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Objective Spreading depolarization (SD) is a transient self-propagating wave of neuronal and glial depolarization coupled with large membrane ionic changes and a subsequent depression of neuronal activity. Spreading depolarization in the cortex is implicated in migraine, stroke, and epilepsy. Conversely, spreading depolarization in the striatum, a brain structure deeply involved in motor control and in Parkinson's disease (PD) pathophysiology, has been poorly investigated. Methods We characterized the participation of glutamatergic and dopaminergic transmission in the induction of striatal spreading depolarization by using a novel approach combining optical imaging, measurements of endogenous DA levels, and pharmacological and molecular analyses. Results We found that striatal spreading depolarization requires the concomitant activation of D1-like DA and N-methyl-d-aspartate receptors, and it is reduced in experimental PD. Chronic l-dopa treatment, inducing dyskinesia in the parkinsonian condition, increases the occurrence and speed of propagation of striatal spreading depolarization, which has a direct impact on one of the signaling pathways downstream from the activation of D1 receptors. Conclusion Striatal spreading depolarization might contribute to abnormal basal ganglia activity in the dyskinetic condition and represents a possible therapeutic target. © 2019 International Parkinson and Movement Disorder Society.

Rhes is one of the most interesting genes regulated by thyroid hormones that, through the inhibition of the striatal cAMP/PKA pathway, acts as a modulator of dopamine neurotransmission. Rhes mRNA is expressed at high levels in the dorsal striatum, with a medial-to-lateral expression gradient reflecting that of both dopamine D2 and adenosine A2A receptors. Rhes transcript is also present in the hippocampus, cerebral cortex, olfactory tubercle and bulb, substantia nigra pars compacta (SNc) and ventral tegmental area of the rodent brain. In line with Rhes-dependent regulation of dopaminergic transmission, data showed that lack of Rhes enhanced cocaine- and amphetamine-induced motor stimulation in mice. Previous studies showed that pharmacological depletion of dopamine significantly reduces Rhes mRNA levels in rodents, non-human primates and Parkinson’s disease (PD) patients, suggesting a link between dopaminergic innervation and physiological Rhes mRNA expression. Rhes protein binds to and activates striatal mTORC1, and modulates L-DOPA-induced dyskinesia in PD rodent models. Finally, Rhes is involved in the survival of mouse midbrain dopaminergic neurons of SNc, thus pointing towards a Rhes-dependent modulation of autophagy and mitophagy processes, and encouraging further investigations about mechanisms underlying dysfunctions of the nigrostriatal system.

d-Amino acids were believed to occur only in bacteria and invertebrates. Today, it is well known that d-amino acids are also present in mammalian tissues in a considerable amount. In particular, high levels of free d-serine (d-Ser) and d-aspartate (d-Asp) are found in the brain. While the functions of d-Ser are well known, many questions remain unanswered regarding the role of d-Asp in the central nervous system. d-Asp is very abundant at the embryonic stage, while it strongly decreases after birth because of the expression of d-aspartate oxidase (Ddo) enzyme, which catalyzes the oxidation of this d-amino acid into oxaloacetate, ammonium, and hydrogen peroxide. Pharmacologically, d-Asp acts as an endogenous agonist of N-methyl d-aspartate and mGlu5 receptors, which are known to control fundamental brain processes, including brain development, synaptic plasticity, and cognition. In this work, we studied a recently generated knockin mouse model (R26ddo/ddo), which was designed to express DDO beginning at the zygotic stage. This strategy enables d-Asp to be almost eliminated in both prenatal and postnatal lives. To understand which biochemical pathways are affected by depletion of d-Asp, in this study, we carried out a metabolomic and lipidomic study of ddo knockin brains at different stages of embryonic and postnatal development, combining nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) techniques. Our study shows that d-Asp deficiency in the brain influences amino acid pathways such as threonine, glycine, alanine, valine, and glutamate. Interestingly, d-Asp is also correlated with metabolites involved in brain development and functions such as choline, creatine, phosphocholine (PCho), glycerophosphocholine (GPCho), sphingolipids, and glycerophospholipids, as well as metabolites involved in brain energy metabolism, such as GPCho, glucose, and lactate.

The mammalian target of rapamycin (mTOR) is a ubiquitously expressed serine/threonine kinase protein complex (mTORC1 or mTORC2) that orchestrates diverse functions ranging from embryonic development to aging. However, its brain tissue-specific roles remain less explored. Here, we have identified that the depletion of the mTOR gene in the mice striatum completely prevented the extrapyramidal motor side effects (catalepsy) induced by the dopamine 2 receptor (D2R) antagonist haloperidol, which is the most widely used typical antipsychotic drug. Conversely, a lack of striatal mTOR in mice did not affect catalepsy triggered by the dopamine 1 receptor (D1R) antagonist SCH23390. Along with the lack of cataleptic effects, the administration of haloperidol in mTOR mutants failed to increase striatal phosphorylation levels of ribosomal protein pS6 (S235/236) as seen in control animals. To confirm the observations of the genetic approach, we used a pharmacological method and determined that the mTORC1 inhibitor rapamycin has a profound influence upon post-synaptic D2R-dependent functions. We consistently found that pretreatment with rapamycin entirely prevented (in a time-dependent manner) the haloperidol-induced catalepsy, and pS6K (T389) and pS6 (S235/236) signaling upregulation, in wild-type mice. Collectively, our data indicate that striatal mTORC1 blockade may offer therapeutic benefits with regard to the prevention of D2R-dependent extrapyramidal motor side effects of haloperidol in psychiatric illness.

A novel molecular target that signals abnormal involuntary movement in Parkinson’s disease treatment is discovered., The therapeutic effects of l-3,4-dihydroxyphenylalanine (l-DOPA) in patients with Parkinson’s disease (PD) severely diminishes with the onset of abnormal involuntary movement, l-DOPA–induced dyskinesia (LID). However, the molecular mechanisms that promote LID remain unclear. Here, we demonstrated that RasGRP1 [(guanine nucleotide exchange factor (GEF)] controls the development of LID. l-DOPA treatment rapidly up-regulated RasGRP1 in the striatum of mouse and macaque model of PD. The lack of RasGRP1 in mice (RasGRP1−/−) dramatically diminished LID without interfering with the therapeutic effects of l-DOPA. Besides acting as a GEF for Ras homolog enriched in the brain (Rheb), the activator of the mammalian target of rapamycin kinase (mTOR), RasGRP1 promotes l-DOPA–induced extracellular signal-regulated kinase (ERK) and the mTOR signaling in the striatum. High-resolution tandem mass spectrometry analysis revealed multiple RasGRP1 downstream targets linked to LID vulnerability. Collectively, the study demonstrated that RasGRP1 is a critical striatal regulator of LID.

Levodopa (L-DOPA) treatment is the main gold-standard therapy for Parkinson disease (PD). Besides good antiparkinsonian effects, prolonged use of this drug is associated to the development of involuntary movements known as L-DOPA-induced dyskinesia (LID). L-DOPA-induced dyskinesia is linked to a sensitization of dopamine (DA) D1 receptors located on spiny projection neurons (SPNs) of the dorsal striatum. Several evidences have shown that the emergence of LID can be related to striatal D1/cAMP/PKA/DARPP-32 and extracellular signal-regulated kinases (ERK1/2) pathway overactivation associated to aberrant N-methyl-d-aspartate (NMDA) receptor function. In addition, within striatum, ERK1/2 is also able to modulate in a D1 receptor-dependent manner the activity of the mammalian target of rapamycin complex 1 (mTORC1) pathway under DA depletion and L-DOPA therapy. Consistently, increased mTORC1 signaling appears during chronic administration of L-DOPA and shows a high correlation with the severity of dyskinesia. Furthermore, the abnormal activation of the D1/PKA/DARPP-32 cascade is paralleled by increased phosphorylation of the GluA1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor at the PKA Ser845 site. The GluA1 promotes excitatory AMPA receptor-mediated transmission and may be implicated in the alterations found at the corticostriatal synapses of dyskinetic animals. In our study, we investigated the role of mTORC1 pathway activation in modulating bidirectional striatal synaptic plasticity in L-DOPA-treated parkinsonian rats. Inhibition of mTORC1 by coadministration of rapamycin to L-DOPA was able to limit the magnitude of LID expression, accounting for a therapeutic effect of this drug. In particular, behavioral data showed that, in L-DOPA-treated rats, rapamycin administration induced a selective decrease of distinct components of abnormal involuntary movements (i.e., axial and orolingual dyskinesia). Furthermore, ex vivo patch clamp and intracellular recordings of SPNs revealed that pharmacological inhibition of mTORC1 also resulted associated with a physiological bidirectional plasticity, when compared to dyskinetic rats treated with L-DOPA alone. This study uncovers the important role of mTORC1 inhibition to prevent the loss of striatal bidirectional plasticity under chronic L-DOPA treatment in rodent models of PD.

Abnormal hippocampal neural plasticity has been implicated in behavioural abnormalities and complex neuropsychiatric conditions, including bipolar disorder (BD). However, the determinants of this neural alteration remain unknown. This work tests the hypothesis that the neurotransmitter serotonin (5-HT) is a key determinant of hippocampal neuroplasticity, and its absence leads to maladaptive behaviour relevant for BD. Depletion of brain 5-HT in Tph2 mutant mice resulted in reduced behavioural despair, reduced anxiety, marked aggression and lower habituation in novel environments, reminiscent of bipolar-associated manic behaviour. Treatment with valproate produced a substantial improvement of the mania-like behavioural phenotypes displayed by Tph2 mutants. Brain-wide fMRI mapping in mutants revealed functional hippocampal hyperactivity in which we also observed dramatically increased neuroplasticity. Importantly, remarkable correspondence between the transcriptomic profile of the Tph2 mutant hippocampus and neurons from bipolar disorder patients was observed. Chronic stress reversed the emotional phenotype and the hippocampal transcriptional landscape of Tph2 mutants. These changes were associated with inappropriate activation of transcriptional adaptive response to stress as assessed by gene set enrichment analyses in the hippocampus of Tph2 mutant mice. These findings delineate 5-HT as a critical determinant in BD associated maladaptive emotional responses and aberrant hippocampal neuroplasticity, and support the use of Tph2−/− mice as a new research tool for mechanistic and therapeutic research in bipolar disorder.

