Therapeutic potential of iron modulating drugs in a mouse model of multiple system atrophy.

Multiple System Atrophy (MSA) is a rare neurodegenerative synucleinopathy which leads to severe disability followed by death within 6-9 years of symptom onset. There is compelling evidence suggesting that biological trace metals like iron and copper play an important role in synucleinopathies like Parkinson's disease and removing excess brain iron using chelators could slow down the disease progression. In human MSA, there is evidence of increased iron in affected brain regions, but role of iron and therapeutic efficacy of iron-lowering drugs in pre-clinical models of MSA have not been studied. We studied age-related changes in iron metabolism in different brain regions of the PLP-αsyn mice and tested whether iron-lowering drugs could alleviate disease phenotype in aged PLP-αsyn mice. Iron content, iron-ferritin association, ferritin protein levels and copper-ceruloplasmin association were measured in prefrontal cortex, putamen, substantia nigra and cerebellum of 3, 8, and 20-month-old PLP-αsyn and age-matched non-transgenic mice. Moreover, 12-month-old PLP-αsyn mice were administered deferiprone or ceruloplasmin or vehicle for 2 months. At the end of treatment period, motor testing and stereological analyses were performed. We found iron accumulation and perturbed iron-ferritin interaction in substantia nigra, putamen and cerebellum of aged PLP-αsyn mice. Furthermore, we found significant reduction in ceruloplasmin-bound copper in substantia nigra and cerebellum of the PLP-αsyn mice. Both deferiprone and ceruloplasmin prevented decline in motor performance in aged PLP-αsyn mice and were associated with higher neuronal survival and reduced density of α-synuclein aggregates in substantia nigra. This is the first study to report brain iron accumulation in a mouse model of MSA. Our results indicate that elevated iron in MSA mice may result from ceruloplasmin dysfunction and provide evidence that targeting iron in MSA could be a viable therapeutic option.

Inactivation of posterior but not anterior dorsomedial caudate-putamen impedes learning with self-administered nicotine stimulus in male rats.

The rodent caudate-putamen is a large heterogeneous neural structure with distinct anatomical connections that differ in their control of learning processes. Previous research suggests that the anterior and posterior dorsomedial caudate-putamen (a- and p-dmCPu) differentially regulate associative learning with a non-contingent nicotine stimulus. The current study used bilateral NMDA-induced excitotoxic lesions to the a-dmCPu and p-dmCPu to determine the functional involvement of a-dmCPu and p-dmCPu in appetitive learning with contingent nicotine stimulus. Rats with a-dmCPu, p-dmCPu, or sham lesions were trained to lever-press for intravenous nicotine (0.03 mg/kg/inf) followed by access to sucrose 30 s later. After 1, 3, 9, and 20 nicotine-sucrose training sessions, appetitive learning in the form of a goal-tracking response was assessed using a non-contingent nicotine-alone test. All rats acquired nicotine self-administration and learned to retrieve sucrose from a receptacle at equal rates. However, rats with lesions to p-dmCPu demonstrated blunted learning of the nicotine-sucrose association. Our primary findings show that rats with lesions to p-dmCPu had a blunted goal-tracking response to a non-contingent nicotine administration after 20 consecutive days of nicotine-sucrose pairing. Our findings extend previous reports to a contingent model of nicotine self-administration and show that p-dmCPu is involved in associative learning with nicotine stimulus using a paradigm where rats voluntarily self-administer nicotine infusions that are paired with access to sucrose-a paradigm that closely resembles learning processes observed in humans.

Neuroinflammation and L-dopa-induced abnormal involuntary movements in 6-hydroxydopamine-lesioned rat model of Parkinson's disease are counteracted by combined administration of a 5-HT1A/1B receptor agonist and A2A receptor antagonist.

