Jan Rodriguez Parkitna

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.

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.

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.

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

ABSTRACT We investigated the efficacy of local intraplantar (i.pl.) injection of peptide and non‐peptide μ‐, δ‐ and κ‐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 ED50 dose range of μ‐ and κ‐agonists required to induce analgesia in neuropathy was much higher than the ED50 for inflammation; moreover, only δ‐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 δ‐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.

Glucocorticoid receptor dimerization is required for survival in septic shock via suppression of interleukin-1 in macrophages

Sepsis is controlled by endogenous glucocorticoids (GCs). Previous studies provided evidence that crosstalk of the monomeric GC receptor (GR) with proinflammatory transcription factors is the crucial mechanism underlying the suppressive GC effect. Here we demonstrate that mice with a dimerization‐deficient GR (GRdim) are highly susceptible to sepsis in 2 different models, namely cecal ligation and puncture and lipopolysaccharide (LPS)‐induced septic shock. TNF‐α is normally regulated in these mice, but down‐regulation of IL‐6 and IL‐1β is diminished. LPS‐treated macrophages derived from GRdim mice are largely resistant to GC actions in vitro in terms of morphology, surface marker expression, and gene expression. Treatment with recombinant IL‐1 receptor antagonist improved survival of GRdim mice and mice lacking the GR in macrophages (GRLysMCre) mice. This suggests that regulation of IL‐1β in macrophages by GCs is pivotal to control sepsis.—Kleiman, A., Hübner, S., Rodriguez Parkitna, J. M., Neumann, A., Hofer, S., Weigand, M. A., Bauer, M., Schmid, W., Schütz, G., Libert, C., Reichardt, H. M., Tuckermann, J. P. Glucocorticoid receptor dimerization is required for survival in septic shock via suppression of interleukin‐1 in macrophages. FASEB J. 26, 722–729 (2012). www.fasebj.org

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 genomewide 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.—Lemberger, T., Parkitna, J. R., Chai, M., Schütz, G., Engblom, D. CREB has a context‐dependent role in activity‐regulated transcription and maintains neuronal cholesterol homeostasis FASEB J. 22, 2872–2879 (2008)

Incentive learning underlying cocaine-seeking requires mGluR5 receptors located on dopamine D1 receptor-expressing neurons

Understanding the psychobiological basis of relapse remains a challenge in developing therapies for drug addiction. Relapse in cocaine addiction often occurs following exposure to environmental stimuli previously associated with drug taking. The metabotropic glutamate receptor, mGluR5, is potentially important in this respect; it plays a central role in several forms of striatal synaptic plasticity proposed to underpin associative learning and memory processes that enable drug-paired stimuli to acquire incentive motivational properties and trigger relapse. Using cell type-specific RNA interference, we have generated a novel mouse line with a selective knock-down of mGluR5 in dopamine D1 receptor-expressing neurons. Although mutant mice self-administer cocaine, we show that reinstatement of cocaine-seeking induced by a cocaine-paired stimulus is impaired. By examining different aspects of associative learning in the mutant mice, we identify deficits in specific incentive learning processes that enable a reward-paired stimulus to directly reinforce behavior and to become attractive, thus eliciting approach toward it. Our findings show that glutamate signaling through mGluR5 located on dopamine D1 receptor-expressing neurons is necessary for incentive learning processes that contribute to cue-induced reinstatement of cocaine-seeking and which may underpin relapse in drug addiction.

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 Ca2+/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.

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.— Parkitna, J. R., Bilbao, A., Rieker, C, Engblom, D., Piechota, M., Nordheim, A., Spanagel, R., Schütz, G. Loss of the serum response factor in the dopamine system leads to hyperactivity. FASEB J. 24, 2427–2435 (2010). www.fasebj.org

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 brains 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.