Dopamine D1 receptor-expressing neurons activity is essential for locomotor and sensitizing effects of a single injection of cocaine.

Dopamine D1 receptors play an important role in the effects of cocaine. Here, we investigated the role of neurons which express these receptors (D1-neurons) in the acute locomotor effects of cocaine and the locomotor sensitization observed after a second injection of this drug, using the previously established two-injection protocol of sensitization. We inhibited D1-neurons using double transgenic mice conditionally expressing the inhibitory Gi-coupled designer receptor exclusively activated by designer drugs (Gi-DREADD) in D1-neurons. Chemogenetic inhibition of D1-neurons by a low dose of clozapine (0.1 mg/kg) decreased the cocaine-induced expression of Fos in striatal neurons. It diminished the basal locomotor activity and acute hyper-locomotion induced by cocaine (20 mg/kg). Clozapine 0.1 mg/kg had no effect by itself and did not alter cocaine effects in wild-type mice. Inhibition of D1-neurons during the first cocaine administration prevented the sensitization of the locomotor response in response to a second cocaine administration 10 days later. On Day 11, inhibition of D1-neurons by clozapine stimulation of Gi-DREADD blocked cocaine-induced locomotion including in sensitized mice, whereas on Day 12, in the absence of clozapine and D1-neurons inhibition, all mice displayed a sensitized response to cocaine. These results show that chemogenetic inhibition of D1-neurons decreases spontaneous and cocaine-induced locomotor activity. It prevents sensitization induction and blocks sensitized locomotion in a two-injection protocol of sensitization but does not reverse established sensitization. Our study further supports the central role of D1-neurons in mediating the acute locomotor effects of cocaine and its sensitization.

Long-lasting tagging of neurons activated by seizures or cocaine administration in Egr1-CreERT2 transgenic mice.

Permanent tagging of neuronal ensembles activated in specific experimental situations is an important objective to study their properties and adaptations. In the context of learning and memory, these neurons are referred to as engram neurons. Here, we describe and characterize a novel mouse line, Egr1-CreERT2 , which carries a transgene in which the promoter of the immediate early gene Egr1 drives the expression of the CreERT2 recombinase that is only active in the presence of tamoxifen metabolite, 4-hydroxy-tamoxifen (4-OHT). Egr1-CreERT2 mice were crossed with various reporter mice, Cre-dependently expressing a fluorescent protein. Without tamoxifen or 4-OHT, no or few tagged neurons were observed. Epileptic seizures induced by pilocarpine or pentylenetetrazol in the presence of tamoxifen or 4-OHT elicited the persistent tagging of many neurons and some astrocytes in the dentate gyrus of hippocampus, where Egr1 is transiently induced by seizures. One week after cocaine and 4-OHT administration, these mice displayed a higher number of tagged neurons in the dorsal striatum than saline/4-OHT controls, with differences between reporter lines. Cocaine-induced tagging required ERK activation and tagged neurons were more likely than others to exhibit ERK phosphorylation or Fos induction after a second injection. Interestingly neurons tagged in saline-treated mice also had an increased propensity to express Fos, suggesting the existence of highly responsive striatal neurons susceptible to be re-activated by cocaine repeated administration, which may contribute to the behavioral adaptations. Our report validates a novel transgenic mouse model for permanently tagging activated neurons and studying long-term alterations of Egr1-expressing cells.

Astrocytic BDNF and TrkB regulate severity and neuronal activity in mouse models of temporal lobe epilepsy.

Astrocytes have emerged as crucial regulators of neuronal network activity, synapse formation, and underlying behavioral and cognitive processes. Despite some pathways have been identified, the communication between astrocytes and neurons remains to be completely elucidated. Unraveling this communication is crucial to design potential treatments for neurological disorders like temporal lobe epilepsy (TLE). The BDNF and TrkB molecules have emerged as very promising therapeutic targets. However, their modulation can be accompanied by several off-target effects such as excitotoxicity in case of uncontrolled upregulation or dementia, amnesia, and other memory disorders in case of downregulation. Here, we show that BDNF and TrkB from astrocytes modulate neuronal dysfunction in TLE models. First, conditional overexpression of BDNF from astrocytes worsened the phenotype in the lithium-pilocarpine mouse model. Our evidences pointed out to the astrocytic pro-BDNF isoform as a major player of this altered phenotype. Conversely, specific genetic deletion of BDNF in astrocytes prevented the increase in the number of firing neurons and the global firing rate in an in vitro model of TLE. Regarding to the TrkB, we generated mice with a genetic deletion of TrkB specifically in hippocampal neurons or astrocytes. Interestingly, both lines displayed neuroprotection in the lithium-pilocarpine model but only the mice with genetic deletion of TrkB in astrocytes showed significantly preserved spatial learning skills. These data identify the astrocytic BDNF and TrkB molecules as promising therapeutic targets for the treatment of TLE.