Several lines of evidence have strongly implicated neuroinflammation in Parkinson's disease (PD) progression and l-dopa-induced dyskinesia. The present study investigated whether early subchronic pretreatment with the serotonin 5-HT1A/1B receptor agonist eltoprazine plus the adenosine A2A receptor antagonist preladenant counteracted l-dopa-induced abnormal involuntary movements (AIMs, index of dyskinesia), and neuroinflammation, in unilateral 6-hydroxydopamine(6-OHDA)-lesioned rat model of PD. The immunoreactivity of glial fibrillary acidic protein (GFAP), and the colocalization of ionized calcium binding adaptor molecule-1 (IBA-1), with interleukin (IL)-1ß, tumor-necrosis-factor-α (TNF-α) and IL-10 were evaluated in the denervated caudate-putamen (CPu) and substantia nigra pars-compacta (SNc). The combined subchronic pretreatment with l-dopa plus eltoprazine and preladenant reduced AIMs induced by acute l-dopa challenge in these rats and decreased GFAP and IBA-1 immunoreactivity induced by the drug in both CPu and SNc, with reduction in IL-1ß in IBA-1-positive cells in both CPu and SNc, and in TNF-α in IBA-1-positive cells in SNc. Moreover, a significant increase in IL-10 in IBA-1-positive cells was observed in SNc. Evaluation of immediate early-gene zif-268 (index of neuronal activation) after l-dopa challenge, showed an increase in its expression in denervated CPu of rats pretreated with l-dopa or l-dopa plus preladenant compared with vehicle, whereas rats pretreated with eltoprazine, with or without preladenant, had lower zif-268 expression. Finally, tyrosine hydroxylase and dopamine transporter examined to evaluate neurodegeneration, showed a significant equal decrease in all experimental groups. The present findings suggest that combination of l-dopa with eltoprazine and preladenant may be promising therapeutic strategy for delaying the onset of dyskinesia, preserving l-dopa efficacy and reducing neuroinflammation markers in nigrostriatal system of 6-OHDA-lesioned rats.

LB100 attenuates methamphetamine-induced behavioral sensitization by inhibiting the Raf1-ERK 1/2 cascade in the caudate putamen.

Methamphetamine (METH) abuse has become a serious social problem. Behavioral sensitization is a common behavioral paradigm used to study the neurobiological mechanism that underlies drug addiction. Our previous study demonstrated that the activity of protein phosphatase 2A (PP2A) and the level of phosphorylated extracellular signal-related kinase 1/2 (p-ERK 1/2) are increased in the caudate putamen (CPu) of METH-sensitive mice. However, the relationship between PP2A and ERK 1/2 in METH-induced behavioral sensitization remains unknown. Some studies have indicated that Raf1 may be involved in this process. In this study, LB100, a PP2A inhibitor for treating solid tumors, was first used to clarify the relationship between PP2A and ERK 1/2. In addition, Western blot was used to examine the levels of p-Raf1 (Ser 259) and p-ERK 1/2 (Thr 202/Tyr 204) in the CPu, hippocampus (Hip) and nucleus accumbens (NAc). Our results showed that 2 mg/kg LB100 significantly attenuated METH-induced behavioral sensitization. Furthermore, Western blot analysis revealed that pretreatment with 2 mg/kg LB100 remarkably reversed METH-induced reduction of p-Raf1, as well as upregulation of p-ERK 1/2 in the CPu. Taken together, these results indicate that PP2A plays an important role in METH-induced behavioral sensitization and phosphorylates ERK 1/2 by dephosphorylating p-Raf1 in the CPu to further regulate METH-induced behavioral sensitization.

Perinatal activation of ERα and ERß but not GPER-1 masculinizes female rat caudate-putamen medium spiny neuron electrophysiological properties.

