Effects of chess-based cognitive remediation training as therapy add-on in alcohol and tobacco use disorders: protocol of a randomised, controlled clinical fMRI trial.

BACKGROUND: Alcohol and tobacco use disorders (AUD, TUD) are frequent, both worldwide and in the German population, and cognitive impairments are known to facilitate instances of relapse. Cognitive training has been proposed for enhancing cognitive functioning and possibly improving treatment outcome in mental disorders. However, these effects and underlying neurobiological mechanisms are not yet fully understood regarding AUD and TUD. Examining the effect of chess-based cognitive remediation training (CB-CRT) on neurobiological, neuropsychological and psychosocial aspects as well as treatment outcomes will provide insights into mechanisms underlying relapse and abstinence and might help to improve health behaviour in affected individuals if used as therapy add-on. METHODS AND ANALYSIS: N=96 individuals with either AUD (N=48) or TUD (N=48) between 18 and 65 years of age will participate in a randomised, controlled clinical functional MRI (fMRI) trial. Two control groups will receive treatment as usual, that is, AUD treatment in a clinic, TUD outpatient treatment. Two therapy add-on groups will receive a 6-week CB-CRT as a therapy add-on. FMRI tasks, neurocognitive tests will be administered before and afterwards. All individuals will be followed up on monthly for 3 months. Endpoints include alterations in neural activation and neuropsychological task performance, psychosocial functioning, and relapse or substance intake. Regarding fMRI analyses, a general linear model will be applied, and t-tests, full factorial models and regression analyses will be conducted on the second level. Behavioural and psychometric data will be analysed using t-tests, regression analyses, repeated measures and one-way analyses of variance. ETHICS AND DISSEMINATION: This study has been approved by the ethics committee of the medical faculty Mannheim of the University of Heidelberg (2017-647N-MA). The findings of this study will be presented at conferences and published in peer-reviewed journals. TRIAL REGISTRATION: The study was registered in the Clinical Trials Register (trial identifier: NCT04057534 at clinicaltrials.gov).

Predicting Participation in and Success of a Concurrent Smoking Cessation Program during Inpatient Treatment for Alcohol Dependence.

BACKGROUND: Predicting participation in and success of smoking cessation programs in alcohol dependent patients has yielded heterogeneous results. Moreover, these findings have rarely been based on prospective studies from clinical routine settings. Identifying predictors in prospective studies could help to tailor interventions that increase participation and success rates of smoking cessation therapies for these patients at a high risk for alcohol- and smoking-related morbidities and mortalities. SUBJECTS AND METHODS: During inpatient alcohol dependence treatment, 99 nicotine dependent patients were recruited. 73 patients chose to participate in a voluntary smoking cessation program. Interviews and questionnaires were used at baseline and at discharge to assess a large set of variables covering smoking and alcohol related factors, general psychopathology, quality of life and personality traits. Multiple logistic regression models were calculated to predict participation in the smoking cessation program and smoking abstinence at follow-up three months after discharge. RESULTS: Participation in the smoking cessation program was predicted by higher stage of change, higher confidence in abstaining from smoking and lower perceived stress. Successful smoking cessation at follow-up was predicted by higher expectations of negative physical feelings due to smoking and lower expectations of temptations to smoke at baseline, and by lower number of daily smoked cigarettes at discharge. CONCLUSION: Despite the small sample size, this prospective study gives a first indication of clinically relevant predictors of participation in and success of a smoking cessation program by exploring many previously reported predictors simultaneously. The findings and their implications for treatment allocation and optimization are discussed.

Activation of the glucocorticoid receptor rapidly triggers calcium-dependent serotonin release in vitro.