Differential enhancement of ERK, PKA and Ca2+ signaling in direct and indirect striatal neurons of Parkinsonian mice.

Parkinson's disease (PD) is characterized by severe locomotor deficits due to the disappearance of dopamine (DA) from the dorsal striatum. The development of PD symptoms and treatment-related complications such as dyskinesia have been proposed to result from complex alterations in intracellular signaling in both direct and indirect pathway striatal projection neurons (dSPNs and iSPNs, respectively) following loss of DA afferents. To identify cell-specific and dynamical modifications of signaling pathways associated with PD, we used a hemiparkinsonian mouse model with 6-hydroxydopamine (6-OHDA) lesion combined with two-photon fluorescence biosensors imaging in adult corticostriatal slices. After DA lesion, extracellular signal-regulated kinase (ERK) activation was increased in response to DA D1 receptor (D1R) or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) stimulation. The cAMP-dependent protein kinase (PKA) pathway contributing to ERK activation displayed supersensitive responses to D1R stimulation after 6-OHDA lesion. This cAMP/PKA supersensitivity was specific of D1R-responding SPNs and resulted from Gαolf upregulation and deficient phosphodiesterase activity. In lesioned striatum, the number of D1R-SPNs with spontaneous Ca2+ transients augmented while Ca2+ response to AMPA receptor stimulation specifically increased in iSPNs. Our work reveals distinct cell type-specific signaling alterations in the striatum after DA denervation. It suggests that over-activation of ERK pathway, observed in PD striatum, known to contribute to dyskinesia, may be linked to the combined dysregulation of DA and glutamate signaling pathways in the two populations of SPNs. These findings bring new insights into the implication of these respective neuronal populations in PD motor symptoms and the occurrence of PD treatment complications.

Cocaine conditioned place preference: unexpected suppression of preference due to testing combined with strong conditioning.

Conditioned place preference (CPP) is widely used for evaluating the rewarding effects of drugs. Like other memories, CPP is proposed to undergo reconsolidation during which it is unstable and sensitive to pharmacological inhibition. Previous studies have shown that cocaine CPP can be apparently erased by extracellular signal-regulated kinase (ERK) pathway inhibition during cocaine reconditioning (re-exposure to the drug-paired environment in the presence of the drug). Here, we show that blockade of D1 receptors during reconditioning prevented ERK activation and induced a loss of CPP. However, we also unexpectedly observed a CPP disappearance in mice that underwent testing and reconditioning with cocaine alone, specifically in strong conditioning conditions. The loss was due to the intermediate test. CPP was not recovered with reconditioning or priming in the short term, but it spontaneously reappeared after a month. When we challenged the D1 antagonist-mediated erasure, we observed that both a high dose of cocaine and a first CPP test were required for this effect. Our results also suggest a balance between D1-dependent ERK pathway activation and an A2a-dependent mechanism in D2 striatal neurons in controlling CPP expression. Our data reveal that, paradoxically, a simple CPP test can induce a complete (but transient) loss of place preference following strong but not weak cocaine conditioning. This study emphasizes the complex nature of CPP memory and the importance of multiple parameters that must be taken into consideration when investigating reconsolidation.

Anatomical and molecular characterization of dopamine D1 receptor-expressing neurons of the mouse CA1 dorsal hippocampus.

