Brain Expression, Physiological Regulation and Role in Motivation and Associative Learning of Peroxisome Proliferator-activated Receptor γ.

Like other members of the superfamily of nuclear receptors, the peroxisome proliferator-activated receptor γ (PPARγ), is a ligand-activated transcription factor known for its insulin-sensitizing actions in the periphery. Despite only sparse evidence for PPARγ in the CNS, many reports suggest direct PPARγ-mediated actions in the brain. This study aimed to (i) map PPARγ expression in rodent brain areas, involved in the regulation of cognitive, motivational, and emotional functions, (ii) examine the regulation of central PPARγ by physiological variables (age, sex, obesity); (iii) chemotypically identify PPARγ-expressing cells in the frontal cortex (FC) and hippocampus (HP); (iv) study whether activation of PPARγ by pioglitazone (Pio) in FC and HP cells can induce target gene expression; and (v) demonstrate the impact of activated PPARγ on learning behavior and motivation. Immunoreactive PPARγ was detectable in specific sub-nuclei/subfields of the FC, HP, nucleus accumbens, amygdala, hypothalamus, thalamus, and granular layers of the cerebellum. PPARγ protein levels were upregulated during aging and in high fat diet-induced obesity. PPARγ mRNA expression was upregulated in the amygdala of females (but not males) that were made obese. Neural precursor cells, mature neurons, and astrocytes in primary FC and HP cultures were shown to express PPARγ. Pioglitazone dose-dependently upregulated PPARγ target genes in manner that was specific to the origin (FC or HP) of the cultures. Lastly, administration of Pio impaired motivation and associative learning. Collectively, we provide evidence for the presence of regulatable PPARγ in the brain and demonstrate their participation the regulation of key behaviors.

miR-323a regulates ERBB4 and is involved in depression.

Major depressive disorder (MDD) is a complex and debilitating illness whose etiology remains unclear. Small RNA molecules, such as micro RNAs (miRNAs) have been implicated in MDD, where they display differential expression in the brain and the periphery. In this study, we quantified miRNA expression by small RNA sequencing in the anterior cingulate cortex and habenula of individuals with MDD and psychiatrically-healthy controls. Thirty-two miRNAs showed significantly correlated expression between the two regions (False Discovery Rate < 0.05), of which four, miR-204-5p, miR-320b, miR-323a-3p, and miR-331-3p, displayed upregulated expression in MDD. We assessed the expression of predicted target genes of differentially expressed miRNAs in the brain, and found that the expression of erb-b2 receptor tyrosine kinase 4 (ERBB4), a gene encoding a neuregulin receptor, was downregulated in both regions, and was influenced by miR-323a-3p in vitro. Finally, we assessed the effects of manipulating miRNA expression in the mouse ACC on anxiety- and depressive-like behaviors. Mice in which miR-323-3p was overexpressed or knocked-down displayed increased and decreased emotionality, respectively. Additionally, these mice displayed significantly downregulated and upregulated expression of Erbb4, respectively. Overall, our findings indicate the importance of brain miRNAs in the pathology of MDD, and emphasize the involvement of miR-323a-3p and ERBB4 in this phenotype. Future studies further characterizing miR-323a-3p and neuregulin signaling in depression are warranted.

Deletion of CRH From GABAergic Forebrain Neurons Promotes Stress Resilience and Dampens Stress-Induced Changes in Neuronal Activity.

Dysregulation of the corticotropin-releasing hormone (CRH) system has been implicated in stress-related psychopathologies such as depression and anxiety. Although most studies have linked CRH/CRH receptor 1 signaling to aversive, stress-like behavior, recent work has revealed a crucial role for distinct CRH circuits in maintaining positive emotional valence and appetitive responses under baseline conditions. Here we addressed whether deletion of CRH, specifically from GABAergic forebrain neurons (Crh CKO-GABA mice) differentially affects general behavior under baseline and chronic stress conditions. Expression mapping in Crh CK O-GABA mice revealed absence of Crh in GABAergic neurons of the cortex and limbic regions including the hippocampus, central nucleus of the amygdala and the bed nucleus of the stria terminals, but not in the paraventricular nucleus of hypothalamus. Consequently, conditional CRH knockout animals exhibited no alterations in circadian and stress-induced corticosterone release compared to controls. Under baseline conditions, absence of Crh from forebrain GABAergic neurons resulted in social interaction deficits but had no effect on other behavioral measures including locomotion, anxiety, immobility in the forced swim test, acoustic startle response and fear conditioning. Interestingly, following exposure to chronic social defeat stress, Crh CKO-GABA mice displayed a resilient phenotype, which was accompanied by a dampened, stress-induced expression of immediate early genes c-fos and zif268 in several brain regions. Collectively our data reveals the requirement of GABAergic CRH circuits in maintaining appropriate social behavior in naïve animals and further supports the ability of CRH to promote divergent behavioral states under baseline and severe stress conditions.

Deciphering the Contributions of CRH Receptors in the Brain and Pituitary to Stress-Induced Inhibition of the Reproductive Axis.

Based on pharmacological studies, corticotropin-releasing hormone (CRH) and its receptors play a leading role in the inhibition of the hypothalamic-pituitary-gonadal (HPG) axis during acute stress. To further study the effects of CRH receptor signaling on the HPG axis, we generated and/or employed male mice lacking CRH receptor type 1 (CRHR1) or type 2 (CRHR2) in gonadotropin-releasing hormone neurons, GABAergic neurons, or in all central neurons and glia. The deletion of CRHRs revealed a preserved decrease of plasma luteinizing hormone (LH) in response to either psychophysical or immunological stress. However, under basal conditions, central infusion of CRH into mice lacking CRHR1 in all central neurons and glia, or application of CRH to pituitary cultures from mice lacking CRHR2, failed to suppress LH release, unlike in controls. Our results, taken together with those of the earlier pharmacological studies, suggest that inhibition of the male HPG axis during acute stress is mediated by other factors along with CRH, and that CRH suppresses the HPG axis at the central and pituitary levels via CRHR1 and CRHR2, respectively.

Chronic CRH depletion from GABAergic, long-range projection neurons in the extended amygdala reduces dopamine release and increases anxiety.

The interplay between corticotropin-releasing hormone (CRH) and the dopaminergic system has predominantly been studied in addiction and reward, while CRH-dopamine interactions in anxiety are scarcely understood. We describe a new population of CRH-expressing, GABAergic, long-range-projecting neurons in the extended amygdala that innervate the ventral tegmental area and alter anxiety following chronic CRH depletion. These neurons are part of a distinct CRH circuit that acts anxiolytically by positively modulating dopamine release.

CRFR1 in AgRP Neurons Modulates Sympathetic Nervous System Activity to Adapt to Cold Stress and Fasting.

Signaling by the corticotropin-releasing factor receptor type 1 (CRFR1) plays an important role in mediating the autonomic response to stressful challenges. Multiple hypothalamic nuclei regulate sympathetic outflow. Although CRFR1 is highly expressed in the arcuate nucleus (Arc) of the hypothalamus, the identity of these neurons and the role of CRFR1 here are presently unknown. Our studies show that nearly half of Arc-CRFR1 neurons coexpress agouti-related peptide (AgRP), half of which originate from POMC precursors. Arc-CRFR1 neurons are innervated by CRF neurons in the hypothalamic paraventricular nucleus, and CRF application decreases AgRP(+)CRFR1(+) neurons' excitability. Despite similar anatomy in both sexes, only female mice selectively lacking CRFR1 in AgRP neurons showed a maladaptive thermogenic response to cold and reduced hepatic glucose production during fasting. Thus, CRFR1, in a subset of AgRP neurons, plays a regulatory role that enables appropriate sympathetic nervous system activation and consequently protects the organism from hypothermia and hypoglycemia.

Fluoxetine treatment prevents the inflammatory response in a mouse model of posttraumatic stress disorder.

Despite intense research efforts the molecular mechanisms affecting stress-vulnerable brain regions in posttraumatic stress disorder (PTSD) remain elusive. In the current study we have applied global transcriptomic profiling to a PTSD mouse model induced by foot shock fear conditioning. We compared the transcriptomes of prelimbic cortex, anterior cingulate cortex (ACC), basolateral amygdala, central nucleus of amygdala, nucleus accumbens (NAc) and CA1 of the dorsal hippocampus between shocked and non-shocked (control) mice, with and without fluoxetine treatment by RNA sequencing. Differentially expressed (DE) genes were identified and clustered for in silico pathway analysis. Findings in relevant brain regions were further validated with immunohistochemistry. DE genes belonging to 11 clusters were identified including increased inflammatory response in ACC in shocked mice. In line with this finding, we noted higher microglial activation in ACC of shocked mice. Chronic fluoxetine treatment initiated in the aftermath of the trauma prevented inflammatory gene expression alterations in ACC and ameliorated PTSD-like symptoms, implying an important role of the immune response in PTSD pathobiology. Our results provide novel insights into molecular mechanisms affected in PTSD and suggest therapeutic applications with anti-inflammatory agents.

Development of a novel IGRA assay to test T cell responsiveness to HBV antigens in whole blood of chronic Hepatitis B patients.

BACKGROUND: Interferon gamma release assays (IGRA) have been developed to support easy and fast diagnosis of diseases like tuberculosis, and CMV in transplant patients. IGRAs focus on cellular immunity especially memory T cells and thus also allow rapid screening prior to complex flow cytometric testing. Here, we describe a novel, sensitive whole blood based cytokine release assay capable of assessing T cell responsiveness to HBV antigens in Hepatitis B patients and assessing hepatitis B vaccination status in healthy individuals. METHODS: Seventy two chronic Hepatitis B patients (CHB), 8 acute hepatitis B patients (AHB) and 80 healthy controls (HC) were tested by ELISA for IFNγ- and IL2-secretion in whole blood after challenge with synthetic peptide libraries of hepatitis B core antigen (HBcAg) or hepatitis B surface antigen (HBsAg). RESULTS: The developed IGRA test reliably differentiated between Hepatitis B patients, vaccinees and unvaccinated healthy controls. Treatment naïve and treated CHB patients showed a weaker IFNγ response to HBcAg (16 ± 5 and 35 ± 28 pg/ml, respectively) compared to the AHB group (82 ± 39 pg/ml), whereas HC remained unresponsive (6 ± 1 pg/ml). IL2 levels after HBcAg challenge were also higher in the AHB group compared to naive and treated CHB as well as HC (47 ± 21 vs. 12 ± 3, 15 ± 10 and 12 ± 9 pg/ml, respectively). HBsAg stimulation led to increased IFNγ and IL2 levels in the AHB group (33 ± 12 and 22 ± 12 pg/ml) and even higher levels in HC due to a high hepatitis B vaccination rate (41 ± 10 and 167 ± 58 pg/ml). Naive and treated CHB patients developed no or only weaker IFNγ or IL2 responses to HBsAg (5 ± 2 and 12 ± 7 pg/ml, for naive CHB, 12 ± 10 and 18 ± 15 pg/ml, for treated CHB). For HC, IL2 release after HBsAg stimulation depicted hepatitis B vaccination status with a diagnostic sensitivity and specificity of 85 % and 90 %. CONCLUSION: Our novel whole blood based cytokine release assay constitutes an easy and robust tool for screening HBV specific cellular immunity as alternative to flow cytometry or ELISPOT assays.

Corticotropin-releasing hormone drives anandamide hydrolysis in the amygdala to promote anxiety.

Corticotropin-releasing hormone (CRH) is a central integrator in the brain of endocrine and behavioral stress responses, whereas activation of the endocannabinoid CB1 receptor suppresses these responses. Although these systems regulate overlapping functions, few studies have investigated whether these systems interact. Here we demonstrate a novel mechanism of CRH-induced anxiety that relies on modulation of endocannabinoids. Specifically, we found that CRH, through activation of the CRH receptor type 1 (CRHR1), evokes a rapid induction of the enzyme fatty acid amide hydrolase (FAAH), which causes a reduction in the endocannabinoid anandamide (AEA), within the amygdala. Similarly, the ability of acute stress to modulate amygdala FAAH and AEA in both rats and mice is also mediated through CRHR1 activation. This interaction occurs specifically in amygdala pyramidal neurons and represents a novel mechanism of endocannabinoid-CRH interactions in regulating amygdala output. Functionally, we found that CRH signaling in the amygdala promotes an anxious phenotype that is prevented by FAAH inhibition. Together, this work suggests that rapid reductions in amygdala AEA signaling following stress may prime the amygdala and facilitate the generation of downstream stress-linked behaviors. Given that endocannabinoid signaling is thought to exert "tonic" regulation on stress and anxiety responses, these data suggest that CRH signaling coordinates a disruption of tonic AEA activity to promote a state of anxiety, which in turn may represent an endogenous mechanism by which stress enhances anxiety. These data suggest that FAAH inhibitors may represent a novel class of anxiolytics that specifically target stress-induced anxiety.

Optogenetic evocation of field inhibitory postsynaptic potentials in hippocampal slices: a simple and reliable approach for studying pharmacological effects on GABAA and GABAB receptor-mediated neurotransmission.

The GABAergic system is the main source of inhibition in the mammalian brain. Consequently, much effort is still made to develop new modulators of GABAergic synaptic transmission. In contrast to glutamatergic postsynaptic potentials (PSPs), accurate monitoring of GABA receptor-mediated PSPs (GABAR-PSPs) and their pharmacological modulation in brain tissue invariably requires the use of intracellular recording techniques. However, these techniques are expensive, time- and labor-consuming, and, in case of the frequently employed whole-cell patch-clamp configuration, impact on intracellular ion concentrations, signaling cascades, and pH buffering systems. Here, we describe a novel approach to circumvent these drawbacks. In particular, we demonstrate in mouse hippocampal slices that selective optogenetic activation of interneurons leads to prominent field inhibitory GABAAR- and GABABR-PSPs in area CA1 which are easily and reliably detectable by a single extracellular recording electrode. The field PSPs exhibit typical temporal and pharmacological characteristics, display pronounced paired-pulse depression, and remain stable over many consecutive evocations. Additionally validating the methodological value of this approach, we further show that the neuroactive steroid 5α-THDOC (5 µM) shifts the inhibitory GABAAR-PSPs towards excitatory ones.

Nectin-3 links CRHR1 signaling to stress-induced memory deficits and spine loss.

Stress impairs cognition via corticotropin-releasing hormone receptor 1 (CRHR1), but the molecular link between abnormal CRHR1 signaling and stress-induced cognitive impairments remains unclear. We investigated whether the cell adhesion molecule nectin-3 is required for the effects of CRHR1 on cognition and structural remodeling after early-life stress exposure. Postnatally stressed adult mice had decreased hippocampal nectin-3 levels, which could be attenuated by CRHR1 inactivation and mimicked by corticotropin-releasing hormone (CRH) overexpression in forebrain neurons. Acute stress dynamically reduced hippocampal nectin-3 levels, which involved CRH-CRHR1, but not glucocorticoid receptor, signaling. Suppression of hippocampal nectin-3 caused spatial memory deficits and dendritic spine loss, whereas enhancing hippocampal nectin-3 expression rescued the detrimental effects of early-life stress on memory and spine density in adulthood. Our findings suggest that hippocampal nectin-3 is necessary for the effects of stress on memory and structural plasticity and indicate that the CRH-CRHR1 system interacts with the nectin-afadin complex to mediate such effects.

Visualizing corticotropin-releasing hormone receptor type 1 expression and neuronal connectivities in the mouse using a novel multifunctional allele.

The corticotropin-releasing hormone (CRH) and its type 1 receptor (CRHR1) play a central role in coordinating the endocrine, autonomic, and behavioral responses to stress. A prerequisite to functionally dissect the complexity of the CRH/CRHR1 system is to unravel the identity of CRHR1-expressing neurons and their connectivities. Therefore, we used a knockin approach to genetically label CRHR1-expressing cells with a tau-lacZ (tZ) reporter gene. The distribution of neurons expressing ß-galactosidase in the brain and the relative intensity of labeling is in full accordance with previously described Crhr1 mRNA expression. Combining the microtubule-binding properties of TAU with the Cre-loxP system allowed to direct the ß-galactosidase to proximal dendrites, and in particular to axons. Thereby, we were able to visualize projections of CRHR1 neurons such as glutamatergic and dopaminergic afferent connections of the striatum and GABAergic CRHR1-expressing neurons located within its patch compartment. In addition, the tZ reporter gene revealed novel details of CRHR1 expression in the spinal cord, skin, and eye. CRHR1 expression in the retina prompted the identification of a new physiological role of CRHR1 related to the visual system. Besides its reporter properties, this novel CRHR1 allele comprises the possibility to conditionally restore or delete CRHR1 via Flp and Cre recombinase, respectively. Finally, the allele is suitable for further manipulations of the CRHR1 locus by recombinase-mediated cassette exchange. Taken together, this novel mouse allele will significantly facilitate the neuroanatomical analysis of CRHR1 circuits and opens up new avenues to address CRHR1 function in more detail.

Assessing behavioural effects of chronic HPA axis activation using conditional CRH-overexpressing mice.

The corticotropin-releasing hormone (CRH) and its cognate receptors have been implicated in the pathophysiology of stress-related disorders. Hypersecretion of central CRH and elevated glucocorticoid levels, as a consequence of impaired feedback control, have been shown to accompany mood and anxiety disorders. However, a clear discrimination of direct effects of centrally hypersecreted CRH from those resulting from HPA axis activation has been difficult. Applying a conditional strategy, we have generated two conditional CRH-overexpressing mouse lines: CRH-COE ( Del ) mice overexpress CRH throughout the body, while CRH-COE ( APit ) mice selectively overexpress CRH in the anterior and intermediate lobe of the pituitary. Both mouse lines show increased basal plasma corticosterone levels and consequently develop signs of Cushing's syndrome. However, while mice ubiquitously overexpressing CRH exhibited increased anxiety-related behaviour, overexpression of CRH in the pituitary did not produce alterations in emotional behaviour. These results suggest that chronic hypercorticosteroidism alone is not sufficient to alter anxiety-related behaviour but rather that central CRH hyperdrive on its own or in combination with elevated glucocorticoids is responsible for the increase in anxiety-related behaviour. In conclusion, the generated mouse lines represent valuable animal models to study the consequences of chronic CRH overproduction and HPA axis activation.

Profiling trait anxiety: transcriptome analysis reveals cathepsin B (Ctsb) as a novel candidate gene for emotionality in mice.

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait "anxiety". We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4).Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse model's behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

Individual stress vulnerability is predicted by short-term memory and AMPA receptor subunit ratio in the hippocampus.

Increased vulnerability to aversive experiences is one of the main risk factors for stress-related psychiatric disorders as major depression. However, the molecular bases of vulnerability, on the one hand, and stress resilience, on the other hand, are still not understood. Increasing clinical and preclinical evidence suggests a central involvement of the glutamatergic system in the pathogenesis of major depression. Using a mouse paradigm, modeling increased stress vulnerability and depression-like symptoms in a genetically diverse outbred strain, and we tested the hypothesis that differences in AMPA receptor function may be linked to individual variations in stress vulnerability. Vulnerable and resilient animals differed significantly in their dorsal hippocampal AMPA receptor expression and AMPA receptor binding. Treatment with an AMPA receptor potentiator during the stress exposure prevented the lasting effects of chronic social stress exposure on physiological, neuroendocrine, and behavioral parameters. In addition, spatial short-term memory, an AMPA receptor-dependent behavior, was found to be predictive of individual stress vulnerability and response to AMPA potentiator treatment. Finally, we provide evidence that genetic variations in the AMPA receptor subunit GluR1 are linked to the vulnerable phenotype. Therefore, we propose genetic variations in the AMPA receptor system to shape individual stress vulnerability. Those individual differences can be predicted by the assessment of short-term memory, thereby opening up the possibility for a specific treatment by enhancing AMPA receptor function.

Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models.

BACKGROUND: Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark of complex and multifactorial psychiatric diseases such as anxiety and mood disorders. About 50-60% of patients with major depression show HPA axis dysfunction, i.e. hyperactivity and impaired negative feedback regulation. The neuropeptide corticotropin-releasing hormone (CRH) and its receptor type 1 (CRHR1) are key regulators of this neuroendocrine stress axis. Therefore, we analyzed CRH/CRHR1-dependent gene expression data obtained from the pituitary corticotrope cell line AtT-20, a well-established in vitro model for CRHR1-mediated signal transduction. To extract significantly regulated genes from a genome-wide microarray data set and to deduce underlying CRHR1-dependent signaling networks, we combined supervised and unsupervised algorithms. RESULTS: We present an efficient variable selection strategy by consecutively applying univariate as well as multivariate methods followed by graphical models. First, feature preselection was used to exclude genes not differentially regulated over time from the dataset. For multivariate variable selection a maximum likelihood (MLHD) discriminant function within GALGO, an R package based on a genetic algorithm (GA), was chosen. The topmost genes representing major nodes in the expression network were ranked to find highly separating candidate genes. By using groups of five genes (chromosome size) in the discriminant function and repeating the genetic algorithm separately four times we found eleven genes occurring at least in three of the top ranked result lists of the four repetitions. In addition, we compared the results of GA/MLHD with the alternative optimization algorithms greedy selection and simulated annealing as well as with the state-of-the-art method random forest. In every case we obtained a clear overlap of the selected genes independently confirming the results of MLHD in combination with a genetic algorithm. With two unsupervised algorithms, principal component analysis and graphical Gaussian models, putative interactions of the candidate genes were determined and reconstructed by literature mining. Differential regulation of six candidate genes was validated by qRT-PCR. CONCLUSIONS: The combination of supervised and unsupervised algorithms in this study allowed extracting a small subset of meaningful candidate genes from the genome-wide expression data set. Thereby, variable selection using different optimization algorithms based on linear classifiers as well as the nonlinear random forest method resulted in congruent candidate genes. The calculated interacting network connecting these new target genes was bioinformatically mapped to known CRHR1-dependent signaling pathways. Additionally, the differential expression of the identified target genes was confirmed experimentally.

Urocortin 3 modulates social discrimination abilities via corticotropin-releasing hormone receptor type 2.

Urocortin 3 (UCN3) is strongly expressed in specific nuclei of the rodent brain, at sites distinct from those expressing urocortin 1 and urocortin 2, the other endogenous ligands of corticotropin-releasing hormone receptor type 2 (CRH-R2). To determine the physiological role of UCN3, we generated UCN3-deficient mice, in which the UCN3 open reading frame was replaced by a tau-lacZ reporter gene. By means of this reporter gene, the nucleus parabrachialis and the premammillary nucleus were identified as previously unknown sites of UCN3 expression. Additionally, the introduced reporter gene enabled the visualization of axonal projections of UCN3-expressing neurons from the superior paraolivary nucleus to the inferior colliculus and from the posterodorsal part of the medial amygdala to the principal nucleus of the bed nucleus of the stria terminalis, respectively. The examination of tau-lacZ reporter gene activity throughout the brain underscored a predominant expression of UCN3 in nuclei functionally connected to the accessory olfactory system. Male and female mice were comprehensively phenotyped but none of the applied tests provided indications for a role of UCN3 in the context of hypothalamic-pituitary-adrenocortical axis regulation, anxiety- or depression-related behavior. However, inspired by the prevalent expression throughout the accessory olfactory system, we identified alterations in social discrimination abilities of male and female UCN3 knock-out mice that were also present in male CRH-R2 knock-out mice. In conclusion, our results suggest a novel role for UCN3 and CRH-R2 related to the processing of social cues and to the establishment of social memories.

Selective enhancement of nutrient-induced insulin secretion by ATP-sensitive K+ channel-blocking imidazolines.

The contribution of ATP-sensitive K(+) channel (K(ATP) channel)-dependent and -independent signaling to the insulinotropic characteristics of imidazolines was explored using perifused mouse islets and beta-cells. Up to a concentration of 100 muM efaroxan had no insulinotropic effect in the presence of a basal glucose concentration, but enhanced the effect of a stimulatory concentration of glucose or nonglucidic nutrients (ketoisocaproate plus glutamine). The secretion by a non-nutrient (40 mM KCl) was not enhanced. At 500 microM, efaroxan stimulated insulin secretion when glucose was basal. Likewise, at 0.1 to 10 microM RX871024 [2-(imidazolin-2-yl)-1-phenylindole] showed a purely enhancing effect, but at 100 microM it elicited a strong KCl-like secretory response in the presence of basal glucose. At 0.1 and 1 microM RX871024 did not significantly depolarize the beta-cell membrane. However, at a purely enhancing drug concentration (10 microM RX871024 or 100 microM efaroxan) K(ATP) channel activity was strongly reduced, the membrane was depolarized, and the cytosolic Ca(2+) concentration was elevated in the presence of basal glucose. Insulin secretion by sulfonylurea receptor (SUR)1 knockout (KO) islets, which have no functional K(ATP) channels, was not increased by efaroxan (100 or 500 microM) or by 10 microM RX871024 but was increased by 100 microM RX871024. The imidazolines phentolamine and alinidine (100 microM) were also ineffective on SUR1 KO islets. It is concluded that a significant K(ATP) channel block is compatible with a purely enhancing effect of the imidazolines on nutrient-induced insulin secretion. Only RX871024 has an additional, nondepolarizing effect, which at a high drug concentration is able to elicit a K(ATP) channel-independent secretion.

Profiling of behavioral changes and hippocampal gene expression in mice chronically treated with the SSRI paroxetine.

INTRODUCTION: Monoamine-based antidepressants inhibit neurotransmitter reuptake within short time. However, it commonly takes several weeks until clinical symptoms start to resolve--indicating the involvement of effects distant from reuptake inhibition. OBJECTIVE: To unravel other mechanisms involved in drug action, a "reverse" pharmacological approach was applied to determine antidepressant-induced alterations of hippocampal gene expression. MATERIALS AND METHODS: The behavioral response to long-term paroxetine administration of male DBA/2Ola mice was assessed by the forced swim test (FST), the modified hole board (mHB), and the dark/light box. Hippocampi of test-naive mice were dissected, and changes in gene expression by paroxetine treatment were investigated by means of microarray technology. RESULTS AND DISCUSSION: Robust effects of paroxetine on passive stress-coping behavior in the FST were observed. Furthermore, anxiolytic properties of long-term antidepressant treatment could be identified in DBA mice in both, the mHB and dark/light box. Analysis of microarray results revealed a list of 60 genes differentially regulated by chronic paroxetine treatment. Preproenkephalin 1 and inhibin beta-A showed the highest level of transcriptional change. Furthermore, a number of candidates involved in neuroplasticity/neurogenesis emerged (e.g., Bdnf, Gfap, Vim, Sox11, Egr1, Stat3). Seven selected candidates were confirmed by in situ hybridization. Additional immunofluorescence colocalization studies of GFAP and vimentin showed more positive cells to be detected in long-term paroxetine-treated DBA mice. CONCLUSION: Candidate genes identified in the current study using a mouse strain validated for its responsiveness to long-term paroxetine treatment add, in our opinion, to unraveling the mechanism of action of paroxetine as a representative for SSRIs.

Expression profiling identifies the CRH/CRH-R1 system as a modulator of neurovascular gene activity.

Corticotropin-releasing hormone receptor type 1 (CRH-R1)-deficient mice display reduced anxiety-like behavior, a chronic corticosterone deficit, and an impaired neuroendocrine stress response caused by disruption of the hypothalamic-pituitary-adrenocortical (HPA) axis. The molecular substrates and pathways of CRH/CRH-R1-dependent signaling mechanisms underlying the behavioral phenotype as well as the consequences of lifelong glucocorticoid deficit remain largely obscure. To dissect involved neuronal circuitries, we performed comparative expression profiling of brains of CRH-R1 mutant and wild-type mice using our custom made MPIP (Max Planck Institute of Psychiatry) 17k cDNA microarray. Microarray analysis yielded 107 genes showing altered expression levels when comparing CRH-R1 knockout mice with wild-type littermates. A significant proportion of differentially expressed genes was related to control of HPA and hypothalamic-pituitary-thyroid (HPT) axes reflecting not only the disturbance of the HPA axis in CRH-R1 mutant mice but also the interplay of both neuroendocrine systems. The spatial analysis of regulated genes revealed a prevalence for genes expressed in the cerebral microvasculature. This phenotype was confirmed by the successful cross-validation of regulated genes in CRH overexpressing mice. Analysis of the cerebral vasculature of CRH-R1 mutant and CRH overexpressing mice revealed alterations of functional rather than structural properties. A direct role of the CRH/CRH-R1 system was supported by demonstrating Crhr1 expression in the adult murine cerebral vasculature. In conclusion, these data suggest a novel, previously unknown role of the CRH/CRH-R1 system in modulating neurovascular gene expression and function.