Exposure to steroid sex hormones such as 17ß-estradiol (estradiol) during early life potentially permanently masculinize neuron electrophysiological phenotype. In rodents, one crucial component of this developmental process occurs in males, with estradiol aromatized in the brain from testes-sourced testosterone. However, it is unknown whether most neuron electrophysiological phenotypes are altered by this early masculinization process, including medium spiny neurons (MSNs) of the rat caudate-putamen. MSNs are the predominant and primary output neurons of the caudate-putamen and exhibit increased intrinsic excitability in females compared to males. Here, we hypothesize that since perinatal estradiol exposure occurs in males, then a comparable exposure in females to estradiol or its receptor agonists would be sufficient to induce masculinization. To test this hypothesis, we injected perinatal female rats with estradiol or its receptor agonists and then later assessed MSN electrophysiology. Female and male rats on postnatal day 0 and 1 were systemically injected with either vehicle, estradiol, the estrogen receptor (ER)α agonist PPT, the ERß agonist DPN, or the G-protein-coupled receptor 1 (GPER-1) agonist G1. On postnatal days 19 ± 2, MSN electrophysiological properties were assessed using whole cell patch clamp recordings. Estradiol exposure abolished increased intrinsic excitability in female compared to male MSNs. Exposure to either an ERα or ERß agonist masculinized female MSN evoked action potential firing rate properties, whereas exposure to an ERß agonist masculinized female MSN inward rectification properties. Exposure to ER agonists minimally impacted male MSN electrophysiological properties. These findings indicate that perinatal estradiol exposure masculinizes MSN electrophysiological phenotype via activation of ERα and ERß.NEW & NOTEWORTHY This study is the first to demonstrate that estradiol and estrogen receptor α and ß stimulation during early development sexually differentiates the electrophysiological properties of caudate-putamen medium spiny neurons, the primary output neuron of the striatal regions. Overall, this evidence provides new insight into the neuroendocrine mechanism by which caudate-putamen neuron electrophysiology is sexually differentiated and demonstrates the powerful action of early hormone exposure upon individual neuron electrophysiology.

Altered mRNA Levels of Stress-Related Peptides in Mouse Hippocampus and Caudate-Putamen in Withdrawal after Long-Term Intermittent Exposure to Tobacco Smoke or Electronic Cigarette Vapour.

Nicotine addiction is a severe public health problem. The aim of this study was to investigate the alterations in key neurotransmissions after 60 days of withdrawal from seven weeks of intermittent cigarette smoke, e-cigarette vapours, or an e-cigarette vehicle. In the nicotine withdrawal groups, increased depressive and anxiety/obsessive-compulsive-like behaviours were demonstrated in the tail suspension, sucrose preference and marble burying tests. Cognitive impairments were detected in the spatial object recognition test. A significant increase in Corticotropin-releasing factor (Crf) and Crf1 mRNA levels was observed, specifically after cigarette withdrawal in the caudate-putamen nucleus (CPu). The nociceptin precursor levels were reduced by cigarette (80%) and e-cigarette (50%) withdrawal in the CPu. The delta opioid receptor showed a significant reduction in the hippocampus driven by the exposure to an e-cigarette solubilisation vehicle, while the mRNA levels doubled in the CPu of mice that had been exposed to e-cigarettes. Withdrawal after exposure to e-cigarette vapour induced a 35% Bdnf mRNA decrease in the hippocampus, whereas Bdnf was augmented by 118% by cigarette withdrawal in the CPu. This study shows that long-term withdrawal-induced affective and cognitive symptoms associated to lasting molecular alterations in peptidergic signalling may determine the impaired neuroplasticity in the hippocampal and striatal circuitry.

Estradiol rapidly modulates excitatory synapse properties in a sex- and region-specific manner in rat nucleus accumbens core and caudate-putamen.