In the hippocampus, a functional role of dopamine D1 receptors (D1R) in synaptic plasticity and memory processes has been suggested by electrophysiological and pharmacological studies. However, comprehension of their function remains elusive due to the lack of knowledge on the precise localization of D1R expression among the diversity of interneuron populations. Using BAC transgenic mice expressing enhanced green fluorescent protein under the control of D1R promoter, we examined the molecular identity of D1R-containing neurons within the CA1 subfield of the dorsal hippocampus. In agreement with previous findings, our analysis revealed that these neurons are essentially GABAergic interneurons, which express several neurochemical markers, including calcium-binding proteins, neuropeptides, and receptors among others. Finally, by using different tools comprising cell type-specific isolation of mRNAs bound to tagged-ribosomes, we provide solid data indicating that D1R is present in a large proportion of interneurons expressing dopamine D2 receptors. Altogether, our study indicates that D1Rs are expressed by different classes of interneurons in all layers examined and not by pyramidal cells, suggesting that CA1 D1R mostly acts via modulation of GABAergic interneurons.

Selective Effects of PDE10A Inhibitors on Striatopallidal Neurons Require Phosphatase Inhibition by DARPP-32

Type 10A phosphodiesterase (PDE10A) is highly expressed in the striatum, in striatonigral and striatopallidal medium-sized spiny neurons (MSNs), which express D1 and D2 dopamine receptors, respectively. PDE10A inhibitors have pharmacological and behavioral effects suggesting an antipsychotic profile, but the cellular bases of these effects are unclear. We analyzed the effects of PDE10A inhibition in vivo by immunohistochemistry, and imaged cAMP, cAMP-dependent protein kinase A (PKA), and cGMP signals with biosensors in mouse brain slices. PDE10A inhibition in mouse striatal slices produced a steady-state increase in intracellular cAMP concentration in D1 and D2 MSNs, demonstrating that PDE10A regulates basal cAMP levels. Surprisingly, the PKA-dependent AKAR3 phosphorylation signal was strong in D2 MSNs, whereas D1 MSNs remained unresponsive. This effect was also observed in adult mice in vivo since PDE10A inhibition increased phospho-histone H3 immunoreactivity selectively in D2 MSNs in the dorsomedial striatum. The PKA-dependent effects in D2 MSNs were prevented in brain slices and in vivo by mutation of the PKA-regulated phosphorylation site of 32 kDa dopamine- and cAMP-regulated phosphoprotein (DARPP-32), which is required for protein phosphatase-1 inhibition. These data highlight differences in the integration of the cAMP signal in D1 and D2 MSNs, resulting from stronger inhibition of protein phosphatase-1 by DARPP-32 in D2 MSNs than in D1 MSNs. This study shows that PDE10A inhibitors share with antipsychotic medications the property of activating preferentially PKA-dependent signaling in D2 MSNs.

PKA-dependent phosphorylation of ribosomal protein S6 does not correlate with translation efficiency in striatonigral and striatopallidal medium-sized spiny neurons.

Ribosomal protein S6 (rpS6), a component of the 40S ribosomal subunit, is phosphorylated on several residues in response to numerous stimuli. Although commonly used as a marker for neuronal activity, its upstream mechanisms of regulation are poorly studied and its role in protein synthesis remains largely debated. Here, we demonstrate that the psychostimulant d-amphetamine (d-amph) markedly increases rpS6 phosphorylation at Ser235/236 sites in both crude and synaptoneurosomal preparations of the mouse striatum. This effect occurs selectively in D1R-expressing medium-sized spiny neurons (MSNs) and requires the cAMP/PKA/DARPP-32/PP-1 cascade, whereas it is independent of mTORC1/p70S6K, PKC, and ERK signaling. By developing a novel assay to label nascent peptidic chains, we show that the rpS6 phosphorylation induced in striatonigral MSNs by d-amph, as well as in striatopallidal MSNs by the antipsychotic haloperidol or in both subtypes by papaverine, is not correlated with the translation of global or 5' terminal oligopyrimidine tract mRNAs. Together, these results provide novel mechanistic insights into the in vivo regulation of the post-translational modification of rpS6 in the striatum and point out the lack of a relationship between PKA-dependent rpS6 phosphorylation and translation efficiency.

Neuroinflammation and Aß accumulation linked to systemic inflammation are decreased by genetic PKR down-regulation.