AIMS: Glucocorticoids rapidly provoke serotonin (5-HT) release in vivo. We aimed to investigate molecular mechanisms of glucocorticoid receptor (GR)-triggered 5-HT release. METHODS: Employing 1C11 cells to model 5-HT neurotransmission, immunofluorescence and Pearson's Correlation Coefficient were used to analyze colocalization of GR, 5-HT, vesicle membrane protein synaptotagmin 1 and vesicle dye FM4-64FX. FFN511 and FM4-64FX dyes as well as calcium imaging were used to visualize vesicular 5-HT release upon application of GR agonist dexamethasone, GR antagonist mifepristone and voltage-gated calcium channel (VGCC) inhibitors. RESULTS: GR, 5-HT, synaptotagmin 1 and FM4-64FX showed overlapping staining patterns, with Pearson's Correlation Coefficient indicating colocalization. Similarly to potassium chloride, dexamethasone caused a release of FFN511 and uptake of FM4-64FX, indicating vesicular 5-HT release. Mifepristone, calcium depletion and inhibition of L-type VGCC significantly diminished dexamethasone-induced vesicular 5-HT release. CONCLUSIONS: In close proximity to 5-HT releasing sites, activated GR rapidly triggers L-type VGCC-dependent vesicular 5-HT release. These findings provide a better understanding of the interrelationship between glucocorticoids and 5-HT release.

ADHD Is Associated with a Higher Risk for Traumatic Events, Self-Reported PTSD, and a Higher Severity of PTSD Symptoms in Alcohol-Dependent Patients.

INTRODUCTION: Alcohol dependence (AD) is frequently associated with attention-deficit/hyperactivity disorder (ADHD) as well as with posttraumatic stress disorder (PTSD). OBJECTIVE: Aim of this study was to investigate whether in patients with AD, ADHD is associated with an increased rate of exposure to potentially traumatic events (PTEs) and PTSD, with increased PTSD severity and higher impact on daily functioning. METHODS: Patients with AD were thoroughly assessed for ADHD during long-term residential treatment. Participants also completed the Posttraumatic Diagnostic Scale (PDS). The PDS is a 49-item self-report instrument that assesses exposure to different PTEs, DSM-IV criteria for PTSD, severity of PTSD, and related functional impairment. RESULTS: Of 341 patients with AD, 66 were diagnosed with ADHD (19%). ADHD was associated with a more frequent exposure to PTEs (88 vs. 65%, p < 0.001). In patients with PTEs (n = 237), odds for PTSD were higher in ADHD versus no-ADHD patients (OR 8.9, 95% CI 3.9-20.5). Furthermore, PTSD severity and functional impairment were increased in ADHD patients. CONCLUSIONS: ADHD in patients with AD is associated with a higher frequency of PTEs and PTSD with more severe and more impairing PTSD symptoms. In alcohol-dependent patients with ADHD, regular screening for PTSD should be considered.

Prevalence Estimates of ADHD in a Sample of Inpatients With Alcohol Dependence.

Objective: ADHD is common in patients with alcohol dependence, but prevalence results are inconsistent. We investigated ADHD prevalence in a complex design to avoid over- or underdiagnosing. Method: Patients with alcohol dependence starting long-term residential treatment were included. A structured interview (Diagnostic Interview for ADHD in Adults [DIVA]) was conducted on all patients. DIVA results indicating childhood or adulthood ADHD were assessed in successive diagnostic interviews by two expert clinicians. Results: 415 of 488 patients had completed the entire diagnostic assessment. ADHD prevalence was 20.5%. DIVA results correlated moderately with experts' diagnoses. In patients with ADHD, a higher comorbid illicit substance use was prevalent and alcohol dependence started earlier and was more severe. Conclusion: This study provides the largest sample on ADHD prevalence in alcohol dependent inpatients. Despite great efforts to avoid overestimation, we found every fifth patient to have ADHD. ADHD diagnosis should not be based solely on a structured interview but should be clinically confirmed.