Estradiol acutely facilitates sex differences in striatum-dependent behaviors. However, little is understood regarding the underlying mechanism. In striatal regions in adult rodents, estrogen receptors feature exclusively extranuclear expression, suggesting that estradiol rapidly modulates striatal neurons. We tested the hypothesis that estradiol rapidly modulates excitatory synapse properties onto medium spiny neurons (MSNs) of two striatal regions, the nucleus accumbens core and caudate-putamen in adult female and male rats. We predicted there would be sex-specific differences in pre- and postsynaptic locus and sensitivity. We further analyzed whether MSN intrinsic properties are predictive of estrogen sensitivity. Estradiol exhibited sex-specific acute effects in the nucleus accumbens core: miniature excitatory postsynaptic current (mEPSC) frequency robustly decreased in response to estradiol in female MSNs, and mEPSC amplitude moderately increased in response to estradiol in both male and female MSNs. This increase in mEPSC amplitude is associated with MSNs featuring increased intrinsic excitability. No MSN intrinsic electrical property associated with changes in mEPSC frequency. Estradiol did not acutely modulate mEPSC properties in the caudate-putamen of either sex. This is the first demonstration of acute estradiol action on MSN excitatory synapse function. This demonstration of sex and striatal region-specific acute estradiol neuromodulation revises our understanding of sex hormone action on striatal physiology and resulting behaviors.NEW & NOTEWORTHY This study is the first to demonstrate rapid estradiol neuromodulation of glutamatergic signaling on medium spiny neurons (MSNs), the major output neuron of the striatum. These findings emphasize that sex is a significant biological variable both in MSN sensitivity to estradiol and in pre- and postsynaptic mechanisms of glutamatergic signaling. MSNs in different regions exhibit diverse responses to estradiol. Sex- and region-specific estradiol-induced changes to excitatory signaling on MSNs explain sex differences partially underlying striatum-mediated behaviors and diseases.

Trial of magnetic resonance-guided putaminal gene therapy for advanced Parkinson's disease.

OBJECTIVE: To investigate the safety and tolerability of convection-enhanced delivery of an adeno-associated virus, serotype-2 vector carrying glial cell line-derived neurotrophic factor into the bilateral putamina of PD patients. METHODS: Thirteen adult patients with advanced PD underwent adeno-associated virus, serotype-2 vector carrying glial cell line-derived neurotrophic factor and gadoteridol (surrogate MRI tracer) coinfusion (450 µL/hemisphere) at escalating doses: 9 × 1010 vg (n = 6); 3 × 1011 vg (n = 6); and 9 × 1011 vg (n = 1). Intraoperative MRI monitored infusion distribution. Patients underwent UPDRS assessment and [18 F]FDOPA-PET scanning preoperatively and 6 and 18 months postoperatively. RESULTS: Adeno-associated virus, serotype-2 vector carrying glial cell line-derived neurotrophic factor was tolerated without clinical or radiographic toxicity. Average putaminal coverage was 26%. UPDRS scores remained stable. Ten of thirteen and 12 of 13 patients had increased [18 F]FDOPA Kis at 6 and 18 months postinfusion (increase range: 5-274% and 8-130%; median, 36% and 54%), respectively. Ki differences between baseline and 6- and 18-month follow-up were statistically significant (P < 0.0002). CONCLUSION: Adeno-associated virus, serotype-2 vector carrying glial cell line-derived neurotrophic factor infusion was safe and well tolerated. Increased [18 F]FDOPA uptake suggests a neurotrophic effect on dopaminergic neurons. © 2019 International Parkinson and Movement Disorder Society.

Tardive dyskinesia is associated with altered putamen Akt/GSK-3ß signaling in nonhuman primates.

BACKGROUND: Tardive dyskinesia is a delayed and potentially irreversible motor complication arising from chronic exposure to antipsychotic drugs. Interaction of antipsychotic drugs with G protein-coupled receptors triggers multiple intracellular events. Nevertheless, signaling pathways that might be associated with chronic unwanted effects of antipsychotic drugs remain elusive. In this study, we aimed to better understand kinase signaling associated with the expression of tardive dyskinesia in nonhuman primates. METHODS: We exposed capuchin monkeys to prolonged haloperidol (n = 10) or clozapine (n = 6) treatments. Untreated animals were used as controls (n = 6). Half of haloperidol-treated animals (5) developed mild tardive dyskinesia similar to that found in humans. Using Western blots and immunochemistry, we measured putamen total and phosphorylated protein kinase levels associated with canonical and noncanonical signaling cascades of G protein-coupled receptors. RESULTS: Antipsychotic drugs enhanced pDARPP-32 and pERK1/2, but no difference ws observed in phosphoprotein kinase levels between dyskinetic and nondyskinetic monkeys. On the other hand, comparison of kinase levels between haloperidol-treated dyskinetic and nondyskinetic monkeys indicated that dyskinetic animals had lower GRK6 and ß-arrestin2 levels. Levels of pAkt and pGSK-3ß were also reduced, but only haloperidol-treated monkeys that developed tardive dyskinesia had reduced pGSK-3ß levels, whereas pAkt levels in dyskinetic animals positively correlated with dyskinetic scores. Interestingly, double immunofluorescence labeling showed that putamen dopamine D3 receptor levels were upregulated and that D3/pAkt colocalization was enriched in haloperidol-treated animals displaying tardive dyskinesia. CONCLUSIONS: Our results suggest that upregulation of putamen dopamine D3 receptor and alterations along the noncanonical GRK6/ß-arrestin2/Akt/GSK-3ß molecular cascade are associated with the development of tardive dyskinesia in nonhuman primates. © 2019 International Parkinson and Movement Disorder Society.