Alzheimer's disease (AD) is a neurodegenerative disorder, marked by senile plaques composed of amyloid-ß (Aß) peptide, neurofibrillary tangles, neuronal loss and neuroinflammation. Previous works have suggested that systemic inflammation could contribute to neuroinflammation and enhanced Aß cerebral concentrations. The molecular pathways leading to these events are not fully understood. PKR is a pro-apoptotic kinase that can trigger inflammation and accumulates in the brain and cerebrospinal fluid of AD patients. The goal of the present study was to assess if LPS-induced neuroinflammation and Aß production could be altered by genetic PKR down regulation. The results show that, in the hippocampus of LPS-injected wild type mice, neuroinflammation, cytokine release and Aß production are significantly increased and not in LPS-treated PKR knock-out mice. In addition BACE1 and activated STAT3 levels, a putative transcriptional regulator of BACE1, were not found increased in the brain of PKR knock-out mice as observed in wild type mice. Using PET imaging, the decrease of hippocampal metabolism induced by systemic LPS was not observed in LPS-treated PKR knock-out mice. Altogether, these findings demonstrate that PKR plays a major role in brain changes induced by LPS and could be a valid target to modulate neuroinflammation and Aß production.

Role of the plasticity-associated transcription factor zif268 in the early phase of instrumental learning.

Gene transcription is essential for learning, but the precise role of transcription factors that control expression of many other genes in specific learning paradigms is yet poorly understood. Zif268 (Krox24/Egr-1) is a transcription factor and an immediate-early gene associated with memory consolidation and reconsolidation, and induced in the striatum after addictive drugs exposure. In contrast, very little is known about its physiological role at early stages of operant learning. We investigated the role of Zif268 in operant conditioning for food. Zif268 expression was increased in all regions of the dorsal striatum and nucleus accumbens in mice subjected to the first session of operant conditioning. In contrast, Zif268 increase in the dorsomedial caudate-putamen and nucleus accumbens core was not detected in yoked mice passively receiving the food reward. This indicates that Zif268 induction in these structures is linked to experiencing or learning contingency, but not to reward delivery. When the task was learned (5 sessions), Zif268 induction disappeared in the nucleus accumbens and decreased in the medial caudate-putamen, whereas it remained high in the lateral caudate-putamen, previously implicated in habit formation. In transgenic mice expressing green fluorescent protein (GFP) in the striatonigral neurons, Zif268 induction occured after the first training session in both GFP-positive and negative neurons indicating an enhanced Zif268 expression in both striatonigral and striatopallidal neurons. Mutant mice lacking Zif268 expression obtained less rewards, but displayed a normal discrimination between reinforced and non-reinforced targets, and an unaltered approach to food delivery box. In addition, their motivation to obtain food rewards, evaluated in a progressive ratio schedule, was blunted. In conclusion, Zif268 participates in the processes underlying performance and motivation to execute food-conditioned instrumental task.

Haloperidol-induced Nur77 expression in striatopallidal neurons is under the control of protein phosphatase 1 regulation by DARPP-32.

Impaired dopaminergic signaling in the striatum is involved in diseases as diverse as Parkinson's disease, addiction, and schizophrenia. An important pathophysiological aspect is the loss of balance between striatopallidal and striatonigral pathways. Nur77 is an orphan nuclear receptor and dopamine-regulated immediate-early gene. Classical antipsychotic drugs widely used in the treatment of schizophrenia, such as haloperidol, increase Nur77 mRNA expression in the striatum. However, little is known about the intracellular signaling pathways involved in Nur77 induction. Here, using pharmacological approaches and transgenic mutant mice, we investigated the mechanisms underlying the up-regulation of Nur77 protein expression in the dorsal striatum after haloperidol injection. In drd1a::EGFP transgenic mice that express GFP in D1 neurons, Nur77 up-regulation induced by haloperidol occurred predominantly in GFP-negative neurons. In Gαolf heterozygous mutant mice, in which cAMP production in response to A2A stimulation is impaired in the striatum, haloperidol effect was not altered. In contrast, in DARPP-32 knock-in mutant mice bearing a T34A point mutation of the site responsible for cAMP-dependent phosphatase 1 inhibition, Nur77 up-regulation by haloperidol was prevented. Haloperidol also induced Nur77 protein in D2 neurons of the nucleus accumbens core of wild type but not T34A knock-in mice. Thus, our results show that expression of Nur77 is induced by haloperidol in D2 receptors-expressing medium-sized spiny neurons, through cAMP-dependent regulation of protein phosphatase 1, which is likely to modulate the effects of other protein kinases. Our results clarify the mechanisms of Nur77 induction by antipsychotic and its possible contribution to extrapyramidal effects.

Transient and rapid activation of Akt/GSK-3ß and mTORC1 signaling by D3 dopamine receptor stimulation in dorsal striatum and nucleus accumbens.

D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time-course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK-3ß in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein-S6 (Ser240/244), and eukaryotic initiation factor-4E binding protein-1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock-out mice lacking D3R. In drd1a-EGFP transgenic mice, quinelorane activated Akt/GSK-3ß in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK-3ß pathway in the two populations of medium-size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease.

RASGRF2 regulates alcohol-induced reinforcement by influencing mesolimbic dopamine neuron activity and dopamine release.

The firing of mesolimbic dopamine neurons is important for drug-induced reinforcement, although underlying genetic factors remain poorly understood. In a recent genome-wide association metaanalysis of alcohol intake, we identified a suggestive association of SNP rs26907 in the ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) gene, encoding a protein that mediates Ca(2+)-dependent activation of the ERK pathway. We performed functional characterization of this gene in relation to alcohol-related phenotypes and mesolimbic dopamine function in both mice and adolescent humans. Ethanol intake and preference were decreased in Rasgrf2(-/-) mice relative to WT controls. Accordingly, ethanol-induced dopamine release in the ventral striatum was blunted in Rasgrf2(-/-) mice. Recording of dopamine neurons in the ventral tegmental area revealed reduced excitability in the absence of Ras-GRF2, likely because of lack of inhibition of the I(A) potassium current by ERK. This deficit provided an explanation for the altered dopamine release, presumably linked to impaired activation of dopamine neurons firing. Functional neuroimaging analysis of a monetary incentive-delay task in 663 adolescent boys revealed significant association of ventral striatal activity during reward anticipation with a RASGRF2 haplotype containing rs26907, the SNP associated with alcohol intake in our previous metaanalysis. This finding suggests a link between the RASGRF2 haplotype and reward sensitivity, a known risk factor for alcohol and drug addiction. Indeed, follow-up of these same boys at age 16 y revealed an association between this haplotype and number of drinking episodes. Together, these combined animal and human data indicate a role for RASGRF2 in the regulation of mesolimbic dopamine neuron activity, reward response, and alcohol use and abuse.

Characterization of dopamine D1 and D2 receptor-expressing neurons in the mouse hippocampus.

The hippocampal formation is part of an anatomical system critically involved in learning and memory. Increasing evidence suggests that dopamine plays an important role in learning and memory as well as in several forms of synaptic plasticity. However, the precise identification of neuronal populations expressing D1 or D2 dopamine receptors within the hippocampus is still lacking. To clarify this issue, we used BAC transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter of dopamine D1 or D2 receptors. In Drd1a-EGFP mice, sparse GFP-expressing neurons were detected among glutamatergic projecting neurons of the granular layer of the dentate gyrus and GABAergic interneurons located in the hilus. A dense immunofluorescence was observed in the outer and medial part of the molecular layer of the dentate gyrus as well as in the inner part of the molecular layer of CA1 corresponding to the terminals of pyramidal neurons of the entorhinal cortex defining the perforant and the temporo-ammonic pathway respectively. Finally, scattered D1 receptor-expressing neurons were also identified as GABAergic interneurons in the CA3/CA1 fields of the hippocampus. In Drd2-EGFP transgenic mice, GFP was exclusively detected in the glutamatergic mossy cells located in the polymorphic layer of the dentate gyrus. This pattern was confirmed in Drd2-Cre mice crossed with NLS-LacZ-Tau(mGFP) :LoxP and RCE:LoxP reporter lines. Our results demonstrate that D1 and D2 receptor-expressing neurons are strictly segregated in the mouse hippocampus. By clarifying the identity of D1 and D2 receptor-expressing neurons in the hippocampus, this study establishes a basis for future investigations aiming at elucidating their roles in the hippocampal network.