[ADHD screening in alcohol dependent subjects : Psychometric characteristics of ADHD self-report scale and Wender Utah Rating Scale short form]. / ADHS-Screening bei Alkoholabhängigen : Psychometrische Eigenschaften der ADHS-Selbstbeurteilungsskala (ADHS-SB) und der Wender-Utah-Rating-Scale Kurzform (WURS­k).

BACKGROUND: Routine screening using self report questionnaires for attention deficit/hyperactivity disorder (ADHD) in patients with alcohol use disorder is recommended due to its high prevalence and the time-consuming and demanding diagnostic assessment. The psychometric properties of such self-report instruments have hardly been investigated in this patient group. METHODS: The performance of the ADHD self-report scale (ADHD-SR) and the German short form of the Wender Utah Rating Scale (WURS-k) was determined in 402 patients with alcohol dependence during long-term residential weaning treatment. The diagnosis of adult ADHD had been confirmed in 85 of these patients using an extensive, standardized diagnostic procedure. RESULTS: The ADHD-SR at a cut-off ≥15 showed the best psychometric properties (sensitivity 75.3%, specificity 94.0%, positive predictive value, PPV 77.1%, negative predictive value, NPV 93.4%). The results of the WURS-k were unsatisfactory at the established cut-off ≥30 and also at lower cut-offs with more false positive screening results compared to the ADHD-SR. The combination of both instruments (ADHD-SR ≥ 15 and/or WURS-k ≥ 30) increased sensitivity (86.9%) albeit at the expense of a higher rate of false positive screening results (specificity 87.7%, PPV 65.2%). CONCLUSION: The ADHD-SR is a suitable instrument for routine screening of ADHD in alcohol dependent patients. To improve sensitivity, a lower cut-off (≥15) should be applied. The WURS-k as a single screening instrument cannot be recommended in alcohol dependent patients. If a higher sensitivity is desired, a combination of ADHD-SR and WURS-k is feasible.

Screening for adult attention-deficit/hyperactivity disorder in alcohol dependent patients: Underreporting of ADHD symptoms in self-report scales.

BACKGROUND: Attention-deficit/hyperactivity disorder (ADHD) is a common comorbid disorder that is frequently overlooked in adults with alcohol use disorder (AUD). Moreover, identifying ADHD in AUD patients is time-consuming and difficult. The aim of this study was to assess the clinical utility of two self-report screening instruments for adult ADHD in AUD patients. METHODS: 404 adults seeking residential treatment for AUD were screened using the Conners' Adult ADHD Rating Scale Screening Self-Rating (CAARS-S-SR) and the Adult ADHD Rating Scale (ASRS). Results were compared with ADHD diagnosis obtained from a stepped approach: first, a structured interview (Diagnostic Interview for ADHD in adults 2.0.; DIVA) was applied; second, probable ADHD diagnoses had to be confirmed by two expert clinicians. RESULTS: At the previously reported cut-off values, ASRS and CAARS-S-SR showed low sensitivities of 57.1 and 70.6%. A high number of false negative results (NPV ASRS: 89.5%; CAARS-S-SR: 92.3%) indicates underreporting of ADHD symptoms. Sensitivity improved at lower cut-off (ASRS ≥ 11; CAARS-S-SR ≥60) or with a combination of both instruments at lower cut-offs. Area Under the Curve (AUC) for the combination of ASRS and CAARS-S-SR was superior to the AUCs of the single questionnaires. CONCLUSIONS: Underreporting of ADHD symptoms in ASRS and CAARS-S-SR of AUD patients requires lower cut-off values to detect the majority of ADHD, albeit at the expense of an increased rate of false-positive results. Cut-off values should be adjusted to the clinical setting. Clinicians should take into consideration that a negative screening result does not necessarily imply absence of ADHD.

Type-1 astrocyte-like stem cells harboring Cacna1d gene deletion exhibit reduced proliferation and decreased neuronal fate choice.