Double dissociation of the anterior and posterior dorsomedial caudate-putamen in the acquisition and expression of associative learning with the nicotine stimulus.

Tobacco use is the leading cause of preventable deaths worldwide. This habit is not only debilitating to individual users but also to those around them (second-hand smoking). Nicotine is the main addictive component of tobacco products and is a moderate stimulant and a mild reinforcer. Importantly, besides its unconditional effects, nicotine also has conditioned stimulus effects that may contribute to the tenacity of the smoking habit. Because the neurobiological substrates underlying these processes are virtually unexplored, the present study investigated the functional involvement of the dorsomedial caudate putamen (dmCPu) in learning processes with nicotine as an interoceptive stimulus. Rats were trained using the discriminated goal-tracking task where nicotine injections (0.4 mg/kg; SC), on some days, were paired with intermittent (36 per session) sucrose deliveries; sucrose was not available on interspersed saline days. Pre-training excitotoxic or post-training transient lesions of anterior or posterior dmCPu were used to elucidate the role of these areas in acquisition or expression of associative learning with nicotine stimulus. Pre-training lesion of p-dmCPu inhibited acquisition while post-training lesions of p-dmCPu attenuated the expression of associative learning with the nicotine stimulus. On the other hand, post-training lesions of a-dmCPu evoked nicotine-like responding following saline treatment indicating the role of this area in disinhibition of learned motor behaviors. These results, for the first time, show functionally distinct involvement of a- and p-dmCPu in various stages of associative learning using nicotine stimulus and provide an initial account of neural plasticity underlying these learning processes.

Transient Adenosine Release Is Modulated by NMDA and GABAB Receptors.

Adenosine is a neuroprotective agent that modulates neurotransmission and is modulated by other neurotransmitters. Spontaneous, transient adenosine is a recently discovered mode of signaling where adenosine is released and cleared from the extracellular space quickly, in less than three seconds. Spontaneous adenosine release is regulated by adenosine A1 and A2a receptors, but regulation by other neurotransmitter receptors has not been studied. Here, we examined the effect of glutamate and GABA receptors on the concentration and frequency of spontaneous, transient adenosine release by measuring adenosine with fast-scan cyclic voltammetry in the rat caudate-putamen. The glutamate NMDA antagonist, 3-(R-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 6.25 mg/kg i.p.), increased the frequency of adenosine transients and the concentration of individual transients, but NMDA (agonist, 50 mg/kg, i.p.) did not change the frequency. In contrast, antagonists of other glutamate receptors had no effect on the frequency or concentration of transient adenosine release, including the AMPA antagonist NBQX (15 mg/kg i.p.) and the mGlu2/3 glutamate receptor antagonist LY 341495 (5 mg/kg i.p.). The GABAB antagonist CGP 52432 (30 mg/kg i.p.) significantly decreased the number of adenosine release events while the GABAB agonist baclofen (4 mg/kg i.p.) increased the frequency of adenosine release. The GABAA antagonist bicuculline (5 mg/kg i.p.) had no significant effects on adenosine. NMDA and GABAB likely act presynaptically, affecting the overall cell excitability for vesicular release. The ability to regulate adenosine with NMDA and GABAB receptors will help control the modulatory effects of transient adenosine release.