N-methyl-d-aspartate receptor (NMDAR) subunit composition strictly commands receptor function and pharmacological responses. Changes in NMDAR subunit composition have been documented in brain disorders such as Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesias (LIDs), where an increase of NMDAR GluN2A/GluN2B subunit ratio at striatal synapses has been observed. A therapeutic approach aimed at rebalancing NMDAR synaptic composition represents a valuable strategy for PD and LIDs. To this, the comprehension of the molecular mechanisms regulating the synaptic localization of different NMDAR subtypes is required. We have recently demonstrated that Rabphilin 3A (Rph3A) is a new binding partner of NMDARs containing the GluN2A subunit and that it plays a crucial function in the synaptic stabilization of these receptors. Considering that protein-protein interactions govern the synaptic retention of NMDARs, the purpose of this work was to analyse the role of Rph3A and Rph3A/NMDAR complex in PD and LIDs, and to modulate Rph3A/GluN2A interaction to counteract the aberrant motor behaviour associated to chronic L-DOPA administration. Thus, an array of biochemical, immunohistochemical and pharmacological tools together with electron microscopy were applied in this study. Here we found that Rph3A is localized at the striatal postsynaptic density where it interacts with GluN2A. Notably, Rph3A expression at the synapse and its interaction with GluN2A-containing NMDARs were increased in parkinsonian rats displaying a dyskinetic profile. Acute treatment of dyskinetic animals with a cell-permeable peptide able to interfere with Rph3A/GluN2A binding significantly reduced their abnormal motor behaviour. Altogether, our findings indicate that Rph3A activity is linked to the aberrant synaptic localization of GluN2A-expressing NMDARs characterizing LIDs. Thus, we suggest that Rph3A/GluN2A complex could represent an innovative therapeutic target for those pathological conditions where NMDAR composition is significantly altered. N-methyl-(D)-aspartate receptor (NMDAR) subunit composition strictly commands receptor function and pharmacological responses. Changes in NMDAR subunit composition have been documented in brain disorders such as Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesias (LIDs), where an increase of NMDAR GluN2A/G1uN2B subunit ratio at striatal synapses has been observed. A therapeutic approach aimed at rebalancing NMDAR synaptic composition represents a valuable strategy for PD and LIDs. To this, the comprehension of the molecular mechanisms regulating the synaptic localization of different NMDAR subtypes is required.We have recently demonstrated that Rabphilin 3A (Rph3A) is a new binding partner of NMDARs containing the GIuN2A subunit and that it plays a crucial function in the synaptic stabilization of these receptors. Considering that protein-protein interactions govern the synaptic retention of NMDARs, the purpose of this work was to analyse the role of Rph3A and Rph3A/NMDAR complex in PD and LIDs, and to modulate Rph3A/GIuN2A interaction to counteract the aberrant motor behaviour associated to chronic L-DOPA administration. Thus, an array of biochemical, immunohistochemical and pharmacological tools together with electron microscopy were applied in this study. Here we found that Rph3A is localized at the striatal postsynaptic density where it interacts with G1uN2A. Notably, Rph3A expression at the synapse and its interaction with GIuN2A-containing NMDARs were increased in parkinsonian rats displaying a dyskinetic profile. Acute treatment of dyskinetic animals with a cell-permeable peptide able to interfere with Rph3A/GIuN2A binding significantly reduced their abnormal motor behaviour.Altogether, our findings indicate that Rph3A activity is linked to the aberrant synaptic localization of GIuN2Aexpressing NMDARs characterizing LIDs. Thus, we suggest that Rph3A/GIuN2A complex could represent an innovative therapeutic target for those pathological conditions where NMDAR composition is significantly altered. (C) 2017 The Authors. Published by Elsevier Inc.

In addition to its role in neuronal survival, the brain neurotrophic factor (BDNF) has been shown to influence serotonin transmission and synaptic plasticity, events strongly implicated in the appearance of l-DOPA-induced dyskinesia (LID), a motor complication occurring in parkinsonian patients after long-term treatment with the dopamine precursor. In order to evaluate a possible influence of BDNF in the appearance of LID, 6-OHDA-lesioned rats received a striatal injection of different concentrations of an adeno-associated viral (AAV) vector over-expressing either BDNF or GFP, as control vector. Eight weeks later, animals started to receive a daily treatment with l-DOPA (4-6mg/kg plus benserazide 4-6mg/kg, s.c.) or saline, and dyskinesias, as well as l-DOPA-induced rotations, were evaluated at several time-points. Moreover, molecular changes in striatal D1 receptor-dependent cAMP/PKA and ERK/mTORC signaling pathways, as well as, sprouting of striatal serotonin axons, were measured. Results showed that the AAV-BDNF vector injection induced striatal over-expression of BDNF, as well as striatal and pallidal serotonin axon hyperinnervation. Moreover, rats that over-expressed BDNF were more prone to develop LID and l-DOPA-induced rotations, compared to the GFP-treated control group. Finally, rats that over-expressed BDNF showed increased levels of striatal D1R-dependent signaling phospho-proteins in response to l-DOPA administration. This study suggests that BDNF over-expression, by inducing changes in pre-synaptic serotonin axonal trophism, is able to exacerbate maladaptive responses to l-DOPA administration.

Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor and non-motor symptoms of Parkinson's Disease (PD), and to L-DOPA-induced dyskinesia. Besides the main agonist L-glutamate, two other amino acids in the atypical D-configuration, D-serine and D-aspartate, activate NMDARs. In the present work, we investigated the effect of dopamine depletion on D-amino acids metabolism in the brain of MPTP-lesioned Macaca mulatta, and in the serum and cerebrospinal fluid of PD patients. We found that MPTP treatment increases D-aspartate and D-serine in the monkey putamen while L-DOPA rescues both D-amino acids levels. Conversely, dopaminergic denervation is associated with selective D-serine reduction in the substantia nigra. Such decrease suggests that the beneficial effect of D-serine adjuvant therapy previously reported in PD patients may derive from the normalization of endogenous D-serine levels and consequent improvement of nigrostriatal hypoglutamatergic transmission at glycine binding site. We also found reduced D-serine concentration in the cerebrospinal fluid of L-DOPA-free PD patients. These results further confirm the existence of deep interaction between dopaminergic and glutamatergic neurotransmission in PD and disclose a possible direct influence of D-amino acids variations in the changes of NMDAR transmission occurring under dopamine denervation and L-DOPA therapy. Dysfunction of NMDA receptor (NMDAR)-mediated transmission is supposed to contribute to the motor and non-motor symptoms of Parkinson’s Disease (PD), and to L-DOPA-induced dyskinesia. Besides the main agonist L-glutamate, two other amino acids in the atypical D-configuration, D-serine and D-aspartate, activate NMDARs. In the present work, we investigated the effect of dopamine depletion on D-amino acids metabolism in the brain of MPTP-lesioned Macaca mulatta, and in the serum and cerebrospinal fluid of PD patients. We found that MPTP treatment increases D-aspartate and D-serine in the monkey putamen while L-DOPA rescues both D-amino acids levels. Conversely, dopaminergic denervation is associated with selective D-serine reduction in the substantia nigra. Such decrease suggests that the beneficial effect of D-serine adjuvant therapy previously reported in PD patients may derive from the normalization of endogenous D-serine levels and consequent improvement of nigrostriatal hypoglutamatergic transmission at glycine binding site. We also found reduced D-serine concentration in the cerebrospinal fluid of L-DOPA-free PD patients. These results further confirm the existence of deep interaction between dopaminergic and glutamatergic neurotransmission in PD and disclose a possible direct influence of D-amino acids variations in the changes of NMDAR transmission occurring under dopamine denervation and L-DOPA therapy.

Background Programmed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as d-serine and d-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of d-amino acids are found. We recently demonstrated that Ddo, the d-aspartate degradation gene, is actively demethylated to ultimately reduce d-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate d-ser levels during brain development has been poorly investigated to date. Methods We performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based d-amino acids level analyses of genes controlling the mammalian brain levels of d-serine and d-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis. Results We report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar d-serine a few days after mouse birth. High amount of 5′-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process. Conclusion The present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of d-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.

The therapeutic benefits of L–3,4–dihydroxyphenylalanine (L-DOPA) in Parkinson disease (PD) patients diminishes with the onset of abnormal involuntary movements (L-DOPA induced dyskinesia), a debilitating motor side effect. L-DOPA induced dyskinesia are due to altered dopaminergic signaling in the striatum, a brain region that controls motor and cognitive functions. However, the molecular mechanisms that promote L-DOPA-induced dyskinesia remain unclear. Here, we have reported that RasGRP1 (also known as CalDAG-GEF-II) physiologically mediated L-DOPA induced dyskinesia in a 6-hydroxy dopamine (6-OHDA) lesioned mouse model of PD. In this study, L-DOPA treatment rapidly upregulated RasGRP1 in the striatum. Our findings showed that RasGRP1 deleted mice (RasGRP1−/−) had drastically diminished L-DOPA-induced dyskinesia, andRasGRP1−/−mice did not interfere with the therapeutic benefits of L-DOPA. In terms of its mechanism, RasGRP1 mediates L-DOPA-induced extracellular regulated kinase (ERK), the mammalian target of rapamycin kinase (mTOR) and the cAMP/PKA pathway and binds directly with Ras-homolog-enriched in the brain (Rheb), which is a potent activator of mTOR, both in vitro and in the intact striatum. High-resolution tandem mass tag mass spectrometry analysis of striatal tissue revealed significant targets, such as phosphodiesterase (Pde1c), Pde2a, catechol-o-methyltransferase (comt), and glutamate decarboxylase 1 and 2 (Gad1 and Gad2), which are downstream regulators of RasGRP1 and are linked to L-DOPA-induced dyskinesia vulnerability. Collectively, the findings of this study demonstrated that RasGRP1 is a major regulator of L-DOPA-induced dyskinesia in the striatum. Drugs or gene-depletion strategies targeting RasGRP1 may offer novel therapeutic opportunities for preventing L-DOPA-induced dyskinesia in PD patients.

