The Journal of Neuroscience : The Official Journal of the Society for Neuroscience

Published by Society for Neuroscience
Online ISSN: 1529-2401
Print ISSN: 0270-6474
Here, we map a quantitative trait locus (QTL) with a large effect on predisposition to barbiturate (pentobarbital) withdrawal to a 0.44 Mb interval of mouse chromosome 1 syntenic with human 1q23.2. We report a detailed analysis of the genes within this interval and show that it contains 15 known and predicted genes, 12 of which demonstrate validated genotype-dependent transcript expression and/or nonsynonymous coding sequence variation that may underlie the influence of the QTL on withdrawal. These candidates are involved in diverse cellular functions including intracellular trafficking, potassium conductance and spatial buffering, and multimolecular complex dynamics, and indicate both established and novel aspects of neurobiological response to sedative-hypnotics. This work represents a substantial advancement toward identification of the gene(s) that underlie the phenotypic effects of the QTL. We identify Kcnj9 as a particularly promising candidate and report the development of a Kcnj9-null mutant model that exhibits significantly less severe withdrawal from pentobarbital as well as other sedative-hypnotics (zolpidem and ethanol) versus wild-type littermates. Reduced expression of Kcnj9, which encodes GIRK3 (Kir3.3), is associated with less severe sedative-hypnotic withdrawal. A multitude of QTLs for a variety of complex traits, including diverse responses to sedative-hypnotics, have been detected on distal chromosome 1 in mice, and as many as four QTLs on human chromosome 1q have been implicated in human studies of alcohol dependence. Thus, our results will be primary to additional efforts to identify genes involved in a wide variety of behavioral responses to sedative-hypnotics and may directly facilitate progress in human genetics.
Static visual cue experiments. A , Experimental design. Subjects started the trial with their hand at the central location (green circle) while both the cue location (gray circle) and final target (yellow circle) were visually presented. Movement initiation was either signified by an acoustic beep (static visual cue condition) or the cue color changing from gray to white (static visual cue attention experiment). The combination of cue (e.g., C3) and target (e.g., T1) uniquely specified the CW force field (Fig. 1 B ), applied once the subjects initiated the movement to the final target. B , For the same final target (T1), if cue C2 was visually presented to the subjects, this uniquely specified the CCW force field, applied once the movement was initiated. C , MPE plotted against block number. The mean across all subjects (solid line) and SE across subjects (shaded region) for each block in both the static visual cue (blue) and static visual cue attention (navy blue) experiments are shown. Although the two force fields produce error in the opposite directions, the sign of errors on trials on which the CCW field was presented have been reversed so that all errors in the direction of the force field are shown as positive. On block 13, the two curl fields were introduced (exposure, gray shaded region), which remained on until block 89, when subjects returned to the null force field. D , Percentage force compensation computed from clamp trials throughout the experiment. The mean Ϯ SE force across subjects over two blocks is plotted as a percentage of the force required for estimated complete compensation. Gray shaded region indicates exposure blocks in which the curl force fields were applied. 
Hand paths during the movements between the central location (green circle) and final target (yellow circle) for the static visual cue and dwell time experiments. The mean (solid line), SE (dark shaded region), and SD (light shaded region) across all subjects for each condition are plotted. The trials on which the CW force field was applied are shown in red, and the trials in which the CCW force field was applied are shown in blue. Pre-exposure, The mean of the last eight trials in the initial null field (block 12). Initial exposure, The first eight trials in the curl force fields (block 13). Final exposure, The last eight trials in the curl field (block 88). Post exposure, The first eight trials in the null field during the washout (aftereffect trials) (block 89). Trials in which the force field is applied are shown with the shaded gray background. A , Paths in the visual static condition. B , Paths in the 1000 ms dwell condition. C , Paths in the 500 ms dwell condition. D , Paths in the 150 ms dwell condition. E , Paths in the 0 ms dwell condition. 
Dwell time contextual effect experiments. A , Experimental design. In the contextual phase, subjects moved from the cue location to the central target while the final target was visually displayed. No forces were applied during this movement at any stage of the experiment. Subjects were then required to remain in the central location for a set dwell time (0, 150, 500, or 1000 ms). After remaining in the central target for the correct time, subjects moved to the final target. The curl force fields were applied during this phase of the trial. For example, the movement from cue C3 and to target T1 uniquely specified the CW force field. B , The movement from cue C2 and to final target T1 uniquely specified the CCW force field. C , Speed profiles for the single force-field condition (black) and dwell time conditions of 0 ms (red), 150 ms (yellow), 500 ms (green), and 1000 ms (cyan) aligned on the contextual movement initiation. The mean and SE of the hand speed profiles are shown across all subjects. D , Speed profiles for the single force-field condition (black) and the dwell time conditions of 0 ms (red), 150 ms (yellow), 500 ms (green), and 1000 ms (cyan) aligned on the peak velocity of the second movement. The mean and SE of the hand speed profiles are shown across all subjects. E , Mean hand path error (MPE) and SE as a function of block, averaged across all subjects for dwell times of 0, 150, 500, and 1000 ms as well as for the single force-field condition (black dashed line). Shaded region indicates exposure blocks in which the curl force fields were applied. Each block in the single force-field condition consisted of nine trials (8 force-field trials and 1 clamp trial) and consecutive blocks were averaged together. This provides an even comparison across blocks with all other experimental conditions as the identical number of force-field trials (of any one field direction) are presented in a block. F , Percentage force compensation computed from clamp trials throughout the experiment. The mean Ϯ SE force over two blocks across subjects is plotted as a percentage of the force required for estimated complete compensation, for dwell times of 0 ms (red), 150 ms (yellow), 500 ms (green), and 1000 ms (cyan), as well as the corresponding values for the single force-field condition (black dashed). Shaded region indicates exposure blocks in which the curl force fields were applied. 
Motion before contextual information cues learning. A , Experimental design. The contextual phase comprised two components. Prior movement, The first movement (cue location to central target) occurred before the complete contextual information (presentation of target), which determined the force-field direction. The trial was initiated with the subject’s hand at one of the cue locations (C3 in this case) while the central location was visually presented. The subject then moved to the central location. Target appearance, Once the subject was within the central location, the target appeared. Adaptation phase, Subjects then moved to the final target, and the force field was applied as soon as subjects initiated the movement. In this case, the previous movement from cue C3 combined with the current target presentation of T1 specified the CW force field. B , The identical previous movement from cue C3 to the central target was performed. However, in the target appearance phase, target T2 was presented. This combination specified the CCW force field on the adaptation phase of the movement. C , Mean MPE (green trace) and SE (green shaded region) across all subjects and blocks during the motion before contextual presentation experiment. For comparison, the mean results for the static visual (blue trace) and 0 ms dwell time (red trace) conditions are shown. Shaded gray region indicates the exposure period in which the two curl force fields were applied. D , Percentage force compensation computed from clamp trials throughout the experiment. The mean Ϯ SE force over two blocks across subjects is plotted as a percentage of the force required for estimated complete compensation. For comparison, the mean results for the static visual normal (blue trace) and 0 ms dwell time (red trace) conditions are shown. Shaded region indicates exposure blocks in which the curl force fields were applied. 
Contextual effects vary with dwell time. A, The mean SE force on clamp trials (in direction toward target T1) as a percentage of the force required for estimated complete compensation. Values for all experiments (on the last third of trials during force-field exposure) are plotted as a function of the mean dwell time that subjects used in these conditions, with SE indicated by horizontal error bars. Note that most of the horizontal error bars are so small that they disappear under the symbols. For comparison, the results from the visual static conditions are plotted as a line (shaded region indicates SE) across all dwell times, because they are not related to a particular dwell time. B, The mean SE MPE over the last block of trials during force-field exposure. C, The mean SE MPE over the first block of trials in the postexposure session in all experiments. D, The mean SE peak velocity across all subjects in all experimental conditions experiments (on the last third of trials during force-field exposure), plotted as a function of dwell time.
Real-world tasks often require movements that depend on a previous action or on changes in the state of the world. Here we investigate whether motor memories encode the current action in a manner that depends on previous sensorimotor states. Human subjects performed trials in which they made movements in a randomly selected clockwise or counterclockwise velocity-dependent curl force field. Movements during this adaptation phase were preceded by a contextual phase that determined which of the two fields would be experienced on any given trial. As expected from previous research, when static visual cues were presented in the contextual phase, strong interference (resulting in an inability to learn either field) was observed. In contrast, when the contextual phase involved subjects making a movement that was continuous with the adaptation-phase movement, a substantial reduction in interference was seen. As the time between the contextual and adaptation movement increased, so did the interference, reaching a level similar to that seen for static visual cues for delays >600 ms. This contextual effect generalized to purely visual motion, active movement without vision, passive movement, and isometric force generation. Our results show that sensorimotor states that differ in their recent temporal history can engage distinct representations in motor memory, but this effect decays progressively over time and is abolished by ∼600 ms. This suggests that motor memories are encoded not simply as a mapping from current state to motor command but are encoded in terms of the recent history of sensorimotor states.
Cells dissociated from the cerebral white matter of immature rats were maintained in monolayer culture. Treatment with platelet-derived growth factor (PDGF) caused a large increase in the numbers of "O2A" oligodendroglial precursor cells (which bind the monoclonal antibody A2B5) and subsequently in the numbers of galactocerebroside (galC)-positive oligodendroglia. A2B5-negative "pre-O2A cells" in cerebral white matter cultures in which O2A cells and oligodendroglia had been killed by antibody-dependent complement-mediated cytolysis were induced by PDGF to proliferate and to differentiate into O2A cells and subsequently into oligodendroglia and type 2 astroglia. The most mature pre-O2A phenotype in these cultures was a small, round, process-bearing cell which expressed vimentin but not glial fibrillary acidic protein or galC. Cells of this phenotype were not observed upon PDGF treatment of immature rat optic nerve monolayer cultures from which O2A cells and oligodendrocytes had been depleted, and PDGF also failed to elicit the accumulation of O2A cells and oligodendroglia in such cultures.
