A comparative analysis of IHC staining patterns with chosen markers for the neurodegeneration process in MSNs did not show an effect of riluzole on delaying the progression of MSN cell death despite an observed enhancement of cell proliferation as visualized by the Ki67 marker. A lack of a riluzole effect was also reflected by the behavioral phenotype associated with MSN degeneration. Moreover, the analysis of dendritic spine morphology did not show differences between mutant and control animals. Discussion Despite the observed increase in newborn cells in the subventricular zone (SVZ) after riluzole administration, our study did not show any differences between riluzole-treated and non-treated mutants, revealing a similar extent of the neurodegenerative phenotype evaluated in 13-week-old TIF-IAD1RCre animals. This could be due to either the treatment paradigm (relatively low dose of riluzole used for this study) or the possibility that the effects were simply too weak to have any functional meaning. Nevertheless, this study is in line with others that question the effectiveness of riluzole in animal models and raise concerns about the utility of this drug due to its rather modest clinical efficacy.