In the central nervous system, CaV 1.2 and CaV 1. 3 constitute the main L-type voltage-gated calcium channels (LTCCs) coupling membrane depolarization to gene transcription. We have previously demonstrated that inducible disruption of Cav1.2 in type-1 astrocyte-like stem cells of the adult dentate gyrus (DG) impairs hippocampal neurogenesis in a cell-autonomous fashion. To address the role of Cav1.3 channels (encoded by the Cacna1d gene), we here generated TgGLAST-CreERT2 /Cacna1dfl/fl /RCE:loxP mice which facilitate inducible deletion of Cacna1d in tandem with induction of EGFP expression in type-1 cells, allowing tracking of recombined cells and their descendants. Neurosphere cultures derived from fluorescence-activated cell sorting sorted Cacna1d-deficient (Cacna1d-/- /EGFP) hippocampal neural precursor cells (NPCs) exhibited a significant decrease in proliferative activity. Further, under differentiation conditions, Cacna1d deficiency conferred an increase in astrogenesis at the expense of neurogenesis. In like manner, type-1 cells lacking Cacna1d showed reduced proliferation in the dentate gyrus (DG) in vivo. Moreover, Cacna1d deficiency resulted in a significant decrease in the number of newly born cells adopting a neuronal fate. Finally, massive excitation induced by repeated electroconvulsive seizures rescued the proliferation defect of Cacna1d-/- /EGFP type-1 cells. Together, the effects of Cacna1d gene deletion closely recapitulate our earlier findings on the role of Cav1.2 channels expressed by type-1 cells. Similar to Cav1.2 channels, Cav1.3 channels on type-1 cells boost type-1 cell proliferation and promote subsequent neuronal fate choice.

Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell-autonomous fashion.

Ca2+ is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha-1C subunit (i.e., Cav 1.2) of the voltage-dependent l-type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Cav 1.2 channels located on astrocyte-like stem cells and their descendants in the development of new granule neurons, we created TgGLAST-CreERT2 /Cacna1cfl/fl /RCE:loxP mice, a transgenic tool that allows cell-type-specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type-1 cells. FACS-sorted Cacna1c-deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c-deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type-1 cells decreased type-1 cell proliferation and reduced the neuronal fate-choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type-1 cells, indicating that there must be Cav 1.2-independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L-type 1.2 channels on type-1 cells. Cav 1.2 channels promote type-1 cell proliferation and push the glia-to-neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Cav 1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell-autonomous fashion.

Cannabinoid type-1 receptor signaling in central serotonergic neurons regulates anxiety-like behavior and sociability.

The endocannabinoid (eCB) system possesses neuromodulatory functions by influencing the release of various neurotransmitters, including γ-aminobutyric acid (GABA) and glutamate. A functional interaction between eCBs and the serotonergic system has already been suggested. Previously, we showed that cannabinoid type-1 (CB1) receptor mRNA and protein are localized in serotonergic neurons of the raphe nuclei, implying that the eCB system can modulate serotonergic functions. In order to substantiate the physiological role of the CB1 receptor in serotonergic neurons of the raphe nuclei, we generated serotonergic 5-hydroxytryptamine (5-HT) neuron-specific CB 1 receptor-deficient mice, using the Cre/loxP system with a tamoxifen-inducible Cre recombinase under the control of the regulatory sequences of the tryptophan hydroxylase 2 gene (TPH2-CreER (T2)), thus, restricting the recombination to 5-HT neurons of the central nervous system (CNS). Applying several different behavioral paradigms, we revealed that mice lacking the CB1 receptor in serotonergic neurons are more anxious and less sociable than control littermates. Thus, we were able to show that functional CB1 receptor signaling in central serotonergic neurons modulates distinct behaviors in mice.

Adult AMPA GLUA1 receptor subunit loss in 5-HT neurons results in a specific anxiety-phenotype with evidence for dysregulation of 5-HT neuronal activity.