We discovered a nonpeptidic compound, TAK-070, that inhibited BACE1, a rate-limiting protease for the generation of Abeta peptides that are considered causative for Alzheimer's disease (AD), in a noncompetitive manner. TAK-070 bound to full-length BACE1, but not to truncated BACE1 lacking the transmembrane domain. Short-term oral administration of TAK-070 decreased the brain levels of soluble Abeta, increased that of neurotrophic sAPPalpha by approximately 20%, and normalized the behavioral impairments in cognitive tests in Tg2576 mice, an APP transgenic mouse model of AD. Six-month chronic treatment decreased cerebral Abeta deposition by approximately 60%, preserving the pharmacological efficacy on soluble Abeta and sAPPalpha levels. These results support the feasibility of BACE1 inhibition with a noncompetitive inhibitor as disease-modifying as well as symptomatic therapy for AD.
Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) is a significant source of disability in the HIV-infected population. Even with stringent adherence to anti-retroviral therapy, >50% of patients living with HIV-1 will develop HAND (Heaton et al., 2010). Because suppression of viral replication alone is not enough to stop HAND progression, there is a need for an adjunctive neuroprotective therapy in this population. To this end, we have developed a small-molecule brain-penetrant inhibitor with activity against mixed-lineage kinase 3 (MLK3), named URMC-099. MLK3 activation is associated with many of the pathologic hallmarks of HAND (Bodner et al., 2002, 2004; Sui et al., 2006) and therefore represents a prime target for adjunctive therapy based on small-molecule kinase inhibition. Here we demonstrate the anti-inflammatory and neuroprotective effects of URMC-099 in multiple murine and rodent models of HAND. In vitro, URMC-099 treatment reduced inflammatory cytokine production by HIV-1 Tat-exposed microglia and prevented destruction and phagocytosis of cultured neuronal axons by these cells. In vivo, URMC-099 treatment reduced inflammatory cytokine production, protected neuronal architecture, and altered the morphologic and ultrastructural response of microglia to HIV-1 Tat exposure. In conclusion, these data provide compelling in vitro and in vivo evidence to investigate the utility of URMC-099 in other models of HAND with the goal of advancement to an adjunctive therapeutic agent.
Humans are known to regulate the timing of interceptive actions by modeling, in a simplified way, Newtonian mechanics. Specifically, when intercepting an approaching ball, humans trigger their movements a bit earlier when the target arrives from above than from below. This bias occurs regardless of the ball's true kinetics, and thus appears to reflect an a priori expectation that a downward moving object will accelerate. We postulate that gravito-inertial information is used to tune visuomotor responses to match the target's most likely acceleration. Here we used the peculiar conditions of parabolic flight--where gravity's effects change every 20 s--to test this hypothesis. We found a striking reversal in the timing of interceptive responses performed in weightlessness compared with trials performed on ground, indicating a role of gravity sensing in the tuning of this response. Parallels between these observations and the properties of otolith receptors suggest that vestibular signals themselves might plausibly provide the critical input. Thus, in addition to its acknowledged importance for postural control, gaze stabilization, and spatial navigation, we propose that detecting the direction of gravity's pull plays a role in coordinating quick reactions intended to intercept a fast-moving visual target.
Pharmacokinetic parameters for MTIP, CP154526, SSR125543A, and R121919
Top, " Hangover anxiety " 12 h after a single large alcohol dose; p 0.01 for 3 g/kg versus vehicle control (n 9 –16). Bottom, Dose-dependent reversal of hangover anxiety observed 12 h after a 3 g/kg alcohol dose by MTIP (main effect of treatment on percentage open time: p 0.00002; n 25 for the control group, 11–15 for each dose group). Differences on post hoc analysis versus the respective control group at p 0.01 or 0.001 are indicated by asterisks (** and ***). For detailed statistics, see Results. 
Nonstressed, spontaneous behaviors unaffected by MTIP treatment
Operant self-administration of alcohol and modulation of this behavior by MTIP. Each lever press resulted in the delivery of 0.1 ml of 10% alcohol solution. No effect was found in genetically nonselected Wistar rats without a history of dependence (left group; n 7). In contrast, a dose-dependent suppression (main treatment effect, p 0.001) was seen in nonselected Wistar animals with a history of dependence (middle group; n 8). A similar dosedependent suppression was also observed in genetically selected, alcohol-preferring msP rats (right group; main treatment effect, p 0.009; n 7). Significant ( p 0.05) increase of self-administration in postdependent, vehicle-treated controls versus nondependent vehicletreated subjects on post hoc comparison is indicated by the number sign (#). Significant suppression of self-administration by MTIP ( p 0.05 and p 0.001, respectively, for individual groups) on post hoc comparison versus corresponding controls is indicated by asterisks (* and **). For detailed statistics, see Results. Error bars indicate SEM. 
We describe a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist with advantageous properties for clinical development, and its in vivo activity in preclinical alcoholism models. 3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-8-(1-ethylpropyl)-2,6-dimethyl-imidazo[1,2-b]pyridazine (MTIP) inhibited 125I-sauvagine binding to rat pituitary membranes and cloned human CRF1 with subnanomolar affinities, with no detectable activity at the CRF2 receptor or other common drug targets. After oral administration to rats, MTIP inhibited 125I-sauvagine binding to rat cerebellar membranes ex vivo with an ED50 of approximately 1.3 mg/kg and an oral bioavailability of 91.1%. Compared with R121919 (2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylamino-pyrazolo[1,5-a]pyrimidine) and CP154526 (N-butyl-N-ethyl-4,9-dimethyl-7-(2,4,6-trimethylphenyl)-3,5,7-triazabicyclo[4.3.0]nona-2,4,8,10-tetraen-2-amine), MTIP had a markedly reduced volume of distribution and clearance. Neither open-field activity nor baseline exploration of an elevated plus-maze was affected by MTIP (1-10 mg/kg). In contrast, MTIP dose-dependently reversed anxiogenic effects of withdrawal from a 3 g/kg alcohol dose. Similarly, MTIP blocked excessive alcohol self-administration in Wistar rats with a history of dependence, and in a genetic model of high alcohol preference, the msP rat, at doses that had no effect in nondependent Wistar rats. Also, MTIP blocked reinstatement of stress-induced alcohol seeking both in postdependent and in genetically selected msP animals, again at doses that were ineffective in nondependent Wistar rats. Based on these findings, MTIP is a promising candidate for treatment of alcohol dependence.
Tracer efflux studies were used to determine the effect of activation of protein kinase C on K channel function in rat brain synaptosomes. Hippocampal synaptosomes were treated with sn-1,2-dioctanoylglycerol (diC8), a synthetic diacylglycerol (DG) analog that activates protein kinase C. DiC8 inhibited depolarization-induced 86Rb efflux through voltage-gated K channels but did not affect the component of efflux corresponding to Ca-activated K channels. In time-course experiments, diC8 inhibited two components of 86Rb efflux: efflux through a rapidly inactivating, voltage-gated K channel (responsible for the "A" current) and that through a slowly inactivating, voltage-gated K channel (believed to be the "delayed rectifier"). Experiments with specific blockers of these voltage-gated K channels supported this observation. Inhibition of K-stimulated 86Rb efflux by diC8 was time dependent: at least 15 sec of preincubation was required before the effect could be observed. The effect of diC8 was concentration dependent: 50 microM diC8 produced a half-maximal inhibition of K-stimulated 86Rb efflux. The idea that the inhibition of synaptosome K channels by diC8 resulted from activation of C kinase was supported by pharmacological evidence. The action of diC8 was mimicked by 1-oleoyl-2-acetylglycerol, another DG analog that activates protein kinase C, but not by deoxy-diC8, a DG analog that does not activate C kinase. Inhibition of C kinase by sphingosine or H-7 prevented the diC8 effect. These studies demonstrate that synaptosomes are a good model in which to study modulation of mammalian CNS K channels.(ABSTRACT TRUNCATED AT 250 WORDS)
In order to develop more selective methods for labeling brain dopamine receptors, this study describes in detail the properties of 2-amino-6,7,-(³H)dihydroxy-1,2,3,4,-tetrahydronaphthalene ((³H) ADTN) binding to dopaminergic sites in rat, calf, and human brain. (³H)ADTN labeled two distinct types of dopaminergic binding sites in the brain striatum of the rat, calf, and human. Very low concentrations of dopamine and dopaminergic catecholamines (with IC50 values of 1 to 10 nM) inhibited the binding of (³H)ADTN to both sites. Neuroleptics, however, inhibited the binding of (³H)ADTN in two distinctly separate concentration ranges, with IC50 values of 0.15 to 40 nM at one site and 100 and 50,000 nM at the other site. The site with high affinity for dopamine and low affinity for neuroleptics had binding properties that corresponded to those of the previously characterized D3 site). The (³H)ADTN binding site with high affinity for neuroleptics demonstrated binding characteristics similar to a site labeled by ³H-Neuroleptics. (³H)Apomorphine appeared to label the same two sites as (³H)ADTN, while (âH)dopamine labeled only the D3 site. Scatchard analysis of (³H)ADTN or (âH)apomorphine binding, under conditions for selective labeling of the low affinity neuroleptic site (D3) and the high affinity site for neuroleptics, detected a density of 70 fmol/mg of protein for each. The density of the D3 site in the calf striatum (170 fmol/mg of protein) was much greater than that of the high affinity neuroleptic site (50 fmol/mg). In the rat, the dissociation constant (KD) of (³H)ADTN was 2 nM for both sites. (³H)Apomorphine, however, had a higher affinity for the D3 site (KD.1.6 nM) than for the high affinity neuroleptic site (KD.4.2 nM).