Comparison of striatal dopamine transporter levels in chronic heroin-dependent and methamphetamine-dependent subjects.

To compare the effects of heroin and methamphetamine (METH) addiction on dopamine transporters (DATs) in the same dose and duration, we assessed DAT levels in the striatum by 99m Tc-TRODAT-1 single-photon emission computed tomography (SPECT) brain images in people with heroin and METH dependence. We recruited 21 healthy human controls, 23 heroin-dependent subjects and 25 METH abusers. The heroin- and METH-dependent subjects exhibited negative urine toxicology after undergoing physiological detoxification. All subjects underwent SPECT brain imaging, and specific tracer uptake ratios (SURs) were assessed bilaterally in the regions of interest. A significant SUR reduction in heroin-dependent subjects and METH-dependent subjects compared with healthy controls was found in the left striatum, right striatum, left caudate nucleus, right caudate nucleus, left putamen and right putamen. There were no significant differences in the heroin group and METH group for the left striatum, right striatum, left caudate nucleus, right caudate nucleus, left putamen and right putamen. The scores of craving, HAMA (Hamilton Anxiety Rating Scale), in heroin abusers were lower than in the METH abusers. Our results show that people with heroin and METH dependence who are currently abstinent had lower DAT levels in the striatum than healthy controls. There were no differences in striatal DAT in heroin and METH users. These results suggest that chronic heroin and METH abuse appears to produce similar effects in striatal DAT in humans. METH users may have more serious craving and anxiety symptoms than heroin users with prolonged abstinence.

Sex differences in behavioral and PKA cascade responses to repeated cocaine administration.

Previous studies have shown sex different patterns in behavioral responses to cocaine. Here, we used between-subject experiment design to study whether sex differences exist in the development of behavioral sensitization and tolerance to repeated cocaine, as well as the role of protein kinase A (PKA) signaling cascade in this process. Ambulatory and rearing responses were recorded in male and female rats after 1 to 14 days of administration of saline or cocaine (15 mg/kg; ip). Correspondent PKA-associated signaling in the nucleus accumbens (NAc) and caudate-putamen (CPu) was measured at each time point. Our results showed that females exhibited higher cocaine-induced behavioral responses and developed behavioral sensitization and tolerance faster than males. Whereas females developed behavioral sensitization to cocaine after 2 days and tolerance after 14 days, male rats developed sensitization after 5 days. In addition, cocaine induced a sexual dimorphic pattern in the progression of neuronal adaptations on the PKA cascade signaling in region (NAc vs. CPu) and time (days of cocaine administration)-dependent manners. In general, more PKA signaling cascade changes were found in the NAc of males on day 5 and in the CPu of females with repeated cocaine injection. In addition, in females, behavioral activities positively correlated with FosB levels in the NAc and CPu and negatively correlated with Cdk5 and p35 in the CPu, while no correlation was observed in males. Our studies suggest that repeated cocaine administration induced different patterns of behavioral and molecular responses in the PKA cascade in male and female rats.

AUTS2 in the nucleus accumbens is essential for heroin-induced behavioral sensitization.

Autism susceptibility candidate 2 (AUTS2) is a gene associated with autism and mental retardation. Recent studies have suggested an association of the AUTS2 gene with heroin dependence, and reduced AUTS2 gene expression may confer increased susceptibility to heroin dependence. However, the functional role of the AUTS2 protein in regulating enduring neuroadaptations in response to heroin exposure has not been established. Here, we investigated the effects of acute and chronic heroin exposure on AUTS2 mRNA and protein expression in the nucleus accumbens (NAc) and caudate-putamen (CPu) to determine whether changes in AUTS2 expression are associated with heroin-induced locomotor sensitization in mice. Moreover, we explored whether AUST2 knockdown affects heroin-induced locomotor sensitization. AUTS2 mRNA and protein expression in the NAc, but not the CPu, was decreased after chronic heroin (1mg/kg) administration. In the NAc, the expression of heroin-induced locomotor sensitization was enhanced through the lentiviral-AUTS2-shRNA-mediated knockdown of AUTS2, while the overexpression of AUTS2 attenuated the locomotor-stimulant effects of heroin. Together, these results indicate that AUTS2 in the NAc, but not the CPu, suppresses the initiation and expression of heroin-induced behavioral sensitization, suggesting that AUST2 may be a potential target for the treatment of heroin dependence.