Both the glutamatergic and serotonergic (5-HT) systems are implicated in the modulation of mood and anxiety. Descending cortical glutamatergic neurons regulate 5-HT neuronal activity in the midbrain raphe nuclei through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors. To analyze the functional role of GLUA1-containing AMPA receptors in serotonergic neurons, we used the Cre-ERT2/loxP-system for the conditional inactivation of the GLUA1-encoding Gria1 gene selectively in 5-HT neurons of adult mice. These Gria1(5-HT-/-) mice exhibited a distinct anxiety phenotype but showed no alterations in locomotion, depression-like behavior, or learning and memory. Increased anxiety-related behavior was associated with significant decreases in tryptophan hydroxylase 2 (TPH2) expression and activity, and subsequent reductions in tissue levels of 5-HT, its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine in the raphe nuclei. However, TPH2 expression and activity as well as monoamine levels were unchanged in the projection areas of 5-HT neurons. Extracellular electrophysiological recordings of 5-HT neurons revealed that, while α1-adrenoceptor-mediated excitation was unchanged, excitatory responses to AMPA were enhanced and the 5-HT1A autoreceptor-mediated inhibitory response to 5-HT was attenuated in Gria1(5-HT-/-) mice. Our data show that a loss of GLUA1 protein in 5-HT neurons enhances AMPA receptor function and leads to multiple local molecular and neurochemical changes in the raphe nuclei that dysregulate 5-HT neuronal activity and induce anxiety-like behavior.

Phenotype of mice with inducible ablation of GluA1 AMPA receptors during late adolescence: relevance for mental disorders.

Adolescence is characterized by important molecular and anatomical changes with relevance for the maturation of brain circuitry and cognitive function. This time period is of critical importance in the emergence of several neuropsychiatric disorders accompanied by cognitive impairment, such as affective disorders and schizophrenia. The molecular mechanisms underlying these changes at neuronal level during this specific developmental stage remains however poorly understood. GluA1-containing AMPA receptors, which are located predominantly on hippocampal neurons, are the primary molecular determinants of synaptic plasticity. We investigated here the consequences of the inducible deletion of GluA1 AMPA receptors in glutamatergic neurons during late adolescence. We generated mutant mice with a tamoxifen-inducible deletion of GluA1 under the control of the CamKII promoter for temporally and spatially restricted gene manipulation. GluA1 ablation during late adolescence induced cognitive impairments, but also marked hyperlocomotion and sensorimotor gating deficits. Unlike the global genetic deletion of GluA1, inducible GluA1 ablation during late adolescence resulted in normal sociability. Deletion of GluA1 induced redistribution of GluA2 subunits, suggesting AMPA receptor trafficking deficits. Mutant animals showed increased hippocampal NMDA receptor expression and no change in striatal dopamine concentration. Our data provide new insight into the role of deficient AMPA receptors specifically during late adolescence in inducing several cognitive and behavioral alterations with possible relevance for neuropsychiatric disorders.

Nongenomic, glucocorticoid receptor-mediated regulation of serotonin transporter cell surface expression in embryonic stem cell derived serotonergic neurons.

Depressive disorders have been linked to the combined dysregulation of the hypothalamus-pituitary-adrenal (HPA)-axis and the serotonergic system. The HPA-axis and serotonergic (5-HT) neurons exert reciprocal regulatory actions. It has been reported that glucocorticoid-glucocorticoid receptor (GR) signaling influences serotonin transporter (5-HTT) transcription but data also points to the fact that 5-HTT expression is regulated nongenomically via redistribution of 5-HTT from the cell surface into intracellular compartments. In order to analyze the acute effects of glucocorticoids on 5-HTT cell surface localization we differentiated serotonergic neurons from mouse embryonic stem (ES) cells derived from the C57BL/6N blastocysts. These postmitotic 5-HT neurons express all relevant serotonergic markers following the application of a growth factor-based differentiation protocol. Increasing concentrations of the GR agonist dexamethasone (Dex) resulted in enhanced, dose-dependent 5-HTT cell surface localization in the presence of the protein synthesis inhibitor cycloheximide already 1h after incubation. Inhibition of GR function by the specific GR-antagonist mifepristone abolished the increase in 5-HTT cell surface localization. Hence, our data account for a nongenomic upregulation of 5-HTT cell surface expression by glucocorticoid-GR interaction which likely constitutes a rapid physiological response to increased levels of glucocorticoids as seen during stress. Taken together, we provide a cellular model to analyze and dissect glucocorticoid-5HTT interactions on a molecular level that corresponds to in vivo animal models using C57BL/6N mice.