The concept of a threshold of dopamine (DA) depletion for onset of Parkinson's disease symptoms, although widely accepted, has, to date, not been determined experimentally in nonhuman primates in which a more rigorous definition of the mechanisms responsible for the threshold effect might be obtained. The present study was thus designed to determine (1) the relationship between Parkinsonian symptom appearance and level of degeneration of the nigrostriatal pathway and (2) the concomitant presynaptic and postsynaptic striatal response to the denervation, in monkeys treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine according to a regimen that produces a progressive Parkinsonian state. The kinetics of the nigrostriatal degeneration described allow the determination of the critical thresholds associated to symptom appearance, these were a loss of 43.2% of tyrosine hydroxylase-immunopositive neurons at the nigral level and losses of 80.3 and 81.6% DA transporter binding and DA content, respectively, at the striatal level. Our data argue against the concept that an increase in DA metabolism could act as an efficient adaptive mechanism early in the disease progress. Surprisingly, the D(2)-like DA receptor binding showed a biphasic regulation in relation to the level of striatal dopaminergic denervation, i.e., an initial decrease in the presymptomatic period was followed by an upregulation of postsynaptic receptors commencing when striatal dopaminergic homeostasis is broken. Further in vivo follow-up of the kinetics of striatal denervation in this, and similar, experimental models is now needed with a view to developing early diagnosis tools and symptomatic therapies that might enhance endogenous compensatory mechanisms.
Parkinson's disease (PD) is an age-related neurodegenerative disease in which the role of reactive oxygen species (ROS) is strongly implicated. The presence of oxidative stress has been detected in human and experimental PD using both direct and indirect indices. Scavenging ROS is, therefore, an important therapeutic avenue for the treatment of PD. Manganic porphyrins are catalytic antioxidants that scavenge a wide range of ROS. In this study, we tested the therapeutic effects of a compound [5,15-bis(methoxycarbonyl)-10,20-bis-trifluoromethyl-porphyrinato manganese (III) chloride (AEOL11207)] belonging to a new generation of lipophilic manganic porphyrins for neuroprotection and oral bioavailability in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism. Groups of adult C57BL/6 mice were administered MPTP with varying subcutaneous or oral dosing regimens of AEOL11207. Neurotoxicity was assessed by measurement of striatal dopamine levels and quantification of tyrosine hydroxylase-positive neurons in the substantial nigra pars compacta one week after the first dose of MPTP. Glutathione depletion, lipid peroxidation, and 3-nitrotyrosine (3-NT) formation were measured as indicators of oxidative stress in the ventral midbrain in vivo. AEOL11207 administered either by subcutaneous or oral routes protected against MPTP-induced dopamine depletion in the striatum as well as dopaminergic neuronal loss, glutathione depletion, lipid peroxidation, and 3-NT formation in the ventral midbrain. Neuroprotection correlated with brain metalloporphyrin concentrations. This is the first demonstration of neuroprotection by an orally active catalytic antioxidant in the MPTP mouse model and suggests its potential clinical utility for the treatment of chronic neurodegenerative diseases such as PD.
Dopamine (DA) has been postulated to play a role in the loss of dopaminergic substantia nigra (SN) neurons in Parkinson's disease because of its propensity to oxidize and form quinones and other reactive oxygen species that can alter cellular function. Moreover, DA depletion can attenuate dopaminergic cell loss in vitro. To test the contribution of DA to SN impairment in vivo, we used DA-deficient mice, which lack the enzyme tyrosine hydroxylase in dopaminergic cells, and mice pharmacologically depleted of DA by alpha-methyl-p-tyrosine pretreatment. Mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that produces parkinsonian pathology in humans, nonhuman primates, and rodents. In contrast to in vitro results, genetic or pharmacologic DA depletion did not attenuate loss of dopaminergic neurons in the SN or dopaminergic neuron terminals in the striatum. These results suggest that DA does not contribute to acute MPTP toxicity in vivo.
Brain levels of MPP+ in SOD-transgenic mice and their nontransgenic littermates 
Administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mammals causes damage to the nigrostriatal dopaminergic pathway similar to that observed in Parkinson's disease. It has been suggested that the mechanism by which MPTP kills dopamine (DA) neurons involves an energy crisis due to the inhibition of mitochondrial complex I. In addition, superoxide radicals (O2-), generated subsequent to the blockade of mitochondrial complex I, may also be involved in MPTP-induced neurotoxicity. Superoxide dismutase (SOD) is a scavenger enzyme that protects cells from the hazard of O2- radicals. To evaluate further the role of O2- radical in MPTP-induced toxicity, we tested the effects of MPTP in transgenic mice with increased SOD activity. In nontransgenic littermates with normal SOD activity, MPTP injection causes a marked reduction in striatal levels of DA and its metabolites as well as in striatal and nigral 3H-DA uptake; these findings are consistent with a loss in dopaminergic neurons. In contrast, in transgenic mice with increased SOD activity, MPTP injection does not cause any significant changes either in levels of DA and metabolites or in 3H-DA uptake. We show that this lack of toxicity is not due to a lower delivery of MPTP to the brain following its intraperitoneal injection, to reduced brain biotransformation of MPTP to N-methyl-4-phenylpyridinium ion (MPP+), to diminished striatal mitochondrial monoamine oxidase B activity, to decreased synaptosomal uptake of MPP+, to lower potency of MPP+ to inhibit the complex I of the mitochondrial electron transport chain, or to faster brain elimination of MPP+. These results suggest that increased SOD activity is, most likely, the protective factor that confers resistance to transgenic mice against MPTP-induced neurotoxicity. Thus, this study provides further evidence that some of the deleterious effects of MPTP may be mediated by O2- radicals. The similarity between the MPTP model and Parkinson's disease further raises the possibility that oxy-radicals may play a significant role in the etiology of this neurodegenerative disorder.
Accumulating evidence suggests that apoptotic and inflammatory factors contribute to the demise of dopaminergic neurons. In this respect, Fas, a member of the tumor necrosis factor receptor family with proapoptotic and inflammatory functions, was reported to be elevated within the striatum and substantia nigra pars compacta (SNc) of Parkinson's disease (PD) patients. Accordingly, the present investigation evaluated the function of Fas in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. Injection of MPTP increased nigral Fas expression, and mice lacking Fas displayed attenuated MPTP-induced SNc dopaminergic loss and microglial activation. In addition, Fas induction was blocked by expression of a dominant-negative c-Jun adenovirus that also protected dopamine neurons from MPTP-induced damage. Together, these data suggest the critical nature of the c-Jun-Fas signaling pathway in MPTP-induced neuronal loss. Although critical for degeneration of the soma, Fas deficiency did not significantly prevent the reduction of dopaminergic terminal fibers within the striatum or normalize the activation of striatal microglia and elevation of the postsynaptic activity marker DeltaFosB induced by denervation. Interestingly, Fas-deficient mice displayed a pre-existing reduction in striatal dopamine levels and locomotor behavior when compared with wild-type mice. Despite the reduced terminals, dopamine levels were not further suppressed by MPTP treatment in mutant mice, raising the possibility of a compensatory response in basal ganglia function in Fas-deficient mice.
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damages the nigrostriatal dopaminergic pathway as seen in Parkinson's disease (PD), a common neurodegenerative disorder with no effective protective treatment. Consistent with a role of glial cells in PD neurodegeneration, here we show that minocycline, an approved tetracycline derivative that inhibits microglial activation independently of its antimicrobial properties, mitigates both the demise of nigrostriatal dopaminergic neurons and the formation of nitrotyrosine produced by MPTP. In addition, we show that minocycline not only prevents MPTP-induced activation of microglia but also the formation of mature interleukin-1beta and the activation of NADPH-oxidase and inducible nitric oxide synthase (iNOS), three key microglial-derived cytotoxic mediators. Previously, we demonstrated that ablation of iNOS attenuates MPTP-induced neurotoxicity. Now, we demonstrate that iNOS is not the only microglial-related culprit implicated in MPTP-induced toxicity because mutant iNOS-deficient mice treated with minocycline are more resistant to this neurotoxin than iNOS-deficient mice not treated with minocycline. This study demonstrates that microglial-related inflammatory events play a significant role in the MPTP neurotoxic process and suggests that minocycline may be a valuable neuroprotective agent for the treatment of PD.
Studies have suggested that there are beneficial effects of exercise in patients with Parkinson's disease, but the underlying molecular mechanisms responsible for these effects are poorly understood. Studies in rodent models provide a means to examine the effects of exercise on dopaminergic neurotransmission. Using intensive treadmill exercise, we determined changes in striatal dopamine in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse. C57BL/6J mice were divided into four groups: (1) saline, (2) saline plus exercise, (3) MPTP, and (4) MPTP plus exercise. Exercise was started 5 d after MPTP lesioning and continued for 28 d. Treadmill running improved motor velocity in both exercise groups. All exercised animals also showed increased latency to fall (improved balance) using the accelerating rotarod compared with nonexercised mice. Using HPLC, we found no difference in striatal dopamine tissue levels between MPTP plus exercise compared with MPTP mice. There was an increase detected in saline plus exercise mice. Analyses using fast-scan cyclic voltammetry showed increased stimulus-evoked release and a decrease in decay of dopamine in the dorsal striatum of MPTP plus exercise mice only. Immunohistochemical staining analysis of striatal tyrosine hydroxylase and dopamine transporter proteins showed decreased expression in MPTP plus exercise mice compared with MPTP mice. There were no differences in mRNA transcript expression in midbrain dopaminergic neurons between these two groups. However, there was diminished transcript expression in saline plus exercise compared with saline mice. Our findings suggest that the benefits of treadmill exercise on motor performance may be accompanied by changes in dopaminergic neurotransmission that are different in the injured (MPTP-lesioned) compared with the noninjured (saline) nigrostriatal system.