Ifenprodil Attenuates Methamphetamine-Induced Behavioral Sensitization and Activation of Ras-ERK-∆FosB Pathway in the Caudate Putamen.

Addiction is a debilitating, chronic psychiatric disorder that is difficult to cure completely owing to the high rate of relapse. Behavioral sensitization is considered to may underlie behavioral changes, such as relapse, caused by chronic abuse of psychomotor stimulants. Thus, its animal models have been widely used to explore the etiology of addiction. Recently, increasing evidence has demonstrated that N-methyl-D-aspartate receptors (NMDARs) play an important role in addiction to psychomotor stimulants. However, the role of GluN2B-containing receptors and their downstream signaling pathway(s) in behavioral sensitization induced by methamphetamine (METH) have not been investigated yet. In this study, we used different doses of ifenprodil (2.5, 5, 10 mg/kg), a selective antagonist of the GluN2B subunit, to investigate the role of GluN2B-containing NMDARs in METH-induced behavioral sensitization. We then examined changes in the levels of Ras, phosphorylated extracellular signal-regulated kinase (pERK)/ERK, and ∆FosB in the caudate putamen (CPu) by western blot. We found that 2.5 or 10 mg/kg ifenprodil significantly attenuated METH-induced behavioral sensitization, whereas the mice treated with a moderate dose of ifenprodil (5 mg/kg) displayed no significant changes. Further results of western blot experiments showed that repeated administration of METH caused the increases in the levels of Ras, pERK/ERK and ∆FosB in the CPu, and these changes were inhibited by only the 2.5 mg/kg dose of ifenprodil. In conclusion, these results demonstrated that 2.5 mg/kg ifenprodil could attenuate METH-induced behavioral sensitization. Moreover, GluN2B-containing NMDARs and their downstream Ras-ERK-∆FosB signaling pathway in the CPu might be involved in METH-induced behavioral sensitization.

N-Acetyl Cysteine May Support Dopamine Neurons in Parkinson's Disease: Preliminary Clinical and Cell Line Data.

BACKGOUND: The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson's disease (PD). METHODS: The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson's Disease Rating Scale (UPDRS) to measure clinical symptoms. RESULTS: The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p<0.05 for all values) in the PD group treated with NAC, and no measurable changes in the control group. UPDRS scores were also significantly improved in the NAC group (mean improvement of 12.9%, p = 0.01). CONCLUSIONS: The results of this preliminary study demonstrate for the first time a potential direct effect of NAC on the dopamine system in PD patients, and this observation may be associated with positive clinical effects. A large-scale clinical trial to test the therapeutic efficacy of NAC in this population and to better elucidate the mechanism of action is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT02445651.

Ovarian steroids alter dopamine receptor populations in the medial preoptic area of female rats: implications for sexual motivation, desire, and behaviour.

Dopamine (DA) transmission in the medial preoptic area (mPOA) plays a critical role in the control of appetitive sexual behaviour in the female rat. We have shown previously that a DA D1 receptor (D1R)-mediated excitatory state appears to occur in females primed with estradiol benzoate (EB) and progesterone (P), whereas a DA D2 receptor (D2R)-mediated inhibitory state appears to occur in females primed only with EB. The present experiment employed three techniques to better understand what changes occur to DA receptors (DARs) in the mPOA under different hormonal profiles. Ovariectomized females were randomly assigned to one of three steroid treatment groups: EB + P (10 and 500 µg, respectively), EB + Oil, or the control (Oil + Oil), with hormone injections administered at 48 and 4 h prior to euthanizing. First, the number of neurons in the mPOA that contained D1R or D2R was assessed using immunohistochemistry. Second, the mPOA and two control areas (the prelimbic cortex and caudate putamen) were analysed for DAR protein levels using western blot, and DAR functional binding levels using autoradiography. Ovarian steroid hormones affected the two DAR subtypes in opposite ways in the mPOA. All three techniques supported previous behavioural findings that females primed with EB have a lower D1R : D2R ratio, and thus a D2R-mediated system, and females primed with EB + P have a higher D1R : D2R ratio, and thus a D1R-mediated system. This provides strong evidence for a DA-driven pathway of female sexual motivation, desire, and behaviour that is modified by different hormone priming regimens.