Genetic fate mapping of type-1 stem cell-dependent increase in newborn hippocampal neurons after electroconvulsive seizures.

Electroconvulsive therapy (ECT) is a uniquely effective treatment for major depressive disorder. An increase in hippocampal neurogenesis is implicated in the recovery from depression. We used an inducible genetic mouse model in which only GFAP-expressing stem-like cells (type-1 cells) and their progeny are selectively labeled with the reporter protein ß-galactosidase to track the process of neurogenesis in the dentate gyrus over 3 months following electroconvulsive seizures (ECS), the mouse equivalent of ECT. All ECS protocols tested induced a transient increase in type-1 cell divisions. While this led to an expansion of the type-1 cell pool after high-frequency ECS sessions for 5 consecutive days (5-ECS), asymmetric divisions drove neurogenesis by giving rise to Doublecortin (DCX)-expressing neuroblasts that matured into NeuN+ neurons. Significantly, the increase in newly generated DCX+ and NeuN+ cells after 5-ECS could be traced back to proliferating type-1 cells. Low-frequency continuation ECS (c-ECS) consisting of five single ECS sessions administered every 2 weeks resulted in a similar increase in newborn neurons as the high-frequency 5-ECS protocol. Moreover, the combination of 5-ECS and c-ECS led to a further significant increase in newborn neurons, suggesting a cellular mechanism responsible for the propitious effects of high-frequency ECT followed by continuation ECT in severely depressed patients. The ability of high- and low-frequency ECS to induce normally quiescent type-1 cells to proliferate and generate new neurons sets it apart from other antidepressant treatments and may underlie the superior clinical efficacy of ECT.

Conditional gene expression systems in the transgenic rat brain.

BACKGROUND: Turning gene expression on and off at will is one of the most powerful tools for the study of gene function in vivo. While several conditional systems were successful in invertebrates, in mice the Cre/loxP recombination system and the tet-controlled transcription activation system are predominant. Both expression systems allow for spatial and temporal control of gene activities, and, in the case of tet regulation, even for the reversible activation/inactivation of gene expression. Although the rat is the principal experimental model in biomedical research, in particular in studies of neuroscience, conditional rat transgenic systems are exceptionally rare in this species. RESULTS: We addressed this lack of technology, and established and thoroughly characterized CreERT2 and tTA transgenic rats with forebrain-specific transgene expression, controlled by the CaMKII alpha promoter. In addition, we developed new universal rat reporter lines for both transcription control systems and established inducible and efficient reporter gene expression in forebrain neurons. CONCLUSIONS: We demonstrate that conditional genetic manipulations in the rat brain are both feasible and practicable and outline advantages and limitations of the Tet and Cre/loxP system in the rat brain.

Tetracycline inducible gene manipulation in serotonergic neurons.

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic ß-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood.

A functional Tph2 C1473G polymorphism causes an anxiety phenotype via compensatory changes in the serotonergic system.

The association of single-nucleotide polymorphisms (SNPs) in the human tryptophan hydroxylase 2 (TPH2) gene with anxiety traits and depression has been inconclusive. Observed inconsistencies might result from the fact that TPH2 polymorphisms have been studied in a genetically heterogeneous human population. A defined genetic background, control over environmental factors, and the ability to analyze the molecular and neurochemical consequences of introduced genetic alterations constitute major advantages of investigating SNPs in inbred laboratory mouse strains. To investigate the behavioral and neurochemical consequences of a functional C1473G SNP in the mouse Tph2 gene, we generated congenic C57BL/6N mice homozygous for the Tph2 1473G allele. The Arg(447) substitution in the TPH2 enzyme resulted in a significant reduction of the brain serotonin (5-HT) in vivo synthesis rate. Despite decreased 5-HT synthesis, we could detect neither a reduction of brain region-specific 5-HT concentrations nor changes in baseline and stress-induced 5-HT release using a microdialysis approach. However, using a [(35)S]GTP-γ-S binding assay and 5-HT(1A) receptor autoradiography, a functional desensitization of 5-HT(1A) autoreceptors could be identified. Furthermore, behavioral analysis revealed a distinct anxiety phenotype in homozygous Tph2 1473G mice, which could be reversed with chronic escitalopram treatment. Alterations in depressive-like behavior could not be detected under baseline conditions or after chronic mild stress. These findings provide evidence for an involvement of functional Tph2 polymorphisms in anxiety-related behaviors, which are likely not caused directly by alterations in 5-HT content or release but are rather due to compensatory changes during development involving functional desensitization of 5-HT(1A) autoreceptors.

Inducible gene manipulations in brain serotonergic neurons of transgenic rats.

The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system.

Inducible gene expression in GFAP+ progenitor cells of the SGZ and the dorsal wall of the SVZ-A novel tool to manipulate and trace adult neurogenesis.

In the adult mammalian brain, neurogenesis originates from astrocyte-like stem cells. We generated a transgenic mouse line in which the tetracycline dependent transactivator (tTA) is expressed under the control of the murine GFAP promoter. In this mouse line, inducible gene expression targets virtually all GFAP-expressing stem-like cells in the dentate gyrus and a subset of GFAP-expressing progenitors located primarily in the dorsal wall/dorsolateral corner of the subventricular zone. Outside the neurogenic zones, astrocytes are infrequently targeted. We introduce a panel of transgenic mice which allow both inducible expression of candidate genes under control of the murine GFAP promoter and, at the same time, lineage tracing of all cells descendant from the original GFAP-positive cell. This new mouse line represents a versatile tool for functional analysis of neurogenesis and lineage tracing.

Inducible gene manipulations in serotonergic neurons.

An impairment of the serotonergic (5-HT) system has been implicated in the etiology of many neuropsychiatric disorders. Despite the considerable genetic evidence, the exact molecular and pathophysiological mechanisms underlying this dysfunction remain largely unknown. To address the lack of instruments for the molecular dissection of gene function in serotonergic neurons we have developed a new mouse transgenic tool that allows inducible Cre-mediated recombination of genes selectively in 5-HT neurons of all raphe nuclei. In this transgenic mouse line, the tamoxifen-inducible CreERT2 recombinase is expressed under the regulatory control of the mouse tryptophan hydroxylase 2 (Tph2) gene locus (177 kb). Tamoxifen treatment efficiently induced recombination selectively in serotonergic neurons with minimal background activity in vehicle-treated mice. These genetic manipulations can be initiated at any desired time during embryonic development, neonatal stage or adulthood. To illustrate the versatility of this new tool, we show that Brainbow-1.0L(TPH2-CreERT2) mice display highly efficient recombination in serotonergic neurons with individual 5-HT neurons labeling with multiple distinct fluorescent colors. This labeling is well suited for visualization and tracing of serotonergic neurons and their network architecture. Finally, the applicability of TPH2-CreERT2 for loxP-flanked candidate gene manipulation is evidenced by our successful knockout induction of the ubiquitously expressed glucocorticoid-receptor exclusively in 5-HT neurons of adult mice. The TPH2-CreERT2 line will allow detailed analysis of gene function in both developing and adult serotonergic neurons.