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces clinical, biochemical, and neuropathological changes reminiscent of those occurring in idiopathic Parkinson's disease (PD). Here we show that a peptide caspase inhibitor, N-benzyloxy-carbonyl-val-ala-asp-fluoromethyl ketone, or adenoviral gene transfer (AdV) of a protein caspase inhibitor, X-chromosome-linked inhibitor of apoptosis (XIAP), prevent cell death of dopaminergic substantia nigra pars compacta (SNpc) neurons induced by MPTP or its active metabolite 1-methyl-4-phenylpyridinium in vitro and in vivo. Because the MPTP-induced decrease in striatal concentrations of dopamine and its metabolites does not differ between AdV-XIAP- and control vector-treated mice, this protection is not associated with a preservation of nigrostriatal terminals. In contrast, the combination of adenoviral gene transfer of XIAP and of the glial cell line-derived neurotrophic factor to the striatum provides synergistic effects, rescuing dopaminergic SNpc neurons from cell death and maintaining their nigrostriatal terminals. These data suggest that a combination of a caspase inhibitor, which blocks death, and a neurotrophic factor, which promotes the specific function of the rescued neurons, may be a promising strategy for the treatment of PD.
We investigated, in mice, the influence of life experience on the vulnerability to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a major neurotoxin that induces a Parkinson's disease-like syndrome in humans, and to cocaine, a potent psychostimulant that promotes drug addiction. Our findings show that adult C57BL/6 mice raised in an enriched environment (EE) for only 2 months are significantly more resistant to both drugs compared with mice raised in a standard environment (SE). Indeed, EE mice showed decreased locomotor activity in response to cocaine (10 and 20 mg/kg) as well as a different pattern of c-fos expression in the striatum compared with SE mice. After MPTP treatment, SE mice showed a 75% loss of dopamine neurons, whereas EE mice showed only a 40% loss. The dopamine transporter plays a key role in mediating the effects of both drugs. We thus investigated the regulation of its expression. EE mice showed less dopamine transporter binding in the striatum and less dopamine transporter mRNA per dopamine neuron at the cellular level as demonstrated by in situ hybridization. In addition, enriched environment promoted an increase in the expression of brain-derived neurotrophic factor in the striatum. These data provide a direct demonstration of the beneficial consequences that a positive environment has in preventing neurodegeneration and in decreasing responsiveness to cocaine. Furthermore, they suggest that the probability of developing neurological disorders such as Parkinson's disease or vulnerability to psychostimulants may be related to life experience.
The present study was designed to elucidate the inflammatory and apoptotic mechanisms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in a model of Parkinson's disease. Our results showed that mutant mice lacking the caspase-11 gene were significantly more resistant to the effects of acute treatment with MPTP than their wild-type mice. Thus, the neurotoxicity of MPTP seems to be mediated by the induction of both mitochondrial dysfunction and free radical generation. Previously, we showed that overexpression of the Apaf-1 dominant-negative inhibitor inhibited the mitochondrial apoptotic cascade in chronic MPTP treatment but not in acute MPTP treatment. The present results indicate that MPTP neurotoxicity may be mediated via activation of the caspase-11 cascade and inflammatory cascade, as well as the mitochondrial apoptotic cascade.
Time-locked response changes during stimulation. A, Population PSTH of all GPe (blue) and GPi (red) neurons during stimulation (SEM, smoothed by a Gaussian window, SD 50 s). B, Example of dynamic time-locked response of a GPe neuron during GPe stimulation. Top, PSTH of response during the whole stimulation period. Middle, Partial PSTHs for different periods. Bottom, Raster plot smoothed over 100 consecutive stimuli. C, D, Compass plots of normalized peaks and amplitudes of PSTHs during the early (C) and late (D) periods. Each neuron is represented by one vector: the direction represents the normalized peak time and the length represents the normalized peak amplitude. The red line represents the mean normalized response. E, Locking index during early versus late periods. Dotted line at diagonal, linear regression is shown as a solid line (R 2 0.55, p 0.001). 
High-frequency stimulation (HFS) in the globus pallidus is used to ameliorate clinical symptoms of Parkinson's disease, dystonia, and other disorders. Previous in vivo studies have shown diverse static effects of stimulation on discharge rates and firing patterns of neurons along the corticobasal ganglia loop. In vitro studies, together with other experimental and theoretical studies, have suggested the involvement of synaptic plasticity in stimulation effects. To explore the effects of HFS on synaptic transmission, we studied the dynamic changes in neuronal activity in vivo, using multielectrode recordings during stimulation in the globus pallidus of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated primates. Stimulation effects evolved over time and were pronounced during the first 10 s of stimulation, where 69% of the 249 recorded neurons changed their firing rate and 61% displayed time-locked firing. The time-locked response faded away in 43% of the responding neurons, and its pattern was altered in the remaining cells: the peak response shifted away in time from the stimulus onset, and its amplitude decreased. Repetition of the stimulation protocol revealed a full resetting of the effect, implying short-term synaptic depression. This evolving response is indicative of the transient plasticity of the corticobasal ganglia network in vivo during HFS. Therefore, short-term depression of synaptic transmission may contribute to the mechanism underlying the effects of stimulation during the resulting steady state, altering the balance of neuronal interactions and interfering with pathological information transmission.
Primary motor cortex (MI) neurons discharge vigorously during voluntary movement. A cardinal symptom of Parkinson's disease (PD) is poverty of movement (akinesia). Current models of PD thus hypothesize that increased inhibitory pallidal output reduces firing rates in frontal cortex, including MI, resulting in akinesia and muscle rigidity. We recorded the simultaneous spontaneous discharge of several neurons in the arm-related area of MI of two monkeys and in the globus pallidus (GP) of one of the two. Accelerometers were fastened to the forelimbs to detect movement, and surface electromyograms were recorded from the contralateral arm of one monkey. The recordings were conducted before and after systemic treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rendering the animals severely akinetic and rigid with little or no tremor. The mean spontaneous MI rates during periods of immobility (four to five spikes/sec) did not change after MPTP; however, in this parkinsonian state, MI neurons discharged in long bursts (sometimes >2 sec long). These bursts were synchronized across many cells but failed to elicit detectable movement, indicating that even robust synchronous MI discharge need not result in movement. These synchronized population bursts were absent from the GP and were on a larger timescale than oscillatory synchrony found in the GP of tremulous MPTP primates, suggesting that MI parkinsonian synchrony arises independently of basal ganglia dynamics. After MPTP, MI neurons responded more vigorously and with less specificity to passive limb movement. Abnormal MI firing patterns and synchronization, rather than reduced firing rates, may underlie PD akinesia and persistent muscle rigidity.
Effects of MP TP administered at 15 mg/kg intraperitoneally every 2 hr for 4 doses on dopamine, DOPAC, and HVA in wild-type and Bcl-2 overexpressing mice. **p 0.01; ***p 0.001 compared with M P TP in controls. 
Effects of MP TP administered at 15 mg / kg intraperitoneally every 2 hr for 4 doses on [ 3 H]mazindol binding in the striatum in wild-type and Bcl-2 overexpressing mice. ***p 0.001 compared with MPTP in controls.
Representative autoradiographs of total [ 3 H]mazindol binding in the striatum in wild-type and Bcl-2 overexpressing mice after acute administration of M P TP. Left, Untreated wild-type mouse. Midleft, MPTPtreated wild-type mouse. Midright, Untreated Bcl-2 transgenic mouse. Right, M P TP-treated Bcl-2 transgenic mouse. 
Effects of MPTP administered at 20 mg / kg intraperitoneally daily for 5 days on dopamine, DOPAC, and H VA in wild-type and Bcl-2 overexpressing mice. **p 0.01 compared with M P TP in controls. 
Effects of MP TP administered at 20 mg / kg intraperitoneally daily for 5 days on [ 3 H]mazindol binding in the striatum in wild-type and Bcl-2 overexpressing mice. ***p 0.001 compared with M P TP in controls. 
The proto-oncogene Bcl-2 rescues cells from a wide variety of insults. Recent evidence suggests that Bcl-2 protects against free radicals and that it increases mitochondrial calcium-buffering capacity. The neurotoxicity of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyride (MPTP) is thought to involve both mitochondrial dysfunction and free radical generation. We therefore investigated MPTP neurotoxicity in both Bcl-2 overexpressing mice and littermate controls. MPTP-induced depletion of dopamine and loss of [3H]mazindol binding were significantly attenuated in Bcl-2 overexpressing mice. Protection was more profound with an acute dosing regimen than with daily MPTP administration over 5 d. 1-Methyl-4-phenylpyridinium (MPP+) levels after MPTP administration were similar in Bcl-2 overexpressing mice and littermates. Bcl-2 blocked MPP+-induced activation of caspases. MPTP-induced increases in free 3-nitrotyrosine levels were blocked in Bcl-2 overexpressing mice. These results indicate that Bcl-2 overexpression protects against MPTP neurotoxicity by mechanisms that may involve both antioxidant activity and inhibition of apoptotic pathways.
Loss of nigrostriatal neurons leads to striatal dopamine deficiency and subsequent development of parkinsonism. The effects of this denervation on D2-like receptors in striatum remain unclear. Most studies have demonstrated increases in striatal dopamine D2-like receptors in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated denervation, but others have found either decreases or no change in binding. To clarify the response to denervation, we have investigated the time-dependent changes in dopamine D2, D3, and D4 receptor protein and mRNA levels in unilaterally MPTP-lesioned baboons. MPTP (0.4 mg/kg) was infused into one internal carotid artery, producing a contralateral hemi-parkinsonian syndrome. After MPTP treatment, the animals were maintained for 17-480 d and then euthanized. MPTP decreased ipsilateral dopamine content by >90%, which did not change with time. Ipsilateral D2-like receptor binding in caudate and putamen initially decreased then increased two- to sevenfold over the first 100 d and returned to near baseline levels by 480 d. Relative levels of D2 mRNA were essentially unchanged over this period. D4 mRNA was not detected. In contrast, D3 mRNA increased sixfold by 2 weeks and then decreased. At the peak period of increase in binding sites, all D2-like receptors were in a micromolar affinity agonist-binding state, implying an increase in uncoupled D2 but not D3 receptor protein. Taken together, these data suggest that MPTP-induced changes in D2-like dopamine receptors are complex and include translational or post-translational mechanisms.