Excitatory amino acids (EAAs) are found present in the nervous and reproductive systems of animals. Numerous studies have demonstrated a regulatory role for Glutamate (Glu), d-aspartate ( d-Asp) and N-methyl- d-aspartate (NMDA) in the control of spermatogenesis. EAAs are able to stimulate the Glutamate receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR). Here in, we assess expression of the main AMPAR subunits, GluA1 and GluA2/3, in the mouse testis and in spermatogonial GC-1 cells. The results showed that both GluA1 and GluA2/3 were localized in mouse testis prevalently in spermatogonia. The subunit GluA2/3 was more highly expressed compared with GluA1 in both the testis and the GC-1 cells. Subsequently, GC-1 cells were incubated with medium containing l-Glu, d-Glu, d-Asp or NMDA to determine GluA1 and GluA2/3 expressions. At 30 minutes and 2 hours of incubation, EAA-treated GC-1 cells showed significantly higher expression levels of both GluA1 and GluA2/3. Furthermore, p-extracellular signal-regulated kinase (ERK), p-Akt, proliferating cell nuclear antigen (PCNA), and Aurora B expressions were assayed in l-Glu-, d-Glu-, and NMDA-treated GC-1 cells. At 30 minutes and 2 hours of incubation, treated GC-1 cells showed significantly higher expression levels of p-ERK and p-Akt. A consequent increase of PCNA and Aurora B expressions was induced by l-Glu and NMDA, but not by d-Glu. Our study demonstrates a direct effect of the EAAs on spermatogonial activity. In addition, the increased protein expression levels of GluA1 and GluA2/3 in EAA-treated GC-1 cells suggest that EAAs could activate ERK and Akt pathways through the AMPAR. Finally, the increased PCNA and Aurora B levels may imply an enhanced proliferative activity.

In mammals, free d-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very low during adulthood as a result of the postnatal expression and activity of the catabolizing enzyme d-aspartate oxidase (DDO). Previous studies have shown that long-lasting exposure to nonphysiological, higher D-Asp concentrations in Ddo knockout (Ddo-/-) mice elicits a precocious decay of synaptic plasticity and cognitive functions, along with a dramatic age-dependent expression of active caspase 3, associated with increased cell death in different brain regions, including hippocampus, prefrontal cortex, and substantia nigra pars compacta. Here, we investigate the yet unclear molecular and cellular events associated with the exposure of abnormally high D-Asp concentrations in cortical primary neurons and in the brain of Ddo-/- mice. For the first time, our in vitro findings document that D-Asp induces in a time-, dose-, and NMDA receptor-dependent manner alterations in JNK and Tau phosphorylation levels, associated with pronounced cell death in primary cortical neurons. Moreover, observations obtained in Ddo-/- animals confirmed that high in vivo levels of D-Asp altered cortical JNK signaling, Tau phosphorylation and enhanced protein SUMOylation, indicating a robust indirect role of DDO activity in regulating these biochemical NMDA receptor-related processes. Finally, no gross modifications in D-Asp concentrations and DDO mRNA expression were detected in the cortex of patients with Alzheimer's disease when compared to age-matched healthy controls. In mammals, free D-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very low during adulthood as a result of the postnatal expression and activity of the catabolizing enzyme D-aspartate oxidase (DDO). Previous studies have shown that long-lasting exposure to nonphysiological, higher D-Asp concentrations in Ddo knockout (Ddo(-/-)) mice elicits a precocious decay of synaptic plasticity and cognitive functions, along with a dramatic age-dependent expression of active caspase 3, associated with increased cell death in different brain regions, including hippocampus, prefrontal cortex, and substantia nigra pars compacta. Here, we investigate the yet unclear molecular and cellular events associated with the exposure of abnormally high D-Asp concentrations in cortical primary neurons and in the brain of Ddo(-/-) mice. For the first time, our in vitro findings document that D-Asp induces in a time-, dose-, and NMDA receptor-dependent manner alterations in JNK and Tau phosphorylation levels, associated with pronounced cell death in primary cortical neurons, Moreover, observations obtained in Ddo(-/-) animals confirmed that high in vivo levels of D-Asp altered cortical JNK signaling, Tau phosphorylation and enhanced protein SUMOylation, indicating a robust indirect role of DDO activity in regulating these biochemical NMDA receptor-related processes. Finally, no gross modifications in D-Asp concentrations and DDO mRNA expression were detected in the cortex of patients with Alzheimer's disease when compared to age-matched healthy controls.

Total concentrations of thyroid hormones T(3) and T(4), and of their free forms, FT(3) and FT(4), D-aspartic acid (D-Asp), D-aspartate oxidase (D-AspO), D-aspartate racemase, H(2)O(2), and ROS (reactive oxygen species) were determined in rats and mice. T(3) and T(4) were 1 and 50 ng/ml, respectively, in serum, and 750 and 40000 ng/g, respectively, in thyroid. Concentrations of the free forms FT(3) and FT(4) were ca. 250 times lower than their respective total concentrations. The endogenous content of D-Asp in thyroid gland was ca. 100 nmol/g tissue, whereas the activity of D-AspO was ca. 80 units/mg thyroid, and that of D-aspartate racemase was ca. 15 units/mg thyroid. H(2)O(2) Concentration in rat and mouse thyroid gland was ca. 290 pmol/g thyroid, and the concentration of ROS was ca. 10 pmol/DCF/min/mg protein. H(2)O(2) is essential for the iodination of the tyrosyl residues to produce mono- and diiodotyrosine that are the precursors for the synthesis of T(3) and T(4). Production of H(2)O(2) in thyroid glands occurs by oxidation of endogenous D-Asp by D-AspO (D-Asp+O(2)+H(2)O-->alpha-oxaloacetate+NH(3)+H(2)O(2)). D-Aspartate racemase catalyzes the in vivo production of D-Asp from L-Asp. Thus, interaction of endogenous D-Asp, D-AspO, and D-aspartate racemase in thyroid gland constitutes an additional biochemical pathway for the production of H(2)O(2) and consequently for the synthesis of thyroid hormones. Total concentrations of thyroid hormones T3 and T4, and of their free forms, FT3 and FT4, D-aspartic acid (D-Asp), D-aspartate oxidase (D-AspO), D-aspartate racemase, H2O 2, and ROS (reactive oxygen species) were determined in rats and mice. T3 and T4 were 1 and 50 ng/ml, respectively, in serum, and 750 and 40000 ng/g, respectively, in thyroid. Concentrations of the free forms FT3 and FT4 were ca. 250 times lower than their respective total concentrations. The endogenous content of D-Asp in thyroid gland was ca. 100 nmol/g tissue, whereas the activity of D-AspO was ca. 80 units/mg thyroid, and that of D-aspartate racemase was ca. 15 units/mg thyroid. H2O2 Concentration in rat and mouse thyroid gland was ca. 290 pmol/g thyroid, and the concentration of ROS was ca. 10 pmol/DCF/min/mg protein. H2O2 is essential for the iodination of the tyrosyl residues to produce mono- and diiodotyrosine that are the precursors for the synthesis of T3 and T4. Production of H 2O2 in thyroid glands occurs by oxidation of endogenous D-Asp by D-AspO (D-Asp+O2+H2O→α-oxaloacetate + NH3+H2O2). D-Aspartate race-mase catalyzes the in vivo production of D-Asp from L-Asp. Thus, interaction of endogenous D-Asp, D-AspO, and D-aspartate racemase in thyroid gland constitutes an additional biochemical pathway for the production of H2O2 and consequently for the synthesis of thyroid hormones. © 2010 Verlag Helvetica Chimica Acta AG.

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the second-generation antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

Adaptation to environmental insults is an evolutionary mechanism essential for survival. The hippocampus participates in controlling adaptive responses to stress and emotional state through the modulation of neuroplasticity events, which are dysregulated in stress-related neuropsychiatric disorders. The neurotransmitter serotonin (5-HT) has been proposed as a pivotal player in hippocampal neuroplasticity in both normal and neuropsychiatric conditions though its role remains still poorly understood. Here, we investigated the impact of 5-HT deficiency on hippocampal activity combining RNA-seq, in vivo neuroimaging, neuroanatomical, biochemical and behavioral experiments on 5-HT depleted mice. We unveil that serotonin is required for appropriate activation of neuroplasticity adaptive mechanisms to environmental insults. Bidirectional deregulation of these programs in serotonin depleted mice is associated with opposite behavioral traits that model core symptoms of bipolar disorder. These findings delineate a previously unreported buffering role of 5-HT in instructing hippocampal activity and emotional responses to environmental demands.

Visual Abstract, Growing evidence shows that the neurotransmitter serotonin (5-HT) modulates the fine-tuning of neuron development and the establishment of wiring patterns in the brain. However, whether serotonin is involved in the maintenance of neuronal circuitry in the adult brain remains elusive. Here, we use a Tph2fl°x conditional knockout (cKO) mouse line to assess the impact of serotonin depletion during adulthood on serotonergic system organization. Data show that the density of serotonergic fibers is increased in the hippocampus and decreased in the thalamic paraventricular nucleus (PVN) as a consequence of brain serotonin depletion. Strikingly, these defects are rescued following reestablishment of brain 5-HT signaling via administration of the serotonin precursor 5-hydroxytryptophan (5-HTP). Finally, 3D reconstruction of serotonergic fibers reveals that changes in serotonin homeostasis affect axonal branching complexity. These data demonstrate that maintaining proper serotonin homeostasis in the adult brain is crucial to preserve the correct serotonergic axonal wiring.