Striatal NOS1 has dimorphic expression and activity under stress and nicotine sensitization.

Nicotine exerts its addictive influence through the meso-cortico-limbic reward system, where the striatum is essential. Nicotine addiction involves different neurotransmitters, nitric oxide (NO) being especially important, since it triggers the release of the others by positive feedback. In the nervous system, NO is mainly produced by nitric oxide synthase 1 (NOS1). However, other subtypes of synthases can also synthesize NO, and little is known about the specific role of each isoform in the process of addiction. In parallel, NOS activity and nicotine addiction are also affected by stress and sexual dimorphism. To determine the specific role of this enzyme, we analyzed both NOS expression and NO synthesis in the striatum of wild-type and NOS1-knocked out (KO) mice of both sexes in situations of nicotine sensitization and stress. Our results demonstrated differences between the caudate-putamen (CP) and nucleus accumbens (NA). With respect to NOS1 expression, the CP is a dimorphic region (27.5% lower cell density in males), but with a stable production of NO, exclusively due to this isoform. Thus, the nitrergic system of CP may not be involved in stress or nicotine addiction. Conversely, the NA is much more variable and strongly involved in both situations: its NO synthesis displays dimorphic variations at both basal (68.5% reduction in females) and stress levels (65.9% reduction in males), which disappear when nicotine is infused. Thus, the KO animals showed an increase in NO production (21.7%) in the NA, probably by NOS3, in an attempt to compensate the lack of NOS1.

Role of Dopamine Type 1 Receptors and Dopamine- and cAMP-Regulated Phosphoprotein Mr 32 kDa in Δ9-Tetrahydrocannabinol-Mediated Induction of ΔFosB in the Mouse Forebrain.

Δ(9)-Tetrahydrocannabinol (THC), the main psychoactive component of marijuana, produces motor and motivational effects via interactions with the dopaminergic system in the caudate-putamen and nucleus accumbens. However, the molecular events that underlie these interactions after THC treatment are not well understood. Our study shows that pretreatment with dopamine D1 receptor (D1R) antagonists before repeated administration of THC attenuated induction of Δ FBJ murine osteosarcoma viral oncogene homolog B (ΔFosB) in the nucleus accumbens, caudate-putamen, amygdala, and prefrontal cortex. Anatomical studies showed that repeated THC administration induced ΔFosB in D1R-containing striatal neurons. Dopamine signaling in the striatum involves phosphorylation-specific effects of the dopamine- and cAMP-regulated phosphoprotein Mr 32 kDa (DARPP-32), which regulates protein kinase A signaling. Genetic deletion of DARPP-32 attenuated ΔFosB expression measured after acute, but not repeated, THC administration in both the caudate-putamen and nucleus accumbens. THC was then acutely or repeatedly administered to wild-type (WT) and DARPP-32 knockout (KO) mice, and in vivo responses were measured. DARPP-32 KO mice exhibited enhanced acute THC-mediated hypolocomotion and developed greater tolerance to this response relative to the WT mice. Agonist-stimulated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding showed that cannabinoid-stimulated G-protein activity did not differ between DARPP-32 KO and WT mice treated with vehicle or repeated THC. These results indicate that D1Rs play a major role in THC-mediated ΔFosB induction in the forebrain, whereas the role of DARPP-32 in THC-mediated ΔFosB induction and modulation of motor activity appears to be more complex.

Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain.

Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [(11)C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300 mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). The association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors.