MicroRNAs are indispensable for the proliferation and differentiation of adult neural progenitor cells in mice.

More than two decades after the discovery of adult neurogenesis in humans, researchers still struggle to elucidate the underlying transcriptional and post-transcriptional mechanisms. RNA interference is a crucially important process in the central nervous system, and its role in adult neurogenesis is poorly understood. In this work, we address the role of Dicer-dependent microRNA biogenesis in neuronal differentiation of adult neural stem cells within the subventricular zone of the mouse brain. Loss of the Dicer1 gene in the tailless (Tlx)-positive cells did not cause the decline in their numbers, but severely affected differentiation. Thus, our findings identify yet another phenomenon associated with microRNA pathway deregulation in adult neural stem cells which might be of relevance both for neuroscience and clinical practice.

Several behavioral traits relevant for alcoholism are controlled by ɣ2 subunit containing GABAA receptors on dopamine neurons in mice.

The risk factors for developing alcohol addiction include impulsivity, high sensitivity to the rewarding action of ethanol, and low sensitivity to its sedative and intoxicating effects. Genetic variation in GABAA receptor subunits, including the ɣ2 subunit (Gabrg2), affects the risk for developing alcoholism. Alcohol directly potentiates GABAA receptors and activates the mesolimbic dopamine system. Here, we deleted Gabrg2 selectively in dopamine cells of adult mice. The deletion resulted in elevated firing of dopamine neurons and made them less sensitive to drugs acting at GABAA receptors. At the behavioral level, the deletion increased exploratory behavior and augmented both correct and incorrect responding in the go/no-go task, a test often used to assay the response inhibition component of impulsivity. In addition, conditioned place preference to alcohol, but not to cocaine or morphine, was increased. Ethanol-induced locomotor activation was enhanced in the mice lacking Gabrg2 on dopaminergic cells, whereas the sedative effect of alcohol was reduced. Finally, the alcohol drinking, but not the alcohol preference, at a high concentration was increased in the mutant mice. In summary, deletion of Gabrg2 on dopamine cells induced several behavioral traits associated with high risk of developing alcoholism. The findings suggest that mice lacking Gabrg2 on dopaminergic cells could be used as models for individuals at high risk for developing alcoholism and that GABAA receptors on dopamine cells are protective against the development of excessive alcohol drinking.

Loss of NMDA receptors in dopamine neurons leads to the development of affective disorder-like symptoms in mice.

The role of changes in dopamine neuronal activity during the development of symptoms in affective disorders remains controversial. Here, we show that inactivation of NMDA receptors on dopaminergic neurons in adult mice led to the development of affective disorder-like symptoms. The loss of NMDA receptors altered activity and caused complete NMDA-insensitivity in dopamine-like neurons. Mutant mice exhibited increased immobility in the forced swim test and a decrease in social interactions. Mutation also led to reduced saccharin intake, however the preference of sweet taste was not significantly decreased. Additionally, we found that while mutant mice were slower to learn instrumental tasks, they were able to reach the same performance levels, had normal sensitivity to feedback and showed similar motivation to exert effort as control animals. Taken together these results show that inducing the loss of NMDA receptor-dependent activity in dopamine neurons is associated with development of affective disorder-like symptoms.

Prostaglandin-dependent modulation of dopaminergic neurotransmission elicits inflammation-induced aversion in mice.

Systemic inflammation causes malaise and general feelings of discomfort. This fundamental aspect of the sickness response reduces the quality of life for people suffering from chronic inflammatory diseases and is a nuisance during mild infections like common colds or the flu. To investigate how inflammation is perceived as unpleasant and causes negative affect, we used a behavioral test in which mice avoid an environment that they have learned to associate with inflammation-induced discomfort. Using a combination of cell-type­specific gene deletions, pharmacology, and chemogenetics, we found that systemic inflammation triggered aversion through MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Further, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor­expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, we demonstrated that inflammation-induced aversion was not an indirect consequence of fever or anorexia but that it constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic motivational circuitry is a key mechanism underlying the negative affect induced by inflammation.

A model of alcohol drinking under an intermittent access schedule using group-housed mice.

Here, we describe a new model of voluntary alcohol drinking by group-housed mice. The model employs sensor-equipped cages that track the behaviors of the individual animals via implanted radio chips. After the animals were allowed intermittent access to alcohol (three 24 h intervals every week) for 4 weeks, the proportions of licks directed toward bottles containing alcohol were 50.9% and 39.6% for the male and female mice, respectively. We used three approaches (i.e., quinine adulteration, a progressive ratio schedule and a schedule involving a risk of punishment) to test for symptoms of compulsive alcohol drinking. The addition of 0.01% quinine to the alcohol solution did not significantly affect intake, but 0.03% quinine induced a greater than 5-fold reduction in the number of licks on the alcohol bottles. When the animals were required to perform increasing numbers of instrumental responses to obtain access to the bottle with alcohol (i.e., a progressive ratio schedule), they frequently reached a maximum of 21 responses irrespective of the available reward. Although the mice rarely achieved higher response criteria, the number of attempts was ∼ 10 times greater in case of alcohol than water. We have developed an approach for mapping social interactions among animals that is based on analysis of the sequences of entries into the cage corners. This approach allowed us to identify the mice that followed other animals in non-random fashions. Approximately half of the mice displayed at least one interaction of this type. We have not yet found a clear correlation between imitative behavior and relative alcohol preference. In conclusion, the model we describe avoids the limitations associated with testing isolated animals and reliably leads to stable alcohol drinking. Therefore, this model may be well suited to screening for the effects of genetic mutations or pharmacological treatments on alcohol-induced behaviors.

Novelty-seeking behaviors and the escalation of alcohol drinking after abstinence in mice are controlled by metabotropic glutamate receptor 5 on neurons expressing dopamine d1 receptors.

BACKGROUND: Novel experiences activate the brain's reward system in a manner similar to drugs of abuse, and high levels of novelty-seeking and sensation-seeking behavior have been associated with increased susceptibility to alcohol and drug abuse. Here, we show that metabotropic glutamate receptor 5 (mGluR5) signaling on dopaminoceptive neurons is necessary for both novelty-seeking behavior and the abstinence-induced escalation of alcohol drinking. METHODS: Mice harboring a transgene expressing microRNA hairpins against mGluR5 messenger RNA under the control of the D1 dopamine receptor gene promoter (mGluR5(KD-D1)) were tested in a battery of behavioral tests measuring learning abilities, anxiety levels, reactions to novelty, operant sensation seeking, and alcohol sensitivity. In addition, we have developed a method to assess long-term patterns of alcohol drinking in mice housed in groups using the IntelliCage system. RESULTS: mGluR5(KD-D1) mice showed no behavioral deficits and exhibited normal anxiety-like behaviors and learning abilities. However, mGluR5(KD-D1) animals showed reduced locomotor activity when placed in a novel environment, and exhibited decreased interaction with a novel object. Moreover, unlike control animals, mutant mice did not perform instrumental responses under the operant sensation-seeking paradigm, although they learned to respond for food normally. When mGluR5(KD-D1) mice were provided access to alcohol, they showed similar patterns of consumption as wild-type animals. However, mutant mice did not escalate their alcohol consumption after a period of forced abstinence, but control mice almost doubled their intake. CONCLUSIONS: These data identify mGluR5 receptors on D1-expressing neurons as a common molecular substrate of novelty-seeking behaviors and behaviors associated with alcohol abuse.

Ablation of serum response factor in dopaminergic neurons exacerbates susceptibility towards MPTP-induced oxidative stress.

The high susceptibility of dopaminergic (DA) neurons to cellular stress is regarded as a primary cause of Parkinson's disease. Here we investigate the role of the serum response factor (SRF), an important regulator of anti-apoptotic responses, for the survival of DA neurons in mice. We show that loss of SRF in DA neurons does not affect their viability and does not influence dopamine-dependent behaviors. However, ablation of SRF causes exacerbated sensitivity to 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), leading to significantly greater loss of DA neurons in the substantia nigra, compared with DA neurons located in the ventral tegmental area. In addition, loss of SRF decreases levels of the anti-apoptotic proteins brain-derived neurotrophic factor (BDNF) and Bcl-2, a plausible underlying cause of increased sensitivity to oxidative stress. These observations support the notion that dysfunction of the SRF-activating mitogen-associated kinase pathway may be part of Parkinson's disease etiology.

Glutamate input to noradrenergic neurons plays an essential role in the development of morphine dependence and psychomotor sensitization.

The brain's noradrenergic system is involved in the development of behaviours induced by drugs of abuse, e.g. dependence and withdrawal, and also reward or psychomotor effects. To investigate how noradrenergic system activity is controlled in the context associated with drug-induced behaviours, we generated a Cre/loxP mouse model in which the essential glutamate NMDA receptor subunit NR1 is ablated in cells expressing dopamine ß-hydroxylase (Dbh). As a result, the noradrenergic cells in NR1DbhCre mice lack the NMDA receptor-dependent component of excitatory post-synaptic currents. The mutant mice displayed no obvious behavioural alterations, had unchanged noradrenaline content and mild increase in dopamine levels in the nucleus accumbens. Interestingly, NR1DbhCre animals did not develop morphine-induced psychomotor sensitization. However, when the morphine injections were preceded by treatment with RX821002, an antagonist of α2-adrenergic receptors, the development of sensitization was restored. Conversely, pretreatment with clonidine, an agonist of α2-adrenergic receptors, blocked development of sensitization in wild-type mice. We also found that while the development of tolerance to morphine was normal in mutant mice, withdrawal symptoms were attenuated. These data reveal that NMDA receptors on noradrenergic neurons regulate development of opiate dependence and psychomotor sensitization, by controlling drug-induced noradrenaline signalling.

Glucocorticoid activity during lung maturation is essential in mesenchymal and less in alveolar epithelial cells.

Corticosteroid treatment is an established therapy for preterm infants, and germline inactivation of the glucocorticoid receptor (GR) gene in the mouse leads to respiratory failure and postnatal lethality. Although glucocorticoids have been thought to critically act in epithelial cells inducing the functional maturation of the lung, inactivation of the GR gene exclusively in the epithelium of the developing murine lung did not impair survival. In contrast, mice lacking GR specifically in mesenchyme-derived cells displayed a phenotype strongly reminiscent of GR knockout animals and died immediately after birth. Detailed analysis of gene expression allows the conclusion that GR acts in cells of the fibroblast lineage controlling their proliferation rate and the composition of the extracellular matrix.

MicroRNA loss enhances learning and memory in mice.

Dicer-dependent noncoding RNAs, including microRNAs (miRNAs), play an important role in a modulation of translation of mRNA transcripts necessary for differentiation in many cell types. In vivo experiments using cell type-specific Dicer1 gene inactivation in neurons showed its essential role for neuronal development and survival. However, little is known about the consequences of a loss of miRNAs in adult, fully differentiated neurons. To address this question, we used an inducible variant of the Cre recombinase (tamoxifen-inducible CreERT2) under control of Camk2a gene regulatory elements. After induction of Dicer1 gene deletion in adult mouse forebrain, we observed a progressive loss of a whole set of brain-specific miRNAs. Animals were tested in a battery of both aversively and appetitively motivated cognitive tasks, such as Morris water maze, IntelliCage system, or trace fear conditioning. Compatible with rather long half-life of miRNAs in hippocampal neurons, we observed an enhancement of memory strength of mutant mice 12 weeks after the Dicer1 gene mutation, before the onset of neurodegenerative process. In acute brain slices, immediately after high-frequency stimulation of the Schaffer collaterals, the efficacy at CA3-to-CA1 synapses was higher in mutant than in control mice, whereas long-term potentiation was comparable between genotypes. This phenotype was reflected at the subcellular and molecular level by the elongated filopodia-like shaped dendritic spines and an increased translation of synaptic plasticity-related proteins, such as BDNF and MMP-9 in mutant animals. The presented work shows miRNAs as key players in the learning and memory process of mammals.