Several studies have suggested that free d-Asp has a crucial role in N-methyl d-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free d-Asp, l-Asp and N-methyl d-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing l-Asp, d-Asp and N-methyl d-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for d-Asp, 0.46 pg/μl for l-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for d-Asp, 1.41 pg/μl for l-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of d-Asp, l-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free d-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses. Several studies have suggested that free D-Asp has a crucial role in N-methyl D-Asp receptor-mediated neurotransmission playing very important functions in physiological and pathological processes. This paper describes the development of an analytical procedure for the direct and simultaneous determination of free D-Asp, L-Asp and N-methyl D-Asp in specimens of different mouse brain tissues using chiral LC-MS/MS in Multiple Reaction Monitoring scan mode. After comparing three procedures and different buffers and extraction solvents, a simple preparation procedure was selected the analytes of extraction. The method was validated by analyzing L-Asp, D-Asp and N-methyl D-Asp recovery at different spiked concentrations (50, 100 and 200 pg/μl) yielding satisfactory recoveries (75-110%), and good repeatability. Limits of detection (LOD) resulted to be 0.52 pg/μl for D-Asp, 0.46 pg/μl for L-Asp and 0.54 pg/μl for NMDA, respectively. Limits of quantification (LOQ) were 1.57 pg/μl for D-Asp, 1.41 pg/μl for L-Asp and 1.64 pg/μl for NMDA, respectively. Different concentration levels were used for constructing the calibration curves which showed good linearity. The validated method was then successfully applied to the simultaneous detection of D-Asp, L-Asp and NMDA in mouse brain tissues. The concurrent, sensitive, fast, and reproducible measurement of these metabolites in brain tissues will be useful to correlate the amount of free D-Asp with relevant neurological processes, making the LC-MS/MS MRM method well suited, not only for research work but also for clinical analyses.

In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinson's disease (PD), Schizophrenia (SCZ), and Bipolar Disorder (BD), when compared to healthy controls (about 200 post-mortem samples). Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the putamen of patients suffering from PD showed a significant 35% reduction of this transcript, compared to healthy subjects. Interestingly, in line with observations obtained in humans, we found 27% decrease in Rhes mRNA levels in the putamen of MPTP-treated primates. Based on the established inhibitory influence of Rhes on dopamine-related responses, we hypothesize that its striatal downregulation in PD patients and animal models of PD might represent an adaptive event of the dopaminergic system to functionally counteract the reduced nigrostriatal innervation.

DNA methylation is often analyzed by reporting the average methylation degree of each cytosine. In this study, we used a single molecule methylation analysis in order to look at the methylation conformation of individual molecules. Using D-aspartate oxidase as a model gene, we performed an in-depth methylation analysis through the developmental stages of three different mouse tissues (brain, lung, and gut), where this gene undergoes opposite methylation destiny. This approach allowed us to track both methylation and demethylation processes at high resolution. The complexity of these dynamics was markedly simplified by introducing the concept of methylation classes (MCs), defined as the number of methylated cytosines per molecule, irrespective of their position. The MC concept smooths the stochasticity of the system, allowing a more deterministic description. In this framework, we also propose a mathematical model based on the Markov chain. This model aims to identify the transition probability of a molecule from one MC to another during methylation and demethylation processes. The results of our model suggest that: 1) both processes are ruled by a dominant class of phenomena, namely, the gain or loss of one methyl group at a time; and 2) the probability of a single CpG site becoming methylated or demethylated depends on the methylation status of the whole molecule at that time.

Abnormal amyloid-β (Aβ) production and deposition is believed to represent one of the main causes of Alzheimer's disease (AD). γ-Secretase is the enzymatic complex responsible for Aβ generation from its precursor protein. Inhibition or modulation of γ-secretase represents an attractive therapeutic approach. CHF5074 is a new γ-secretase modulator that has been shown to inhibit brain plaque deposition and to attenuate memory deficit in adult AD transgenic mice after chronic treatment. To date, it is not known whether the positive behavioral effects of this compound also occur in young transgenic mice without plaque deposition. Here, we evaluated the effects of acute and subchronic treatment with CHF5074 on contextual and recognition memory and on hippocampal synaptic plasticity in plaque-free Tg2576 mice. We found that at 5 months of age, contextual memory impairment was significantly attenuated after acute subcutaneous administration of 30 mg/kg CHF5074. At 6 months of age, recognition memory impairment was fully reversed after a 4-week oral treatment in the diet (≈60 mg/kg/day). These cognitive effects were associated with a reversal of long-term potentiation (LTP) impairment in the hippocampus. A significant reduction in brain intraneuronal AβPP/Aβ levels and hyperphosphorylated tau, but no change in soluble or oligomeric Aβ levels was detected in Tg2576 mice showing functional recovery following CHF5074 treatment. We conclude that the beneficial effects of CHF5074 treatment in young transgenic mice occurred at a stage that precedes plaque formation and were associated with a reduction in intraneuronal AβPP/Aβ and hyperphosphorylated tau.

The endocannabinoid anandamide (AEA) plays a crucial role in emotional control, and inhibition of its degradation by the fatty acid amide hydrolase (FAAH) has a potent antianxiety effect. The mechanism by which the magnification of AEA activity reduces anxiety is still largely undetermined. By using FAAH mutant mice and both intraperitoneal and intracerebroventricular administration of the FAAH inhibitor (3'-(aminocarbonyl)[1,1'-biphenyl]-3-yl)-cyclohexylcarbamate (URB597), we found that enhanced AEA signaling reversed, via central cannabinoid CB1 receptors (CB1Rs), the anxious phenotype of mice exposed to social defeat stress. This behavioral effect was associated with preserved activity of CB1Rs regulating GABA transmission in the striatum, whereas these receptors were dramatically down-regulated by stress in control animals. The hypothalamic-pituitary-adrenal (HPA) axis was not involved in the antistress effects of FAAH inhibition, although the HPA axis is a biological target of endogenous AEA. We also provided some physiological indications that striatal CB1Rs regulating GABA synapses are not the receptor targets of FAAH inhibition, which rather resulted in the stimulation of striatal CB1Rs regulating glutamate transmission. Collectively, our findings suggest that preservation of cannabinoid CB1 receptor function within the striatum is a possible synaptic correlate of the antianxiety effects of FAAH inhibition.

Caffeine is the most commonly self-administered psychoactive substance worldwide. At usual doses, the effects of caffeine on vigilance, attention, mood and arousal largely depend on the modulation of central adenosine receptors. The present review article describes the action of caffeine within the striatum, to provide a possible molecular mechanism at the basis of the psychomotor and reinforcing properties of this pharmacological agent. The striatum is in fact a subcortical area involved in sensorimotor, cognitive, and emotional processes, and recent experimental findings showed that chronic caffeine consumption enhances the sensitivity of striatal GABAergic synapses to the stimulation of cannabinoid CB1 receptors. The endocannabinoid system is involved in the psychoactive effects of many compounds, and adenosine A2A receptors (the main receptor target of caffeine) elicit a permissive effect towards CB1 receptors, thus suggesting that A2A-CB1 receptor interaction plays a major role in the generation and maintenance of caffeine reinforcing behavior. Aim of this review is to describe the effects of caffeine on striatal neurotransmission with special reference to the modulation of the endocannabinoid system.

DYT1 dystonia is a severe form of inherited dystonia, characterized by involuntary twisting movements and abnormal postures. It is linked to a deletion in the dyt1 gene, resulting in a mutated form of the protein torsinA. The penetrance for dystonia is incomplete, but both clinically affected and non-manifesting carriers of the DYT1 mutation exhibit impaired motor learning and evidence of altered motor plasticity. Here, we characterized striatal glutamatergic synaptic plasticity in transgenic mice expressing either the normal human torsinA or its mutant form, in comparison to non-transgenic (NT) control mice. Medium spiny neurons recorded from both NT and normal human torsinA mice exhibited normal long-term depression (LTD), whereas in mutant human torsinA littermates LTD could not be elicited. In addition, although long-term potentiation (LTP) could be induced in all the mice, it was greater in magnitude in mutant human torsinA mice. Low-frequency stimulation (LFS) can revert potentiated synapses to resting levels, a phenomenon termed synaptic depotentiation. LFS induced synaptic depotentiation (SD) both in NT and normal human torsinA mice, but not in mutant human torsinA mice. Since anti-cholinergic drugs are an effective medical therapeutic option for the treatment of human dystonia, we reasoned that an excess in endogenous acetylcholine could underlie the synaptic plasticity impairment. Indeed, both LTD and SD were rescued in mutant human torsinA mice either by lowering endogenous acetylcholine levels or by antagonizing muscarinic M1 receptors. The presence of an enhanced acetylcholine tone was confirmed by the observation that acetylcholinesterase activity was significantly increased in the striatum of mutant human torsinA mice, as compared with both normal human torsinA and NT littermates. Moreover, we found similar alterations of synaptic plasticity in muscarinic M2/M4 receptor knockout mice, in which an increased striatal acetylcholine level has been documented. The loss of LTD and SD on one hand, and the increase in LTP on the other, demonstrate that a 'loss of inhibition' characterizes the impairment of synaptic plasticity in this model of DYT1 dystonia. More importantly, our results indicate that an unbalanced cholinergic transmission plays a pivotal role in these alterations, providing a clue to understand the ability of anticholinergic agents to restore motor deficits in dystonia. DYT1 dystonia is a severe form of inherited dystonia, characterized by involuntary twisting movements and abnormal postures. It is linked to a deletion in the dyt1 gene, resulting in a mutated form of the protein torsinA. The penetrance for dystonia is incomplete, but both clinically affected and non-manifesting carriers of the DYT1 mutation exhibit impaired motor learning and evidence of altered motor plasticity. Here, we characterized striatal glutamatergic synaptic plasticity in transgenic mice expressing either the normal human torsinA or its mutant form, in comparison to non-transgenic (NT) control mice. Medium spiny neurons recorded from both NT and normal human torsinA mice exhibited normal long-term depression (LTD), whereas in mutant human torsinA littermates LTD could not be elicited. In addition, although long-term potentiation (LTP) could be induced in all the mice, it was greater in magnitude in mutant human torsinA mice. Low-frequency stimulation (LFS) can revert potentiated synapses to resting levels, a phenomenon termed synaptic depotentiation. LFS induced synaptic depotentiation (SD) both in NT and normal human torsinA mice, but not in mutant human torsinA mice. Since anti-cholinergic drugs are an effective medical therapeutic option for the treatment of human dystonia, we reasoned that an excess in endogenous acetylcholine could underlie the synaptic plasticity impairment. Indeed, both LTD and SD were rescued in mutant human torsinA mice either by lowering endogenous acetylcholine levels or by antagonizing muscarinic M1 receptors. The presence of an enhanced acetylcholine tone was confirmed by the observation that acetylcholinesterase activity was significantly increased in the striatum of mutant human torsinA mice, as compared with both normal human torsinA and NT littermates. Moreover, we found similar alterations of synaptic plasticity in muscarinic M2/M4 receptor knockout mice, in which an increased striatal acetylcholine level has been documented. The loss of LTD and SD on one hand, and the increase in LTP on the other, demonstrate that a 'loss of inhibition' characterizes the impairment of synaptic plasticity in this model of DYT1 dystonia. More importantly, our results indicate that an unbalanced cholinergic transmission plays a pivotal role in these alterations, providing a clue to understand the ability of anticholinergic agents to restore motor deficits in dystonia.

Background Enhanced supraspinal glutamate levels following nerve injury are associated with pathophysiological mechanisms responsible for neuropathic pain. Chronic pain can interfere with specific brain areas involved in glutamate-dependent neuropsychological processes, such as cognition, memory, and decision-making. The medial prefrontal cortex (mPFC) is thought to play a critical role in pain-related depression and anxiety, which are frequent co-morbidities of chronic pain. Using an animal model of spared nerve injury (SNI) of the sciatic nerve, we assess bio-molecular modifications in glutamatergic synapses in the mPFC that underlie neuropathic pain-induced plastic changes at 30 days post-surgery. Moreover, we examine the effects of palmitoylethanolamide (PEA) administration on pain-related behaviours, as well as the cortical biochemical and morphological changes that occur in SNI animals. Results At 1 month, SNI was associated with mechanical and thermal hypersensitivity, as well as depression-like behaviour, cognitive impairments, and obsessive-compulsive activities. Moreover, we observed an overall glutamate synapse modification in the mPFC, characterized by changes in synaptic density proteins and amino acid levels. Finally, with regard to the resolution of pain and depressive-like syndrome in SNI mice, PEA restored the glutamatergic synapse proteins and changes in amino acid release. Conclusions Given the potential role of the mPFC in pain mechanisms, our findings may provide novel insights into neuropathic pain forebrain processes and indicate PEA as a new pharmacological tool to treat neuropathic pain and the related negative affective states. Graphical Abstract Palmitoylethanolamide: a new pharmacological tool to treat neuropathic pain and the related negative affective states. Electronic supplementary material The online version of this article (doi:10.1186/s13041-015-0139-5) contains supplementary material, which is available to authorized users.

Free d- aspartate and d- serine occur at substantial levels in the mammalian brain. d- Serine is a physiological endogenous co-agonist for synaptic N-Methyl d -Aspartate (NMDA) receptors (NMDARs), and is involved in the pathophysiology of schizophrenia. Much less is known about the biological meaning of d- aspartate. d- Aspartate is present at high levels in the embryo brain and strongly decreases at post-natal phases. Temporal reduction of d- aspartate levels depends on the post-natal onset of d- aspartate oxidase (DDO), an enzyme able to selectively catabolize this d- amino acid. Pharmacological evidence indicates that d- aspartate binds to and activates NMDARs. Characterization of genetic and pharmacological mouse models with abnormally higher levels of d- aspartate has evidenced that increased d- aspartate enhances hippocampal NMDAR-dependent synaptic plasticity, dendritic morphology and spatial memory. In line with the hypothesis of a hypofunction of NMDARs in the pathogenesis of schizophrenia, it has been shown that increased d- aspartate levels also improve brain connectivity, produce corticostriatal adaptations resembling those observed after chronic haloperidol treatment, and protects against prepulse inhibition deficits and abnormal circuits activation induced by psychotomimetic drugs. In healthy humans, genetic variation predicting reduced expression of DDO in post-mortem prefrontal cortex is associated with greater prefrontal gray matter and activity during working memory. On the other side, evaluation of d- aspartate content in post-mortem patients with schizophrenia has shown a significant reduction of this d- amino acid in the prefrontal cortex and striatum. Generation of mouse models with reduced embryonic levels of d- aspartate may disclose unprecedented role for d- aspartate in developmental brain processes associated with vulnerability to psychotic-like symptoms.

Ras homolog enriched in striatum (Rhes) is highly expressed in striatal medium spiny neurons (MSNs) of rodents. In the present study, we characterized the expression of Rhes mRNA across species, as well as its functional role in other striatal neuron subtypes. Double in situ hybridization analysis showed that Rhes transcript is selectively localized in striatal cholinergic interneurons (ChIs), but not in GABAergic parvalbumin- or in neuropeptide Y-positive cell populations. Rhes is closely linked to dopamine-dependent signaling. Therefore, we recorded ChIs activity in basal condition and following dopamine receptor activation. Surprisingly, instead of an expected dopamine D2 receptor (D2R)-mediated inhibition, we observed an aberrant excitatory response in ChIs from Rhes knockout mice. Conversely, the effect of D1R agonist on ChIs was less robust in Rhes mutants than in controls. Although Rhes deletion in mutants occurs throughout the striatum, we demonstrate that the D2R response is altered specifically in ChIs, since it was recorded in pharmacological isolation, and prevented either by intrapipette BAPTA or by GDP-β-S. Moreover, we show that blockade of Cav2.2 calcium channels prevented the abnormal D2R response. Finally, we found that the abnormal D2R activation in ChIs was rescued by selective PI3K inhibition thus suggesting that Rhes functionally modulates PI3K/Akt signaling pathway in these neurons. Our findings reveal that, besides its expression in MSNs, Rhes is localized also in striatal ChIs and, most importantly, lack of this G-protein, significantly alters D2R modulation of striatal cholinergic excitability.

Vitamin A and its derivatives, retinoids, are involved in the regulation of gene expression by binding two nuclear receptor families, retinoic acid receptors and retinoid X receptors. Retinoid receptors are highly expressed in the striatum, revealing an involvement of this system in the control of movement as demonstrated by previous observations in knockout mice. To further assess the role of retinoids in adult striatal function, the present study investigated the effect of vitamin A deprivation on rat motor activity and coordination, the rate of synthesis and release of dopamine, the functioning of D1 and D2 receptors and their expression in the striatum. Moreover, the content of acetylcholine in the striatum was measured. Results show that 24 weeks of postnatal vitamin A deprivation induced severe locomotor deficits and impaired motor coordination. Vitamin A deprivation rats showed a significant hyperactivity following D1 receptor stimulation by R(+)-6-chloro-7,8-dihydroxy-1-phenyil-2,3,4,5-tetrahydro-1H-3-benzazepine or amphetamine and reduced catalepsy in response to haloperidol treatment. This different response to the above drugs is not due to a change in striatal DA release or synthesis between vitamin A deprivation and control animals. In situ hybridization experiments showed identical level of expression for the D1 and D2 receptor transcripts. On the other hand, the striatal tissue content of acetylcholine was reduced significantly by about 30% starting from the initial manifestation of motor deficits. We suggest that the locomotor impairment could be imputable to the dysfunction in striatal cholinergic interneurons. Our results stress the basic role of vitamin A in the maintenance of basal ganglia motor function in the adult rat brain. Vitamin A and its derivatives, retinoids, are involved in the regulation of gene expression by binding two nuclear receptor families, retinoic acid receptors and retinoid X receptors. Retinoid receptors are highly expressed in the striatum, revealing an involvement of this system in the control of movement as demonstrated by previous observations in knockout mice. To further assess the role of retinoids in adult striatal function, the present study investigated the effect of vitamin A deprivation on rat motor activity and coordination, the rate of synthesis and release of dopamine, the functioning of D1 and D2 receptors and their expression in the striatum. Moreover, the content of acetylcholine in the striatum was measured. Results show that 24 weeks of postnatal vitamin A deprivation induced severe locomotor deficits and impaired motor coordination. Vitamin A deprivation rats showed a significant hyperactivity following D1 receptor stimulation by R(+)-6-chloro-7,8-dihydroxy-1-phenyil-2,3,4,5-tetrahydro-1H-3-benzazepine or amphetamine and reduced catalepsy in response to haloperidol treatment. This different response to the above drugs is not due to a change in striatal DA release or synthesis between vitamin A deprivation and control animals. In situ hybridization experiments showed identical level of expression for the D1 and D2 receptor transcripts. On the other hand, the striatal tissue content of acetylcholine was reduced significantly by about 30% starting from the initial manifestation of motor deficits. We suggest that the locomotor impairment could be imputable to the dysfunction in striatal cholinergic interneurons. Our results stress the basic role of vitamin A in the maintenance of basal ganglia motor function in the adult rat brain. (c) 2006 Published by Elsevier Ltd on behalf of IBRO.

Reduced glutamate-mediated synaptic transmission has been implicated in the pathophysiology of schizophrenia. Because antipsychotic agents might exert their beneficial effects against schizophrenic symptoms by strengthening excitatory transmission in critical dopaminoceptive brain areas, in the present study we have studied the effects of acute and chronic haloperidol treatment on striatal synaptic plasticity. Repetitive stimulation of corticostriatal terminals in slices induced either long-term depression or long-term potentiation (LTP) of excitatory transmission in control rats, whereas it invariably induced NMDA receptor-dependent LTP in animals treated chronically with haloperidol. Haloperidol effects were mimicked and occluded in mice lacking both D2L and D2S isoforms of dopamine D2 receptors (D2R-/-), in mice lacking D2L receptors and expressing normal levels of D2S receptors (D2R-/-;D2L-/-), and in mice lacking D2L receptors and overexpressing D2S receptors (D2L-/-). These data indicate that the blockade of D2L receptors was responsible for the LTP-favoring action of haloperidol in the striatum. In contrast, overexpression of D2S receptors uncovered a facilitatory role of this receptor isoform in LTP formation because LTP recorded from D2L-/- mice, but not those recorded from wild-type, D2R-/-, and D2R-/-;D2L-/- mice, was insensitive to the pharmacological blockade of D1 receptors.The identification of the cellular, molecular, and receptor mechanisms involved in the action of haloperidol in the brain is essential to understand how antipsychotic agents exert their beneficial and side effects.

The typical neuroleptic haloperidol increases the state of phosphorylation and activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines. Here we show that the increases in TH phosphorylation produced by haloperidol at Ser31 and Ser40, two sites critically involved in the regulation of enzymatic activity, are abolished in dopamine D2 receptor-null mice and mimicked by the selective dopamine D2 receptor antagonist, eticlopride. Moreover, the ability of haloperidol and eticlopride to stimulate phosphorylation at both seryl residues is prevented by treatment with SL327, a compound that blocks activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). We also show that chronic administration of haloperidol reduces the basal levels of phosphoSer31-TH and decreases the ability of the drug to stimulate Ser40 phosphorylation. These results provide a model accounting for the stimulation exerted by haloperidol on dopamine synthesis. According to this model, haloperidol increases TH activity via blockade of dopamine D2 receptors, disinhibition of dopaminergic projection neurons and ERK1/2-dependent phosphorylation of TH at Ser31 and Ser40. These studies also show that lower levels of phosphorylated TH are associated with chronic neuroleptic treatment and may be related to depressed dopaminergic transmission in nigrostriatal neurons.

Compelling evidence indicates that the long (D2L) and the short (D2S) isoform of dopamine (DA) D2 receptors serve distinct physiological functions in vivo. To address the involvement of these isoforms in the control of synaptic transmission in the striatum, we measured the sensitivity to D2 receptor stimulation of glutamate- and GABA-mediated currents recorded from striatal neurons of three mutant mice, in which the expression of D2L and D2S receptors was either ablated or variably altered. Our data indicate that both isoforms participate in the presynaptic inhibition of GABA transmission in the striatum, while the D2-receptor-dependent modulation of glutamate release preferentially involves the D2S receptor. Accordingly, the inhibitory effects of the DA D2 receptor agonist quinpirole (10 microM) on GABA(A)-mediated spontaneous inhibitory postsynaptic currents (IPSCs)correlate with the total number of D2 receptor sites in the striatum, irrespective of the specific receptor isoform expressed. In contrast, glutamate-mediated spontaneous excitatory postsynaptic currents (EPSCs) were significantly inhibited by quinpirole only when the total number of D2 receptor sites, normally composed by both D2L and D2S receptors in a ratio favoring the D2L isoform, was modified to express only the D2S isoform at higher than normal levels. Understanding the physiological roles of DA D2 receptors in the striatum is essential for the treatment of several neuropsychiatric conditions, such as Parkinson's disease, Tourette's syndrome, schizophrenia, and drug addiction. Compelling evidence indicates that the long (D2L) and the short (D2S) isoform of dopamine (DA) D2 receptors serve distinct physiological functions in vivo. To address the involvement of these isoforms in the control of synaptic transmission in the striatum, we measured the sensitivity to D2 receptor stimulation of glutamate- and GABA-mediated currents recorded from striatal neurons of three mutant mice, in which the expression of D2L and D2S receptors was either ablated or variably altered. Our data indicate that both isoforms participate in the presynaptic inhibition of GABA transmission in the striatum, while the D2-receptor-dependent modulation of glutamate release preferentially involves the D2S receptor. Accordingly, the inhibitory effects of the DA D2 receptor agonist quinpirole (10 μM) on GABA A-mediated spontaneous inhibitory postsynaptic currents (IPSCs)correlate with the total number of D2 receptor sites in the striatum, irrespective of the specific receptor isoform expressed. In contrast, glutamate-mediated spontaneous excitatory postsynaptic currents (EPSCs) were significantly inhibited by quinpirole only when the total number of D2 receptor sites, normally composed by both D2L and D2S receptors in a ratio favoring the D2L isoform, was modified to express only the D2S isoform at higher than normal levels. Understanding the physiological roles of DA D2 receptors in the striatum is essential for the treatment of several neuropsychiatric conditions, such as Parkinson's disease, Tourette's syndrome, schizophrenia, and drug addiction. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

The endogenous cannabinoid system is involved in the regulation of the central reward pathway. Running wheel and sucrose consumption have rewarding and reinforcing properties in rodents, and share many neurochemical and behavioral characteristics with drug addiction. In this study, we investigated whether running wheel or sucrose consumption altered the sensitivity of striatal synapses to the activation of cannabinoid CB1 receptors. We found that cannabinoid CB1 receptor-mediated presynaptic control of striatal inhibitory postsynaptic currents was remarkably potentiated after these environmental manipulations. In contrast, the sensitivity of glutamate synapses to CB1 receptor stimulation was unaltered, as well as that of GABA synapses to the stimulation of presynaptic GABAB receptors. The sensitization of cannabinoid CB1 receptor-mediated responses was slowly reversible after the discontinuation of running wheel or sucrose consumption, and was also detectable following the mobilization of endocannabinoids by metabotropic glutamate receptor 5 stimulation. Finally, we found that the upregulation of cannabinoid transmission induced by wheel running or sucrose had a crucial role in the protective effects of these environmental manipulations against the motor and synaptic consequences of stress. The endogenous cannabinoid system is involved in the regulation of the central reward pathway. Running wheel and sucrose consumption have rewarding and reinforcing properties in rodents, and share many neurochemical and behavioral characteristics with drug addiction. In this study, we investigated whether running wheel or sucrose consumption altered the sensitivity of striatal synapses to the activation of cannabinoid CB1 receptors. We found that cannabinoid CB1 receptor-mediated presynaptic control of striatal inhibitory postsynaptic currents was remarkably potentiated after these environmental manipulations. In contrast, the sensitivity of glutamate synapses to CB1 receptor stimulation was unaltered, as well as that of GABA synapses to the stimulation of presynaptic GABAB receptors. The sensitization of cannabinoid CB1 receptor-mediated responses was slowly reversible after the discontinuation of running wheel or sucrose consumption, and was also detectable following the mobilization of endocannabinoids by metabotropic glutamate receptor 5 stimulation. Finally, we found that the upregulation of cannabinoid transmission induced by wheel running or sucrose had a crucial role in the protective effects of these environmental manipulations against the motor and synaptic consequences of stress. Neuropsychopharmacology (2010) 35, 374-387; doi: 10.1038/npp.2009.141; published online 23 September 2009

Interleukin-1β (IL-1β) is involved in mood alterations associated with inflammatory illnesses and with stress. The synaptic basis of IL-1β-induced emotional disturbances is still unknown. To address the possible involvement of the endocannabinoid system in IL-1β-induced anxiety, we performed behavioral and neurophysiological studies in mice exposed to stress or to intracerebroventricular injections of this inflammatory cytokine or of its antagonist. We found that a single intracerebroventricular injection of IL-1β caused anxiety in mice, and abrogated the sensitivity of cannabinoid CB1 receptors (CB1Rs) controlling GABA synapses in the striatum. Identical behavioral and synaptic results were obtained following social defeat stress, and intracerebroventricular injection of IL-1 receptor antagonist reverted both effects. IL-1β-mediated inhibition of CB1R function was secondary to altered cholesterol composition within membrane lipid rafts, and required intact function of the transient receptor potential vanilloid 1 (TRPV1) channel, another element of the endocannabinoid system. Membrane lipid raft disruption and inhibition of cholesterol synthesis, in fact, abrogated IL-1β–CB1R coupling, and TRPV1−/− mice were indeed insensitive to the synaptic and behavioral effects of both IL-1β and stress. On the other hand, cholesterol enrichment of striatal slices mimicked the synaptic effects of IL-1β on CB1Rs only in control mice, while the same treatment was ineffective in slices prepared from TRPV1−/− mice. The present investigation identifies a previously unrecognized interaction between a major proinflammatory cytokine and the endocannabinoid system in the pathophysiology of anxiety.

The role of brain-derived neurotrophic factor (BDNF) in emotional processes suggests an interaction with the endocannabinoid system. Here, we addressed the functional interplay between BDNF and cannabinoid CB(1) receptors (CB(1)Rs) in the striatum, a brain area in which both BDNF and CB(1)s play a role in the emotional consequences of stress and of rewarding experiences. BDNF potently inhibited CB(1)R function in the striatum, through a mechanism mediated by altered cholesterol metabolism and membrane lipid raft function. The effect of BDNF was restricted to CB(1)Rs controlling GABA-mediated IPSCs (CB(1)R((GABA))), whereas CB(1)Rs modulating glutamate transmission and GABA(B) receptors were not affected. The action of BDNF on CB(1)R((GABA)) function was tyrosine kinase dependent and was complete even after receptor sensitization with cocaine or environmental manipulations activating the dopamine (DA)-dependent reward system. In mice lacking one copy of the BDNF gene (BDNF(+/−)), CB(1)R((GABA)) responses were potentiated and were preserved from the action of haloperidol, a DA D(2) receptor (D(2)R) antagonist able to fully abolish CB(1)R((GABA)) function in rewarded animals. Haloperidol also enhanced BDNF levels in the striatum, suggesting that this neurotrophin may act as a downstream effector of D(2)Rs in the modulation of cannabinoid signaling. Accordingly, 5 d cocaine exposure both reduced striatal BDNF levels and increased CB(1)R((GABA)) activity, through a mechanism dependent on D(2)Rs. The present study identifies a novel mechanism of CB(1)R regulation mediated by BDNF and cholesterol metabolism and provides some evidence that DA D(2)R-dependent modulation of striatal CB(1)R activity is mediated by this neurotrophin.

We recently reported that the small G-protein Rhes has the properties of a SUMO-E3 ligase and mediates mutant huntingtin (mHtt) cytotoxicity. We now demonstrate that Rhes is a physiologic regulator of sumoylation, which is markedly reduced in the corpus striatum of Rhes-deleted mice. Sumoylation involves activation and transfer of small ubiquitin-like modifier (SUMO) from the thioester of E1 to the thioester of Ubc9 (E2) and final transfer to lysines on target proteins, which is enhanced by E3s. We show that E1 transfers SUMO from its thioester directly to lysine residues on Ubc9, forming isopeptide linkages. Conversely, sumoylation on E1 requires transfer of SUMO from the thioester of Ubc9. Thus, the process regarded as “autosumoylation” reflects intermolecular transfer between E1 and Ubc9, which we designate “cross-sumoylation.” Rhes binds directly to both E1 and Ubc9, enhancing cross-sumoylation as well as thioester transfer from E1 to Ubc9.

Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein,we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32kDa(DARPP32)at Thr75 residue, but preserved D2 receptor (D2R) function. However, although we demonstrated that striatal D1 receptor (D1R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinaseA(PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D1Rs since haloperidol, by blocking the tonic inhibition of D2R, unmasked a normal activation of striatal adenosine A2A receptor-mediated cAMP/PKA/DARPP32 cascade in mutants.Most importantly, our studies highlighted that amphetamine, nomifensine,andbupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation ofD1R/cAMP/PKA/DARPP32 signaling in response to increased striatal extracellular dopamine levels. Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32kDa (DARPP32) at Thr75 residue, but preserved D2 receptor (D2R) function. However, although we demonstrated that striatal D1 receptor (D 1R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8- dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D1Rs since haloperidol, by blocking the tonic inhibition of D2R, unmasked a normal activation of striatal adenosine A2A receptor-mediated cAMP/PKA/DARPP32cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these drugs in DAT-CI mutants depends on selective aberrant phasic activation of D1R/cAMP/PKA/ DARPP32 signaling in response to increased striatal extracellular dopamine levels. Copyright © 2010 the authors.

The D-amino acid, D-aspartate, is abundant in the developing brain, yet its function is unknown. Addition of d-aspartate to hippocampal or cortical slices prepared from 8- to 9-day-old rats stimulated polyphosphoinositide (PI) hydrolysis to a slightly greater extent than L-glutamate. The action of D-aspartate was concentration-dependent with an apparent EC(50) value of 1 mM and a maximal stimulation of 6- and 20-fold in cortical and hippocampal slices, respectively. Stimulation of PI hydrolysis by D-aspartate was largely reduced by pharmacological blockade of mGlu5 metabotropic glutamate receptors with 2-methyl-6-(phenylethynyl)pyridine. These findings suggest that D-aspartate behaves as an endogenous agonist of mGlu5 receptors during early postnatal life.

Since their discovery in the mammalian CNS,d-aspartate andd-serine have aroused a strong interest with regard to their role as putative neuromodulatory molecules. Whereas the functional role ofd-serine as an endogenous coagonist of NMDA receptors (NMDARs) has been elucidated, the biological significance ofd-aspartate in the brain is still mostly unclear. In the present study, we demonstrated that nonphysiological high levels ofd-aspartate (1) increasedin vivoNMDAR activity, (2) attenuated prepulse inhibition deficits induced by amphetamine and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate], (3) produced striatal adaptations of glutamate synapses resembling those observed after chronic haloperidol treatment, and (4) enhanced hippocampal NMDAR-dependent memory. This evidence was obtained using two different experimental strategies that produced an abnormal increase of endogenousd-aspartate levels in the mouse: a genetic approach based on the targeted deletion of thed-aspartate oxidase gene and a pharmacological approach based on oral administration ofd-aspartate. This work providesin vivoevidence of a neuromodulatory role exerted byd-aspartate on NMDAR signaling and raises the intriguing hypothesis that also thisd-amino acid, liked-serine, could be used as a therapeutic agent in the treatment of schizophrenia-related symptoms.

Caffeine, the psychoactive ingredient of coffee and of many soft drinks, is frequently abused by humans especially during stressful live events. The endocannabinoid system is involved in the central effects of many psychoactive compounds and of stress. Whether caffeine alters the cannabinoid system and interferes with stress-induced synaptic alterations is however unknown. We have studied electrophysiologically the sensitivity of cannabinoid receptors modulating synaptic transmission in the striatum of mice exposed to caffeine in their drinking solution. Chronic caffeine assumption sensitized GABAergic synapses to the presynaptic effect of cannabinoid CB1 receptor stimulation by exo- and endocannabinoids. Caffeine was conversely unable to affect the sensitivity of cannabinoid receptors modulating glutamate transmission. The synaptic effects of caffeine were slowly reversible after its removal from the drinking solution. Furthermore, although exposure to caffeine for only 24h did not produce measurable changes of the sensitivity of cannabinoid CB1 receptors, it was able to contrast the down-regulation of CB1 receptor-mediated responses after social defeat stress. Our data suggest that the cannabinoid system is implicated in the psychoactive properties of caffeine and in the ability of caffeine to reduce the pathological consequences of stress.

In the present study, we demonstrated that D-aspartate acts as an in vitro and in vivo neuromodulatory molecule upon hippocampal NMDAR transmission. Accordingly, we showed that this D-amino acid, widely expressed during embryonic phase, was able to strongly influence hippocampus-related functions at adulthood. Thus, while up-regulated levels of D-aspartate increased LTP and spatial memory in four-month old adult mice, the prolonged deregulation of this molecule in thirteen-month old animals induced a substantial acceleration of age-dependent decay of synaptic plasticity and cognitive functions. Moreover, we highlighted a role for D-aspartate in enhancing NMDAR-dependent synaptic plasticity through an inducible "turn-on/turn-off-like mechanism". Strikingly, we also showed that D-aspartate, when administered to aged mice, strongly rescued their physiological synaptic decay and attenuated their cognitive deterioration. In conclusion, our data suggest a tantalizing hypothesis for which this in-embryo-occurring D-amino acid, might disclose plasticity windows in the aging brain.

Exposure to stressful events has a myriad of consequences in animals and in humans, and triggers synaptic adaptations in many brain areas. Stress might also alter cannabinoid-receptor-mediated transmission in the brain, but no physiological study has addressed this issue so far. In the present study, we found that social defeat stress, induced in mice by exposure to aggression, altered cannabinoid CB1-receptor-mediated control of synaptic transmission in the striatum. In fact, the presynaptic inhibition of GABAergic IPSCs induced by the cannabinoid CB1receptor agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol] was reduced after a single stressful episode and fully abolished after 3 and 7 d of stress exposure. Repeated psychoemotional stress also impaired the sensitivity of GABA synapses to endocannabinoids mobilized by group I metabotropic glutamate receptor stimulation, whereas the cannabinoid CB1-mediated control of glutamate transmission was unaffected by repeated exposure to an aggressor. Corticosteroids released in response to the activation of the hypothalamic–pituitary–adrenal axis played a major role in the synaptic defects observed in stressed animals, because these alterations were fully prevented by pharmacological blockade of glucocorticoid receptors and were mimicked by corticosterone injections. The recovery of stress-induced synaptic defects was favored when stressed mice were given access to a running wheel or to sucrose consumption, which function as potent natural rewards. A similar rescuing effect was obtained by a single injection of cocaine, a psychostimulant with strong rewarding properties. Targeting cannabinoid CB1receptors or endocannabinoid metabolism might be a valuable option to treat stress-associated neuropsychiatric conditions.

Cocaine is a psychostimulant and a drug widely abused by humans. Cocaine elicits its effects primarily by blocking the activity of the dopamine (DA) transporter, leading to elevated levels of extracellular DA in areas receiving dopaminergic innervation, with the consequent activation of DA receptors. Cocaine, however, also elevates other neurotransmitter levels, leading to a general activation of interconnected brain circuitries. Studies aimed at unraveling the molecular mechanisms underlying the effects of cocaine have shown a leading role of DA D1 receptors in the cascade of cellular events elicited by this drug. In this study, we have analyzed the acute response to cocaine in animals deleted for the expression of DA D2 receptors (D2R), an inhibitor of DA signaling. Importantly, we show that although D1 receptor-mediated functions are preserved and even enhanced in D2R-/- mutants, the behavioral response to acute cocaine administration is severely altered. In addition, c-fos response to acute cocaine administration, in contrast to wild-type mice, is absent in D2R-/- mutants. Our findings show that the absence of D2R, very likely through a presynaptic mechanism, uncovers an inhibitory signaling pathway normally masked by the activity of this receptor on brain circuitries engaged by abused drugs. Cocaine is a psychostimulant and a drug widely abused by humans. Cocaine elicits its effects primarily by blocking the activity of the dopamine (DA) transporter, leading to elevated levels of extracellular DA in areas receiving dopaminergic innervation, with the consequent activation of DA receptors. Cocaine, however, also elevates other neurotransmitter levels, leading to a general activation of interconnected brain circuitries. Studies aimed at unraveling the molecular mechanisms underlying the effects of cocaine have shown a leading role of DA D1 receptors in the cascade of cellular events elicited by this drug. In this study, we have analyzed the acute response to cocaine in animals deleted for the expression of DA D2 receptors (D2R), an inhibitor of DA signaling. Importantly, we show that although D1 receptor-mediated functions are preserved and even enhanced in D2R-/- mutants, the behavioral response to acute cocaine administration is severely altered. In addition, c-fos response to acute cocaine administration, in contrast to wild-type mice, is absent in D2R-/- mutants. Our findings show that the absence of D2R, very likely through a presynaptic mechanism, uncovers an inhibitory signaling pathway normally masked by the activity of this receptor on brain circuitries engaged by abused drugs. © 2007 by The National Academy of Sciences of the USA.

Activation of the cAMP/PKA pathway in the dopaminoceptive neurons of the striatum has been proposed to mediate the actions of various classes of drugs of abuse. Here, we show that, in the mouse nucleus accumbens and dorsal striatum, acute administration of morphine resulted in an increase in the state of phosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at Thr34, without affecting phosphorylation at Thr75. The ability of morphine to stimulate Thr34 phosphorylation was prevented by blockade of dopamine D1 receptors. DARPP-32 knockout mice and T34A DARPP-32 mutant mice displayed a lower hyperlocomotor response to a single injection of morphine than wild-type controls. In contrast, in T75A DARPP-32 mutant mice, morphine-induced psychomotor activation was indistinguishable from that of wild-type littermates. In spite of their reduced response to the acute hyperlocomotor effect of morphine, DARPP-32 knockout mice and T34A DARPP-32 mutant mice were able to develop behavioral sensitization to morphine comparable to that of wild-type controls and to display morphine conditioned place preference. These results demonstrate that dopamine D1 receptor-mediated activation of the cAMP/DARPP-32 cascade in striatal medium spiny neurons is involved in the psychomotor action, but not in the rewarding properties, of morphine.

Background The advanced stages of addiction are characterized by compulsive drug-seeking and drug-taking behaviors despite the loss of the hedonic effect of drug consumption. A pathology of habit forming systems might underlie these features of addiction. Methods We have compared use-dependent plasticity of corticostriatal synapses in saline- and cocaine-treated rats by means of single neuron electrophysiological recordings. Results High-frequency stimulation of cortical afferents induced long-term potentiation (LTP) of corticostriatal synapses in treated and untreated animals. Saline- and acute–cocaine-treated rats, however, showed synaptic depotentiation in response to subsequent low-frequency stimulation of the same pathway, whereas chronic cocaine-treated animals were refractory to this process. Depotentiation was also absent in control slices bathed with cocaine, dopamine, or with the D1 receptor agonist SKF38393. The effect of cocaine on depotentiation was prevented by D1 but not D2 dopamine receptor antagonists and was mimicked by pharmacological inhibition of cyclin-dependent kinase 5, to enhance D1-receptor–associated intracellular signaling. Conclusions These results provide the first evidence that cocaine blocks the reversal of LTP in brain circuits. This alteration might be important for the persistence of addictive behavior despite efforts to abstain.

An increase of extracellular dopamine (DA) concentration is a major neurobiological substrate of the addictive properties of drugs of abuse. In this article we investigated the contribution of the DA D2 receptor (D2R) in the control of this response. Extracellular DA levels were measured in the striatum of mice lacking D2R expression (D2R-/-) by in vivo microdialysis after administration of the psychostimulant cocaine and the opioid morphine. Interestingly, the increase in extracellular DA induced by both drugs was strikingly higher in D2R-/- than in wild-type littermates. This indicates that D2Rs play a key role in the modulation of DA release in response to drugs of abuse. Furthermore, this observation prompted us to investigate the dopaminergic autoreceptor function in the absence of D2 receptor in D2R-/- mice. Results obtained using complementary microdialysis and voltammetry analyses show that the autoreceptor function regulating DA release is totally abolished in the absence of D2R, despite unchanged DA uptake and basal DA efflux. Finally, we propose that the short isoform D2S receptor of the D2 receptors is the one controlling change in DA release induced by drugs of abuse. Indeed, the neurochemical effects of cocaine and morphine are unchanged in animals with a selective deletion of the long isoform D2L receptor. Thus, deregulated expression of D2R isoforms might be involved in the vulnerability of an individual to drug abuse An increase of extracellular dopamine (DA) concentration is a major neurobiological substrate of the addictive properties of drugs of abuse. In this article we investigated the contribution of the DA D2 receptor (D2R) in the control of this response. Extracellular DA levels were measured in the striatum of mice lacking D2R expression (D2R-/-) by in vivo microdialysis after administration of the psychostimulant cocaine and the opioid morphine. Interestingly, the increase in extracellular DA induced by both drugs was strikingly higher in D2R-/- than in wildtype littermates. This indicates that D2Rs play a key role in the modulation of DA release in response to drugs of abuse. Furthermore, this observation prompted us to investigate the dopaminergic autoreceptor function in the absence of D2 receptor in D2R-/- mice. Results obtained using complementary microdialysis and voltammetry analyses show that the autoreceptor function regulating DA release is totally abolished in the absence of D2R, despite unchanged DA uptake and basal DA efflux. Finally, we propose that the short isoform D2S receptor of the D2 receptors is the one controlling change in DA release induced by drugs of abuse. Indeed, the neurochemical effects of cocaine and morphine are unchanged in animals with a selective deletion of the long isoform D2L receptor. Thus, deregulated expression of D2R isoforms might be involved in the vulnerability of an individual to drug abuse.

Two different isoforms of the dopamine D2 receptor exist in vivo, named D2L and D2S. These isoforms are derived by alternative splicing of the sixth exon of the gene (Dal Toso et al., 1989; Giros et al., 1989). D2L is the longer isoform composed of 444 amino acids (aa) in rat (Monsma et al., 1989; Chio et al., 1990) and mouse (Montmayeur et al., 1991) and 443 in human (Dal Toso et al., 1989; Giros et al., 1989; Grandy et al., 1989). D2S is instead composed of 415 aa in rat (Bunzow et al., 1988) and mouse (Montmayeur et al., 1991) and 414 in human (Dal Toso et al., 1989; Giros et al., 1989).

In this study we report on the effects of N-methyl-D-aspartate (NMDA)- and dopamine (DA)-receptor manipulation on the modulation of one-trial inhibitory avoidance response and the encoding of spatial information, as assessed with a non-associative task. Further, a comparison with the well-known effects of the manipulation of these two receptor systems on locomotor activity is outlined. It is well assessed that NMDA-receptor blockage induces a stimulatory action on locomotor activity similar to that exerted by DA agonists. There is evidence showing that the nucleus accumbens is involved in the response induced by both NMDA antagonists and DA agonists. We show results indicating a functional interaction between these two neural systems in modulating locomotor activity, with D2 DA-receptor antagonists (sulpiride and haloperidol) being more effective than the D1 antagonist (SCH 23390) in blocking MK-801-induced locomotion. A different profile is shown in the effects of NMDA antagonists and DA agonists in the modulation of memory processes. In one-trial inhibitory avoidance response, NMDA antagonists (MK-801 and CPP) impair the response on test day, while DA agonists exert a facilitatory effect; furthermore, sub-effective doses of both D1 (SKF 23390) and D2 (quinpirole) are able to attenuate the impairing effect in a way similar to that induced by NMDA antagonists. The effects of NMDA- and DA-acting drugs on the response to spatial novelty, as assessed with a task designed to study the ability of animals to react to discrete spatial changes, are in good accord with the effects observed on passive avoidance. The results show that NMDA as well as DA antagonists, at low doses, selectively impair the reactivity of mice to spatial changes. In a last series of experiments, the possible role of NMDA receptors located in the nucleus accumbens was investigated regarding reactivity to spatial novelty. The experiments gave apparently contrasting results: while showing an impairing effect of focal administrations of NMDA antagonists in the nucleus accumbens on reactivity to spatial novelty, no effect of ibotenic acid lesions of the same structure was observed. In this study we report on the effects of N-methyl-D-aspartate (NMDA)- and dopamine (DA)-receptor manipulation on the modulation of one-trial inhibitory avoidance response and the encoding of spatial information, as assessed with a non-associative task. Further, a comparison with the well-known effects of the manipulation of these two receptor systems on locomotor activity is outlined. It is well assessed that NMDA-receptor blockage induces a stimulatory action on locomotor activity similar to that exerted by DA agonists. There is evidence showing that the nucleus accumbens is involved in the response induced by both NMDA antagonists and DA agonists. We show results indicating a functional interaction between these two neural systems in modulating locomotor activity, with D2 DA-receptor antagonists (sulpiride and haloperidol) being more effective than the D1 antagonist (SCH 23390) in blocking MK-801-induced locomotion. A different profile is shown in the effects of NMDA antagonists and DA agonists in the modulation of memory processes. In one-trial inhibitory avoidance response, NMDA antagonists (MK-801 and CPP) impair the response on test day, while DA agonists exert a facilitatory effect; furthermore, sub-effective doses of both D1 (SKF 23390) and D2 (quinpirole) are able to attenuate the impairing effect in a way similar to that induced by NMDA antagonists. The effects of NMDA- and DA-acting drugs on the response to spatial novelty, as assessed with a task designed to study the ability of animals to react to discrete spatial changes, are in good accord with the effects observed on passive avoidance. The results show that NMDA as well as DA antagonists, at low doses, selectively impair the reactivity of mice to spatial changes. In a last series of experiments, the possible role of NMDA receptors located in the nucleus accumbens was investigated regarding reactivity to spatial novelty. The experiments Save apparently contrasting results: while showing an impairing effect of focal administrations of NMDA antagonists in the nucleus accumbens on reactivity to spatial novelty, no effect of ibotenic acid lesions of the same structure was observed.