Effects of the cell type-specific ablation of the cAMP-responsive transcription factor in noradrenergic neurons on locus coeruleus firing and withdrawal behavior after chronic exposure to morphine.

Repeated exposure to opiates leads to cellular and molecular changes and behavioral alterations reflecting a state of dependence. In noradrenergic neurons, cyclic AMP (cAMP)-dependent pathways are activated during opiate withdrawal, but their contribution to the activity of locus coeruleus noradrenergic neurons and behavioral manifestations remains controversial. Here, we test whether the cAMP-dependent transcription factors cAMP responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) in noradrenergic neurons control the cellular markers and the physical signs of morphine withdrawal in mice. Using the Cre/loxP system we ablated the Creb1 gene in noradrenergic neurons. To avoid adaptive effects because of compensatory up-regulation of CREM, we crossed the conditional Creb1 mutant mice with a Crem-/- line. We found that the enhanced expression of tyrosine hydroxylase normally observed during withdrawal was attenuated in CREB/CREM mutants. Moreover, the withdrawal-associated cellular hyperactivity and c-fos expression was blunted. In contrast, naloxone-precipitated withdrawal signs, such as jumping, paw tremor, tremor and mastication were preserved. We conclude by a specific genetic approach that the withdrawal-associated hyperexcitability of noradrenergic neurons depends on CREB/CREM activity in these neurons, but does not mediate several behavioral signs of morphine withdrawal.

Loss of the serum response factor in the dopamine system leads to hyperactivity.

The serum response factor (SRF) is a key regulator of neural development and cellular plasticity, which enables it to act as a regulator of long-term adaptations in neurons. Here we performed a comprehensive analysis of SRF function in the murine dopamine system. We found that loss of SRF in dopaminoceptive, but not dopaminergic, neurons is responsible for the development of a hyperactivity syndrome, characterized by reduced body weight into adulthood, enhanced motor activity, and deficits in habituation processes. Most important, the hyperactivity also develops when the ablation of SRF is induced in adult animals. On the molecular level, the loss of SRF in dopaminoceptive cells is associated with altered expression of neuronal plasticity-related genes, in particular transcripts involved in calcium ion binding, formation of the cytoskeleton, and transcripts encoding neuropeptide precursors. Furthermore, abrogation of SRF causes specific deficits in activity-dependent transcription, especially a complete lack of psychostimulant-induced expression of the Egr genes. We inferred that alterations in SRF-dependent gene expression underlie the observed hyperactive behavior. Thus, SRF depletion in dopaminoceptive neurons might trigger molecular mechanisms responsible for development of psychopathological conditions involving hyperactivity.

Cocaine-evoked synaptic plasticity: persistence in the VTA triggers adaptations in the NAc.

Addictive drugs hijack mechanisms of learning and memory that normally underlie reinforcement of natural rewards and induce synaptic plasticity of glutamatergic transmission in the mesolimbic dopamine (DA) system. In the ventral tegmental area (VTA), a single exposure to cocaine efficiently triggers NMDA receptor-dependent synaptic plasticity in DA neurons, whereas plasticity in the nucleus accumbens (NAc) occurs only after repeated injections. Whether these two forms of plasticity are independent or hierarchically organized remains unknown. We combined ex vivo electrophysiology in acute brain slices with behavioral assays modeling drug relapse in mice and found that the duration of the cocaine-evoked synaptic plasticity in the VTA is gated by mGluR1. Overriding mGluR1 in vivo made the potentiation in the VTA persistent. This led to synaptic plasticity in the NAc, which contributes to cocaine-seeking behavior after protracted withdrawal. Impaired mGluR1 function in vulnerable individuals could represent a first step in the recruitment of the neuronal network that underlies drug addiction.

Generation of Cre recombinase-expressing transgenic mice using bacterial artificial chromosomes.

Generation of genetically modified mice is one of the primary methods for understanding gene function. In particular, approaches that allow for restricting the effects of a mutation to defined cell-types are fundamental for understanding the roles of genes in specific cells or tissues. The Cre/loxP recombination system is the most robust approach to produce cell-type-specific gene inactivation. When the Cre recombinase is expressed from a transgene containing a tissue-type-specific promoter it will delete genomic segments flanked by loxP sequences in this tissue only. In this regard, the selectivity and reproducibility of Cre expression is absolutely critical for the result. To meet these requirements large constructs based on bacterial artificial chromosomes (BACs) have been successfully used. Here we present a protocol for the generation of constructs in which the Cre recombinase or a tamoxifen-inducible Cre fusion protein, are inserted at the translation start sequence of a BAC-derived gene. We describe all the critical steps, including construct-design, recombineering, and preparation of the transgene-containing genomic fragment for pronuclear injection and identification of "founder" animals among the resulting offspring. In our experience, the use of this protocol typically results in specific and transgene copy number-dependent expression of the Cre recombinase.

Local peripheral opioid effects and expression of opioid genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory pain.

We investigated the efficacy of local intraplantar (i.pl.) injection of peptide and non-peptide mu-, delta- and kappa-opioid receptor agonists in rat models of inflammatory and neuropathic pain. Locally applied agonists dose-dependently reduced formalin-induced flinching of the inflamed paw and induced antiallodynic and antihyperalgesic effects in sciatic nerve ligation-induced neuropathic pain. These effects were mediated by peripheral opioid receptors localized at the side of tissue/nerve injury, as was demonstrated by selective and non-selective opioid receptors antagonists. The ED(50) dose range of mu- and kappa-agonists required to induce analgesia in neuropathy was much higher than the ED(50) for inflammation; moreover, only delta-agonists were effective in the same dose range in both pain models. Additionally, effective antinociception was achieved at a lower dose of peptide, compared to non-peptide, opioids. Such findings support the use of the peripheral administration of opioid peptides, especially delta-agonists, in treating chronic pain. Furthermore, in order to assess whether adaptations in the expression of opioid genes could underlie the clinical observation of reduced opioid effectiveness in neuropathic pain, we analyzed the abundance of opioid transcripts in the spinal cord and dorsal root ganglia (DRG) during the neuropathy and inflammation. Nerve injury down-regulated mRNA for all types of opioid receptors in the DRG, which is predicted to decrease in the synthesis of opioid receptors to possibly account for the reduced effectiveness of locally administered opioids in neuropathy. The obtained results differentiate inflammatory and neuropathic pain and provide a novel insight into the peripheral effectiveness of opioids in both types of pain.

Loss of the Ca2+/calmodulin-dependent protein kinase type IV in dopaminoceptive neurons enhances behavioral effects of cocaine.

The persistent nature of addiction has been associated with activity-induced plasticity of neurons within the striatum and nucleus accumbens (NAc). To identify the molecular processes leading to these adaptations, we performed Cre/loxP-mediated genetic ablations of two key regulators of gene expression in response to activity, the Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) and its postulated main target, the cAMP-responsive element binding protein (CREB). We found that acute cocaine-induced gene expression in the striatum was largely unaffected by the loss of CaMKIV. On the behavioral level, mice lacking CaMKIV in dopaminoceptive neurons displayed increased sensitivity to cocaine as evidenced by augmented expression of locomotor sensitization and enhanced conditioned place preference and reinstatement after extinction. However, the loss of CREB in the forebrain had no effect on either of these behaviors, even though it robustly blunted acute cocaine-induced transcription. To test the relevance of these observations for addiction in humans, we performed an association study of CAMK4 and CREB promoter polymorphisms with cocaine addiction in a large sample of addicts. We found that a single nucleotide polymorphism in the CAMK4 promoter was significantly associated with cocaine addiction, whereas variations in the CREB promoter regions did not correlate with drug abuse. These findings reveal a critical role for CaMKIV in the development and persistence of cocaine-induced behaviors, through mechanisms dissociated from acute effects on gene expression and CREB-dependent transcription.

Glutamate receptors on dopamine neurons control the persistence of cocaine seeking.

Cocaine strengthens excitatory synapses onto midbrain dopamine neurons through the synaptic delivery of GluR1-containing AMPA receptors. This cocaine-evoked plasticity depends on NMDA receptor activation, but its behavioral significance in the context of addiction remains elusive. Here, we generated mice lacking the GluR1, GluR2, or NR1 receptor subunits selectively in dopamine neurons. We report that in midbrain slices of cocaine-treated mice, synaptic transmission was no longer strengthened when GluR1 or NR1 was abolished, while in the respective mice the drug still induced normal conditioned place preference and locomotor sensitization. In contrast, extinction of drug-seeking behavior was absent in mice lacking GluR1, while in the NR1 mutant mice reinstatement was abolished. In conclusion, cocaine-evoked synaptic plasticity does not mediate concurrent short-term behavioral effects of the drug but may initiate adaptive changes eventually leading to the persistence of drug-seeking behavior.

CREB has a context-dependent role in activity-regulated transcription and maintains neuronal cholesterol homeostasis.

Induction of specific gene expression patterns in response to activity confers functional plasticity to neurons. A principal role in the regulation of these processes has been ascribed to the cAMP responsive element binding protein (CREB). Using genome-wide expression profiling in mice lacking CREB in the forebrain, accompanied by deletion of the cAMP responsive element modulator gene (CREM), we here show that the role of these proteins in activity-induced gene expression is surprisingly selective and highly context dependent. Thus, only a very restricted subset of activity-induced genes (i.e., Gadd45b or Nr4a2) requires these proteins for their induction in the hippocampus after kainic acid administration, while they are required for most of the cocaine-induced expression changes in the striatum. Interestingly, in the absence of CREB, CREM is able to rescue activity-regulated transcription, which strengthens the notion of overlapping functions of the two proteins. In addition, we show that cholesterol metabolism is dysregulated in the brains of mutant mice, as reflected coordinated expression changes in genes involved in cholesterol synthesis and neuronal accumulation of cholesterol. These findings provide novel insights into the role of CREB and CREM in stimulus-dependent transcription and neuronal homeostasis.

Effects of glycogen synthase kinase 3beta and cyclin-dependent kinase 5 inhibitors on morphine-induced analgesia and tolerance in rats.

Repeated administration of morphine is associated with the development of tolerance, yet the mechanism underlying this phenomenon is still poorly understood. Recent evidence implicating glycogen synthase kinase 3 (GSK3) in opioid receptor signaling pathways has prompted us to investigate its role in morphine tolerance. Administration of 10 mg/kg morphine i.p. to Wistar rats twice daily for 8 days resulted in complete tolerance to its analgesic effects as measured by the tail-flick test. When injections of morphine were preceded by intrathecal (i.t.) administration of either an inhibitor of GSK3 [(3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763) or 6-bromoindirubin-3'oxime] or an inhibitor of cyclin-dependent kinase (Cdk), roscovitine, development of tolerance to morphine analgesia was completely abolished. In addition, a single i.t. injection of either kinase inhibitor was able to restore in a dose-dependent manner the analgesic effect of morphine in morphine-tolerant rats. None of the inhibitors in doses used in the present study had analgesic effects of their own nor an effect on the analgesic potency of morphine. Repeated i.t. administration of either inhibitor had caused an increase in abundance of GSK-3beta phosphorylated at Ser(9) in the dorsal lumbar part of the spinal cord of rats that were chronically treated with morphine. Furthermore, reversal of morphine tolerance by a single injection of either inhibitor was always associated with increased abundance of phospho-GSK3beta. In conclusion, our data indicate that chronic morphine treatment activates a highly efficient pathway by means of which Cdk5 regulates GSK3beta activity.