Striatal preprotachykinin (PPT) gene expression and [(3)H]mazindol binding were examined in monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Some animals (n = 5) became moderately to severely parkinsonian after receiving large doses of MPTP over 9-30 d and remained symptomatic for a relatively short time (3 weeks to 3 months; acutely symptomatic group). A second group of animals (n = 5) received low doses of MPTP (1.5-12 months), developed cognitive impairments but displayed no gross motor deficits (asymptomatic group), and were killed 3-12 months after their final dose of MPTP. Other animals became moderately to severely parkinsonian after receiving escalating doses of MPTP (>6 months; n = 4) or high doses of MPTP (<1 month; n = 1) and remained symptomatic for 2.5-5.75 years (chronically symptomatic group). All MPTP-treated animals had extensive losses of [(3)H]mazindol binding in dorsal striatal sensorimotor regions with asymptomatic animals generally having a lesser degree of damage. However, PPT mRNA levels differed sharply among treatment groups. Symptomatic animals (acutely and chronically parkinsonian) had significantly decreased PPT mRNA levels in most striatal regions. In asymptomatic animals, PPT mRNA expression was not significantly different from that measured in control animals, despite decreases in [(3)H]mazindol binding in some striatal regions of similar magnitude to those observed in symptomatic animals. These observations suggest that PPT gene expression may be directly related to expression of parkinsonian motor symptomatology regardless of duration of MPTP exposure, duration of the parkinsonism, or extent of dopamine denervation. These results imply that the direct striatal output circuit may have a greater contribution to expression of parkinsonian symptomatology than proposed previously.
We examined whether selective activation of mGlu4 metabotropic glutamate receptors attenuates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal damage in mice. C57BL mice were treated with a single dose of MPTP (30 mg/kg, i.p.) preceded, 30 min earlier, by a systemic injection of the mGlu4 receptor enhancer N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC). PHCCC was injected either subcutaneously in cremophor EL or intraperitoneally in saline containing 50% DMSO. PHCCC treatment (3 or 10 mg/kg) significantly reduced MPTP toxicity, as assessed by measurements of the striatal levels of dopamine and its metabolites and by tyrosine hydroxylase, dopamine transporter, and glial fibrillary acidic protein immunostaining in the corpus striatum and substantia nigra. In another set of experiments, a higher cumulative dose of MPTP (80 mg/kg divided into four injections with 2 h of interval) was injected to mGlu4-/- mice and their Sv129/CD1 wild-type controls. A higher dose was used in these experiments because Sv129/CD1 mice are less sensitive to MPTP toxicity. Systemic administration of PHCCC was protective in wild-type mice but failed to affect nigrostriatal damage in mGlu4-/- mice. Finally, unilateral infusion of PHCCC in the external globus pallidus protected the ipsilateral nigrostriatal pathway against MPTP toxicity. These data support the view that mGlu4 receptors are potential targets for the experimental treatment of parkinsonism.
We combined the use of knock-out mice and subtype-selective antagonists [2-methyl-6-(phenylethynyl)pyridine (MPEP) and (E)-2-methyl-6-(2-phenylethenyl)-pyridine (SIB1893)] to examine whether endogenous activation of mGlu5 metabotropic glutamate receptors contributes to the pathophysiology of nigro-striatal damage in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of parkinsonism. High doses of MPTP (four injections of 20 mg/kg, i.p., every 2 hr) induced a high mortality rate and a nearly total degeneration of the nigro-striatal pathway in wild-type mice. mGlu5 knock-out mice were less sensitive to MPTP toxicity, as shown by a higher survival and a milder nigro-striatal damage. Protection against MPTP (80 mg/kg) toxicity was also observed after MPEP injections (four injections of 5 mg/kg, i.p., 30 min before each MPTP injection). MPEP treatment did not further increase neuroprotection against 80 mg/kg of MPTP in mGlu5 knock-out mice, indicating that the drug acted by inhibiting mGlu5 receptors. In wild-type mice, MPEP was also neuroprotective when challenged against lower doses of MPTP (either 30 mg/kg, single injection, or four of 10 mg/kg injections). The action of MPEP was mimicked by SIB1893 but not by the mGlu1 receptor antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester. MPEP did not change the kinetics of 1-methyl-4-phenylpyridinium ion formation in the striatum of mice injected with MPTP. We conclude that mGlu5 receptors act as amplifiers of MPTP toxicity and that mGlu5 receptor antagonists may limit the extent of nigro-striatal damage in experimental models of parkinsonism.
Epidemiological studies have strongly linked caffeine consumption with a reduced risk of developing Parkinson's disease (PD) in men. Interestingly, in women, this inverse association is present only in those who have not taken postmenopausal estrogens, suggesting an interaction between the influences of estrogen and caffeine use on the risk of PD. To explore a possible biological basis for this interaction, we systematically investigated how the neuroprotective effect of caffeine is influenced by gender, ovariectomy (OVX), and then exogenous estrogen in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD. (1) Caffeine treatment produced a dose-dependent attenuation of MPTP-induced striatal dopamine loss in both young and retired breeder (RB) male, but not female, mice. (2) In female mice (both young and RB), caffeine was less potent or altogether ineffective as a neuroprotectant after sham surgery compared to OVX or after OVX plus estrogen replacement compared to OVX plus placebo treatment. (3) Estrogen treatment also prevented the protection of caffeine against dopamine loss in young male mice. (4) Consistent with the putative protective effect of estrogen, female and OVX plus estrogen mice were relatively resistant to MPTP toxicity compared to male and OVX plus placebo mice, respectively. (5) There was no overall difference in brain levels of caffeine and its metabolites between OVX plus placebo and OVX plus estrogen mice. Together, these results suggest that estrogen can occlude and thereby prevent the neuroprotective effect of caffeine in a model of PD neurodegeneration, supporting a biological basis for the interaction between estrogen and caffeine in modifying the risk of PD.
Current physiological studies emphasize the role of neuronal oscillations and synchronization in the pathophysiology of Parkinson's disease; however, little is known about their specific roles in the neuronal substrate of dopamine replacement therapy (DRT). We investigated oscillatory activity and correlations throughout the different states of levodopa-naive parkinsonism as well as "Off-On" and dyskinetic states of DRT in the external globus pallidum (GPe) of tremulous (vervet) and rigid-akinetic (macaque) monkeys and in the internal globus pallidum (GPi) of the vervet monkey. We found that, although oscillatory activity of cells and interneuronal correlation in both pallidal segments increases after induction of parkinsonism with 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) and decreases in response to DRT, important differences exist between the two pallidal segments. In the GPi, the fraction of oscillatory cells and relative power of oscillations were significantly higher than in the GPe, and the dominant frequency was within the range of 7.5-13.5 Hz compared with a range of 4.5-7.5 Hz within the GPe. The interneuronal correlations were mostly oscillatory in the GPi, whereas at least half are non-oscillatory in the GPe. We demonstrate that the tremor characteristics after exposure to DRT do not resemble those of the normal or the levodopa-naive state. Moreover, although DRT reverses the MPTP-induced neuronal changes (rate, pattern, and pairwise correlations), the balance between GPe and GPi fails to restore. We therefore suggest that this imbalance reflects additional abnormal organization of the basal ganglia networks in response to dopamine replacement and may constitute the physiological substrate of the limitations and side effects of chronic DRT.
Effective sample clustering by informative genes. Affymetrix U74A probe sets were filtered by dChip to yield a 2102 member gene list for use in sample clustering. The expression patterns of these genes in the 12 arrays (saline and chronic MPTP treatments) segregated arrays into the proper treatment groups. Although all arrays are correctly partitioned by injury status, the analysis showed that the MML group was more closely related to the control group, possibly indicating some transcriptional recovery at this later time point. Numbers represent the four samples (arrays) in each of the three groups.
PCA of MPTP-treated samples. PCA of the Affymetrix GeneChip data indicates that each treatment group's samples occupy nonoverlapping areas in the two-dimensional space defined by PC1 and PC2. The samples can also be seen to fall into two different pairings: one in which the controls and MPTP late samples (f) share the bottom left area and the MPTP early samples (p) are alone in the top right area, and another in which the controls () are alone in the far left area, whereas the two MPTP groups occupy the right side of the space. These are precisely the two different pairings seen in hierarchical clustering. 
Validation by qRTPCR of selected gene expression changes in the MPTP- treated SN
Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death.
High-frequency stimulation of the globus pallidus (GP) has emerged as a successful tool for treating Parkinson's disease and other motor disorders. However, the mechanism governing its therapeutic effect is still under debate. To shed light on the basic mechanism of deep brain stimulation (DBS), we performed microstimulation in the GP of a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated monkey while recording with other microelectrodes in the same nucleus. We used robust methods to reduce the stimulus artifact, and 600-3000 repetitions of a single stimulus and of high-frequency short trains (10-40 stimuli), enabling high temporal resolution analysis of neural responses. Low-frequency stimulation yielded a typical three-stage response: short-term (2-3 msec duration) activity, followed by mid-term (15-25 msec) inhibition, and occasionally longer-term (30-40 msec) excitation. Trains of high-frequency stimuli elicited complex locking of the response to the stimuli in most neurons. The locking displayed a stereotypic temporal structure consisting of three short-duration (1-2 msec) phases: an initial (mean latency = 2.9 msec) excitation followed by an inhibition (4.6 msec) and a second excitation (6.3 msec). The change in the mean firing rate was mixed; the majority of the neurons displayed partial inhibition during the stimulus train. Slow inhibitory and excitatory multiphase changes in the firing rate were observed after the stimulus trains. The activity of neurons recorded simultaneously displayed rate correlations but no spike-to-spike correlations. Our results suggest that the effect of DBS on the GP is not complete inhibition but rather a complex reshaping of the temporal structure of the neuronal activity within that nucleus.
Previous physiological studies have revealed changes in firing rates and synchronization of pallidal neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) primate model of Parkinson's disease. Several primate and human studies have demonstrated that dopamine replacement therapy (DRT) reverses the changes in the pallidal firing rates; however, the effects of DRT on pallidal synchronization have never been explored. To do so, we recorded the simultaneous activity of pallidal neurons of a vervet monkey before and after induction of severe parkinsonism by systemic MPTP treatment. We subsequently recorded the pallidal activity before and after daily administration of oral DRT. We extended the time scale of our correlation studies to +/-5 sec to allow detection of long-duration synchronized neuronal activity. After MPTP treatment, firing rates decreased in the external segment of the globus pallidus (GP(e)) and increased in the internal segment (GP(i)). A reversal of these rate changes occurred during the "on" periods of DRT. The percentage of correlated pairs increased from 16.7% in the normal state to 46.9% after MPTP treatment and was restored to nearly normal values (25% correlated pairs) under the influence of DRT. These changes in rate and correlation were observed at both the population level and at the level of units recorded continuously before, during, and after the clinical transition from "off" to "on" periods. We conclude that changes in both pallidal discharge rates and synchronization are correlated with the clinical manifestations of parkinsonism and its pharmacological treatment.
In Parkinson's disease (PD), neurogenesis is impaired in the subventricular zone (SVZ) of postmortem human PD brains, in primate nonhuman and rodent models of PD. The vital role of Wingless-type MMTV integration site (Wnt)/β-catenin signaling in the modulation of neurogenesis, neuroprotection, and synaptic plasticity coupled to our recent findings uncovering an active role for inflammation and Wnt/β-catenin signaling in MPTP-induced loss and repair of nigrostriatal dopaminergic (DAergic) neurons prompted us to study the impact of neuroinflammation and the Wnt/β-catenin pathway in the response of SVZ neuroprogenitors (NPCs) in MPTP-treated mice. In vivo experiments, using bromodeoxyuridine and cell-specific markers, and ex vivo time course analyses documented an inverse correlation between the reduced proliferation of NPCs and the generation of new neuroblasts with the phase of maximal exacerbation of microglia reaction, whereas a shift in the microglia proinflammatory phenotype correlated with a progressive NPC recovery. Ex vivo and in vitro experiments using microglia-NPC coculture paradigms pointed to NADPH-oxidase (gpPHOX(91)), a major source of microglial ROS, and reactive nitrogen species as candidate inhibitors of NPC neurogenic potential via the activation of glycogen synthase 3 (pGSK-3β(Tyr216)), leading to loss of β-catenin, a chief downstream transcriptional effector. Accordingly, MPTP/MPP(+) (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) caused β-catenin downregulation and pGSK-3β(Tyr216) overexpression, whereas manipulation of Wnt/β-catenin signaling with RNA interference-mediated GSK-3β knockdown or GSK-3β antagonism reversed MPTP-induced neurogenic impairment ex vivo/in vitro or in vivo. Reciprocally, pharmacological modulation of inflammation prevented β-catenin downregulation and restored neurogenesis, suggesting the possibility to modulate this endogenous system with potential consequences for DAergic neuroprotection and self-repair.
Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the degeneration of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). Activation of the mixed lineage kinase and c-Jun N-terminal kinase (JNK) has been reported in models of PD. Our focus was to discern whether distinct pathways were activated in cell-specific manner within the SNpc. We now demonstrate the selective phosphorylation of p38 MAP kinase within the dopaminergic neurons, whereas JNK activation occurs predominantly in the microglia. p38 activation results in downstream phosphorylation of p53 and increased p53 mediated transcription of Bax and Puma in the ventral midbrain. Treatment with p38 inhibitor, SB239063 protected primary dopaminergic neurons derived from human progenitor cells from MPP(+) mediated cell death and prevented the downstream phosphorylation of p53 and its translocation to the nucleus in vivo, in the ventral midbrain. The increased staining of phosphorylated p38 in the surviving neurons of SNpc in human brain sections from patients with PD and in MPTP treated mice but not in the ventral tegmental area provides further evidence suggesting a role for p38 in the degeneration of dopaminergic neurons of SNpc. We thus demonstrate the cell specific activation of MAP kinase pathways within the SNpc after MPTP treatment emphasizing the role of multiple signaling cascades in the pathogenesis and progression of the disease. Selective inhibitors of p38 may therefore, help preserve the surviving neurons in PD and slow down the disease progression.
The effect of MDMA on motor activity ( a), motor disability ( b), chorea ( c), dystonia ( d), and dyskinesia ( e) induced by a high dose of L-DOPA (25 mg/kg) in the presence of 12.5 mg/kg carbidopa. Doubling the concentration of L-DOPA did not increase the motor activity significantly but led to a partial reversal of the inhibitory effect of MDMA (12 mg/kg). There was an earlier expression of motor disability and dyskinesia after the doubling of the L-DOPA dose, and a partial reversal of the anti-dyskinetic effect of MDMA. 
Ecstasy [3,4-methylenedioxymethamphetamine (MDMA)] was shown to prolong the action of L-3,4-dihydroxyphenylalanine (L-DOPA) while suppressing dyskinesia in a single patient with Parkinson's disease (PD). The clinical basis of this effect of MDMA is unknown but may relate to its actions on either dopaminergic or serotoninergic systems in brain. In normal, drug-naive common marmosets, MDMA administration suppressed motor activity and exploratory behavior. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated, L-DOPA-primed common marmosets, MDMA transiently relieved motor disability but over a period of 60 min worsened motor symptoms. When given in conjunction with L-DOPA, however, MDMA markedly decreased dyskinesia by reducing chorea and to a lesser extent dystonia and decreased locomotor activity to the level observed in normal animals. MDMA similarly alleviated dyskinesia induced by the selective dopamine D2/3 agonist pramipexole. The actions of MDMA appeared to be mediated through 5-HT mechanisms because its effects were fully blocked by the selective serotonin reuptake inhibitor fluvoxamine. Furthermore, the effect of MDMA on L-DOPA-induced motor activity and dyskinesia was partially inhibited by 5-HT1a/b antagonists. The ability of MDMA to inhibit dyskinesia results from its broad spectrum of action on 5-HT systems. Serotoninergic receptors appear to play an important modulatory role in l-DOPA-induced dyskinesia, and this study may provide a framework for the use of serotoninergic agents in the treatment of L-DOPA-induced dyskinesia.
To investigate the role of the basal ganglia in parkinsonian tremor, we recorded hand tremor and simultaneous activity of several neurons in the external and internal segments of the globus pallidus (GPe and GPi) in two vervet monkeys, before and after systemic treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and development of parkinsonism with tremor of 5 and 11 Hz. In healthy monkeys, only 11% (20/174) of the GPe cells and 3% (1/29) of the GPi cells displayed significant 3-19 Hz oscillations. After MPTP treatment, 39% (107/271) of the GPe cells and 43% (26/61) of the GPi cells developed significant oscillations. Oscillation frequencies of single cells after MPTP treatment were bimodally distributed around 7 and 13 Hz. For 10% of the oscillatory cells that were recorded during tremor periods, there was a significant tendency for the tremor and neuronal oscillations to appear simultaneously. Cross-correlation analysis revealed a very low level of correlated activity between pallidal neurons in the normal state; 95.6% (477/499) of the pairs were not correlated, and oscillatory cross-correlograms were found in only 1% (5/499) of the pairs. After MPTP treatment, the correlations increased dramatically, and 40% (432/1080) of the cross-correlograms had significant oscillations, centered around 13-14 Hz. Phase shifts of the cross-correlograms of GPe pairs, but not of GPi, were clustered around 0 degrees. The results illustrate that MPTP treatment changes the pattern of activity and synchronization in the GPe and GPi. These changes are related to the symptoms of Parkinson's disease and especially to the parkinsonian tremor.
Parkinson's disease (PD) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity are both associated with dopaminergic neuron death in the substantia nigra (SN). Apoptosis has been implicated in this cell loss; however, whether or not it is a major component of disease pathology remains controversial. Caspases are a major class of proteases involved in the apoptotic process. To evaluate the role of caspases in PD, we analyzed caspase activation in MPTP-treated mice, in cultured dopaminergic cells, and in postmortem PD brain tissue. MPTP was found to elicit not only the activation of the effector caspase-3 but also the initiators caspase-8 and caspase-9, mitochondrial cytochrome c release, and Bid cleavage in the SN of wild-type mice. These changes were attenuated in transgenic mice neuronally expressing the general caspase inhibitor protein baculoviral p35. These mice also displayed increased resistance to the cytotoxic effects of the drug. MPTP-associated toxicity in culture was found temporally to involve cytochrome c release, activation of caspase-9, caspase-3, and caspase-8, and Bid cleavage. Caspase-9 inhibition prevented the activation of both caspase-3 and caspase-8 and also inhibited Bid cleavage, but not cytochrome c release. Activated caspase-8 and caspase-9 were immunologically detectable within MPP(+)-treated mesencephalic dopaminergic neurons, dopaminergic nigral neurons from MPTP-treated mice, and autopsied Parkinsonian tissue from late-onset sporadic cases of the disease. These data demonstrate that MPTP-mediated activation of caspase-9 via cytochrome c release results in the activation of caspase-8 and Bid cleavage, which we speculate may be involved in the amplification of caspase-mediated dopaminergic cell death. These data suggest that caspase inhibitors constitute a plausible therapeutic for PD.
Distribution pattern of the phenotype "neostriatal dopamine depletion after MPTP administration." (C57BL/6 BALB/c)F 2 hybrids were treated with four doses of 15 mg MPTPHCl/g body weight in 2 hr intervals, and neostriatal DA levels were determined postmortem by HPLC, with electrochemical detection 7 d later. Absolute numbers of animals are shown in 10% intervals of DA depletion separately for males (n 179) and females (n 132). "Depletion" is defined as dopamine loss in percentage compared with controls. DA depletion was significantly higher in males than in females (t test; t 16.1; df 309; p 0.001). Saline-treated controls, which are C57BL/6 BALB/c)F 2 hybrids, had neostriatal dopamine levels of 15.23 0.41 and 15.28 0.40 for males (n 23) and females (n 24), respectively (nanograms of DA per milligram of wet tissue weight; means SEM). 
The degree of dopamine depletion by genotype for the D9Mit4 marker on Chr. 9 as a function of genotype at another locus (D3Mit147) on Chr. 3. An interaction is demonstrated by the fact that the phenotypic effect of the Chr. 9 QTL reverses direction as a function of the Chr. 3 modifier locus genotype at D3Mit147. Neither locus had a significant main effect in the MapManager analysis, which does not assess interactions, but emerged as significant only in the PAIRSCAN analysis mainly because of the strength of the interaction shown above ( p 0.0008). 
Loci identified using a conservative significance level by separate QTL analyses for each gender
Parkinson's disease (PD) is a neurodegenerative disorder characterized by the dysfunction of the nigrostriatal dopaminergic pathway. Although its etiology is not yet fully understood, an interaction of genetic predisposition and environmental factors is frequently discussed. The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) can evoke PD-like symptoms and neuropathological changes in various species, including mice. It was found repeatedly that mouse strains differ in their susceptibility to MPTP, which might serve as a model for genetic predisposition to neurodegeneration of the nigrostriatal system. In the present study, F2 intercross mice, derived from parental strains with high (C57BL/6J) versus low (BALB/cJ) MPTP susceptibility, were treated with MPTP and phenotyped for dopamine (DA) loss in the neostriatum, a highly sensitive marker of nigrostriatal dysfunction. A subsequent quantitative trait loci analysis revealed a gender-dependent locus for DA loss on chromosome 15 and a putative locus on chromosome 13. A number of potential candidate genes, including the membrane dopamine transporter, are located in the respective areas. Several mechanisms that are possibly involved in the control of the action of MPTP on the nigrostriatal system are discussed.
Glutathione peroxidase (GSHPx) is a critical intracellular enzyme involved in detoxification of hydrogen peroxide (H(2)O(2)) to water. In the present study we examined the susceptibility of mice with a disruption of the glutathione peroxidase gene to the neurotoxic effects of malonate, 3-nitropropionic acid (3-NP), and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Glutathione peroxidase knock-out mice showed no evidence of neuropathological or behavioral abnormalities at 2-3 months of age. Intrastriatal injections of malonate resulted in a significant twofold increase in lesion volume in homozygote GSHPx knock-out mice as compared to both heterozygote GSHPx knock-out and wild-type control mice. Malonate-induced increases in conversion of salicylate to 2,3- and 2, 5-dihydroxybenzoic acid, an index of hydroxyl radical generation, were greater in homozygote GSHPx knock-out mice as compared with both heterozygote GSHPx knock-out and wild-type control mice. Administration of MPTP resulted in significantly greater depletions of dopamine, 3,4-dihydroxybenzoic acid, and homovanillic acid in GSHPx knock-out mice than those seen in wild-type control mice. Striatal 3-nitrotyrosine (3-NT) concentrations after MPTP were significantly increased in GSHPx knock-out mice as compared with wild-type control mice. Systemic 3-NP administration resulted in significantly greater striatal damage and increases in 3-NT in GSHPx knock-out mice as compared to wild-type control mice. The present results indicate that a knock-out of GSHPx may be adequately compensated under nonstressed conditions, but that after administration of mitochondrial toxins GSHPx plays an important role in detoxifying increases in oxygen radicals.
During embryonic development, axons from sensory neurons in the olfactory epithelium (OE) extend into the olfactory bulb (OB) where they synapse with projection neurons and form glomerular structures. To determine whether glycans play a role in these processes, we analyzed mice deficient for the glycosyltransferase beta1,3-N-acetylglucosaminyltransferase 1 (beta3GnT1), a key enzyme in lactosamine glycan synthesis. Terminal lactosamine expression, as shown by immunoreactivity with the monoclonal antibody 1B2, is dramatically reduced in the neonatal null OE. Postnatal beta3GnT1-/- mice exhibit severely disorganized OB innervation and defective glomerular formation. Beginning in embryonic development, specific subsets of odorant receptor-expressing neurons are progressively lost from the OE of null mice, which exhibit a postnatal smell perception deficit. Axon guidance errors and increased neuronal cell death result in an absence of P2, I7, and M72 glomeruli, indicating a reduction in the repertoire of odorant receptor-specific glomeruli. By approximately 2 weeks of age, lactosamine is unexpectedly reexpressed in sensory neurons of null mice through a secondary pathway, which is accompanied by the regrowth of axons into the OB glomerular layer and the return of smell perception. Thus, both neonatal OE degeneration and the postnatal regeneration are lactosamine dependent. Lactosamine expression in beta3GnT1-/- mice is also reduced in pheromone-receptive vomeronasal neurons and dorsal root ganglion cells, suggesting that beta3GnT1 may perform a conserved function in multiple sensory systems. These results reveal an essential role for lactosamine in sensory axon pathfinding and in the formation of OB synaptic connections.
The beat frequency of the myogenic heart of the tobacco hawkmoth, Manduca sexta, markedly increases at adult emergence in response to 2 blood-borne peptide neurohormones, known as the cardioacceleratory peptides (CAP1 and CAP2). Three independent lines of evidence are presented supporting the hypothesis that the CAPs exert their cardiostimulatory effects on the insect myocardium through a change in the intracellular levels of inositol 1,4,5-trisphosphate (InsP3). I show that (1) InsP3 levels increase in response to CAP2 in a timely fashion, (2) exogenous application of InsP3 mimics the effects of CAP2 application, and (3) a blocker of InsP3 metabolism inhibits the effect of CAP2. These results provide strong support for the hypothesis that InsP3 is likely to be the second messenger in the regulation of heart beat activity by CAP2. Besides establishing the nature of the signaling system between CAP2 and the heart, these data also identify a novel role for InsP3, namely, the control of contraction frequency in a myogenic muscle. Given the widespread distribution of cellular systems employing InsP3 as a second messenger, it is suggested that InsP3 may also be involved in the long-term regulation of rhythmic activity in other spontaneously contractile muscles and endogenously active cells.
Type 1 inositol (1,4,5)-trisphosphate receptors (InsP3R1s) play a major role in neuronal calcium (Ca2+) signaling. The InsP3R1s are phosphorylated by protein kinase A (PKA), but the functional consequences of InsP3R1 phosphorylation and the mechanisms that control the phosphorylated state of neuronal InsP3R1s are poorly understood. In a yeast two-hybrid screen of rat brain cDNA library with the InsP3R1-specific bait, we isolated the protein phosphatase 1alpha (PP1alpha). In biochemical experiments, we confirmed the specificity of the InsP3R1-PP1alpha association and immunoprecipitated the InsP3R1-PP1 complex from rat brain synaptosomes and from the neostriatal lysate. We also established that the association with PP1 facilitates dephosphorylation of PKA-phosphorylated InsP3R1 by the endogenous neostriatal PP1 and by the recombinant PP1alpaha. We demonstrated that exposure of neostriatal slices to 8-bromo-cAMP, dopamine, calyculin A, or cyclosporine A, but not to 10 nM okadaic acid, promotes the phosphorylation of neostriatal InsP3R1 by PKA in vivo. We discovered that PKA activates and PP1alpha inhibits the activity of recombinant InsP3R1 reconstituted into planar lipid bilayers. We found that phosphorylation of InsP3R1 by PKA induces at least a fourfold increase in the sensitivity of InsP3R1 to activation by InsP3 without shifting the peak of InsP3R1 bell-shaped Ca2+ dependence. Based on these data, we suggest that InsP3R1 may participate in cross talk between cAMP and Ca2+ signaling in the neostriatum and possibly in other regions of the brain.
The inositol 1,4,5-trisphosphate (InsP3) receptor type I (InsP3R-I) is the principle channel for intracellular calcium (Ca2+) release in many cell types, including central neurons. It is regulated by endogenous compounds like Ca2+ and ATP, by protein partners, and by posttranslational modification. We report that the InsP3R-I is modified by O-linked glycosylation of serine or threonine residues with beta-N-acetylglucosamine (O-GlcNAc). The level of O-GlcNAcylation can be altered in vitro by the addition of the enzymes which add [OGT (O-GlcNActransferase)] or remove (O-GlcNAcase) this sugar or by loading cells with UDP-GlcNAc. We monitored the effects of this modification on InsP3R function at the single-channel level and on intracellular Ca2+ transients. Single-channel activity was monitored with InsP3R incorporated into bilayers; Ca2+ signaling was monitored using cells loaded with a Ca2+-sensitive fluorophore. We found that channel activity was decreased by the addition of O-GlcNAc and that this decrease was reversed by removal of the sugar. Similarly, cells loaded with UDP-GlcNAc had an attenuated response to uncaging of InsP3. These results show that O-GlcNAcylation is an important regulator of the InsP3R-I and suggest a mechanism for neuronal dysfunction under conditions in which O-GlcNAc is high, such as diabetes or physiological stress.
The opisthotonos (opt) mutation arose spontaneously in a C57BL/Ks-db2J colony and is the only known, naturally occurring allele of opt. This mutant mouse was first identified based on its ataxic and convulsive phenotype. Genetic and molecular data presented here demonstrate that the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) protein, which serves as an IP3-gated channel to release calcium from intracellular stores, is altered in the opt mutant. A genomic deletion in the IP3R1 gene removes two exons from the IP3R1 mRNA but does not interrupt the translational reading frame. The altered protein is predicted to have lost several modulatory sites and is present at markedly reduced levels in opt homozygotes. Nonetheless, a strong calcium release from intracellular stores can be elicited in cerebellar Purkinje neurons treated with the metabotropic glutamate receptor (mGluR) agonist quisqualate (QA). QA activates Group 1 mGluRs linked to GTP-binding proteins that stimulate phospholipase C and subsequent production of the intracellular messenger IP3, leading to calcium mobilization via the IP3R1 protein. The calcium response in opt homozygotes shows less attenuation to repeated QA application than in control littermates. These data suggest that the convulsions and ataxia observed in opt mice may be caused by the physiological dysregulation of a functional IP3R1 protein.
We developed new biochemical approaches to demonstrate the presence of inositol 1,4,5-triphosphate (InsP3)-gated calcium channels in presynaptic plasma membranes (SPM) and their involvement in the presynaptic receptor-mediated Ca2+ influx into nerve terminals. In perfusion experiments using SPM vesicles preloaded with 45Ca2+, InsP3 elicited the release of 45CA2+ into perfusates in a saturable manner. The InsP3- evoked 45Ca2+ release from resealed SPM vesicles was more potent than that from resealed vesicles using any other subcellular fractions. Here we also report the involvement of InsP3-gated mechanisms in the presynaptic receptor-mediated Ca2+ influx into synaptosomes (nerve terminals) by use of such resealed vesicles reconstituted with purified Gi1.
In many excitatory glutamatergic synapses, both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are closely distributed on the postsynaptic membrane. However, the functional significance of the close distribution of the two types of glutamate receptors has not been fully clarified. In this study, we examined the functional interaction between iGluR and mGluR at parallel fiber (PF)--> Purkinje cell synapses in the generation of inositol 1,4,5-trisphosphate (IP3), a key second messenger that regulates many important cellular functions. We visualized local IP3 dynamics in Purkinje cells using the green fluorescent protein-tagged pleckstrin homology domain (GFP-PHD) as a fluorescent IP3 probe. Purkinje cells were transduced with Sindbis virus encoding GFP-PHD and imaged with a two-photon laser scanning microscope. Translocation of GFP-PHD from the plasma membrane to the cytoplasm attributable to an increase in IP3 concentration was observed on PF stimulation in fine dendrites of Purkinje cells. Surprisingly, this PF-induced IP3 production was blocked not only by the group I mGluR antagonist but also by the AMPA receptor (AMPAR) antagonist. The PF-induced IP3 production was blocked by either the inhibition of G-protein activation by GDP-betaS or intracellular Ca2+ buffering by BAPTA. These results show that IP3 production is mediated cooperatively by group I mGluR and AMPAR through G-protein activation and Ca2+ influx at PF--> Purkinje cell synapses, identifying the robust cross talk between iGluR and mGluR for the generation of IP3 signals.
Ionic Ca2+ functions as a second messenger to control several intracellular processes. It also influences intercellular communication. The release of Ca2+ from intracellular stores through the inositol 1,4,5-trisphosphate receptor (InsP3R) occurs in both excitable and nonexcitable cells. In Drosophila, InsP3R activity is required in aminergic interneurons during pupal development for normal flight behavior. By altering intracellular Ca2+ and InsP3 levels through genetic means, we now show that signaling through the InsP3R is required at multiple steps for generating the neural circuit required in air puff-stimulated Drosophila flight. Decreased Ca2+ release in aminergic neurons during development of the flight circuit can be compensated by reducing Ca2+ uptake from the cytosol to intracellular stores. However, this mode of increasing intracellular Ca2+ is insufficient for maintenance of flight patterns over time periods necessary for normal flight. Our study suggests that processes such as maintenance of wing posture and formation of the flight circuit require InsP3 receptor function at a slow timescale and can thus be modulated by altering levels of cytosolic Ca2+ and InsP3. In contrast, maintenance of flight patterns probably requires fast modulation of Ca2+ levels, in which the intrinsic properties of the InsP3R play a pivotal role.
The type 1 metabotropic glutamate receptor (mGluR1) is through to act via the phosphoinositide (PI) system with the associated formation of inositol 1,4,5-trisphosphate (IP3) and Ca2+ release. Utilizing immunohistochemistry and in situ hybridization, we have localized protein and mRNA, respectively, for the mGluR1 and the IP3 receptor (IP3R). We have also localized glutamate-linked PI turnover by autoradiography with 3H-cytidine. We observe a striking contrast in localizations of mGluR1 and IP3R both for protein and mRNA. For instance, mGluR1 occurs in the apparent absence of IP3R in neurons of the stratum oriens of the CA1 hippocampus, islands of Calleja, anterodorsal nucleus of thalamus, lateral nucleus of hypothalamus, and the granular cell layer and the deep nuclei of cerebellum. mGluR1 actions in these brain regions may primarily be mediated through the protein kinase C limb of the PI system, as they contain moderate amounts of 3H-phorbol ester binding. The subthalamic nucleus, red nucleus, and Darkshevich's nucleus, which possess high levels of mGluR1, are devoid of both IP3R immunoreactivity and 3H-phorbol ester binding. These reciprocal localizations suggest that mGluR1 actions in many brain areas may not primarily involve IP3, reflecting instead influences on protein kinase C or other second messengers.
Here, we show that cultured Purkinje cells from inositol 1,4,5-trisphosphate receptor type 1 knock-out (IP3R1KO) mice exhibited abnormal dendritic morphology. Interestingly, despite the huge amount of IP3R1 expression in Purkinje cells, IP3R1 in granule cells, not in the Purkinje cells, was responsible for the shape of Purkinje cell dendrites. We also found that BDNF application rescued the dendritic abnormality of IP3R1KO Purkinje cells, and that the increase in BDNF expression in response to activation of AMPA receptor (AMPAR) and metabotropic glutamate receptor (mGluR) was impaired in IP3R1KO cerebellar granule cells. In addition, we observed abnormalities in the dendritic morphology of Purkinje cells and in the ultrastructure of parallel fiber-Purkinje cell (PF-PC) synapses in IP3R1KO mice in vivo. We concluded that activation of AMPAR and mGluR increases BDNF expression through IP3R1-mediated signaling in cerebellar granule cells, which contributes to the dendritic outgrowth of Purkinje cells intercellularly, possibly by modifying PF-PC synaptic efficacy.
Distribution of immunoreactivity for IP,R, PLC& and PLCr in perikarya and neuropil of the primate basal ganglia 
The neurochemical organization of the basal ganglia has been studied extensively with respect to neurotransmitters, neuropeptides, and their receptors. The chemoarchitecture of the striatum has been found particularly striking, because it distinguishes many substances by their relative distributions within the striosome and matrix compartments of the striatum. Very little is yet known about the differential distribution of second messenger systems in the basal ganglia, however, and no information is available about whether the distribution of second messenger systems is related to the prominent neurochemical compartmentalization of the striatum. We have examined the distribution of the phosphoinositide second messenger system in the primate basal ganglia and substantia nigra, as detected with polyclonal antisera against the inositol 1,4,5-trisphosphate receptor (IP3R), and monoclonal antisera against phospholipase C beta (PLC beta) and phospholipase C gamma (PLC gamma). In the striatum, immunostaining for each of the three proteins was present predominantly in medium-sized neuronal perikarya and in the neuropil. Circumscribed zones of enhanced IP3R, PLC beta, and PLC gamma immunoreactivity appeared in a background of generally weaker staining, and these zones corresponded to striosomes as identified by calbinidin D28k and substance P immunostaining in adjacent sections. Thus, the richest representation of the phosphoinositide system in the primate striatum appears to be in striosomes. In the substantia nigra pars compacta, neurons and neuropil were immunopositive, but in the substantia nigra pars reticulata and in each segment of the globus pallidus, immunostaining was mainly confined to the neuropil. Perikaryal PCL gamma immunoreactivity in the absence of detectable PLC beta or IP3R immunolabeling was found in the magnocellular neurons embedded in the medullary layer between the putamen and the globus pallidus. These observations demonstrate that the phosphoinositide second messenger system is selectively enhanced in neuronal subsystems of the basal ganglia, including striosomes, and suggest that signaling by phosphoinositide pathways elicits discrete effects on input-output processing by the basal ganglia.
Ca2+ signals associated with action potentials (APs) and metabotropic glutamate receptor (mGluR) activation exert distinct influences on neuronal activity and synaptic plasticity. However, it is not clear how these two types of Ca2+ signals are differentially regulated by neurotransmitter inputs in a single neuron. We investigated this issue in dopaminergic neurons of the ventral midbrain using brain slices. Intracellular Ca2+ was assessed by measuring Ca2+-sensitive K+ currents or imaging the fluorescence of Ca2+ indicator dyes. Tonic activation of metabotropic neurotransmitter receptors (mGluRs, alpha1 adrenergic receptors, and muscarinic acetylcholine receptors), attained by superfusion of agonists or weak, sustained (approximately 1 s) synaptic stimulation, augmented AP-induced Ca2+ transients. In contrast, Ca2+ signals elicited by strong, transient (50-200 ms) activation of mGluRs with aspartate iontophoresis were suppressed by superfusion of agonists. These opposing effects on Ca2+ signals were both mediated by an increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels, because they were blocked by heparin, an IP3 receptor antagonist, and reproduced by photolytic application of IP3. Evoking APs repetitively at low frequency (2 Hz) caused inactivation of IP3 receptors and abolished IP3 facilitation of single AP-induced Ca2+ signals, whereas facilitation of Ca2+ signals triggered by bursts of APs (five at 20 Hz) was attenuated by less than half. We further obtained evidence suggesting that the psychostimulant amphetamine may augment burst-induced Ca2+ signals via both depression of basal firing and production of IP3. We propose that intracellular IP3 tone provides a mechanism to selectively amplify burst-induced Ca2+ signals in dopaminergic neurons.
Top-cited authors
Eric J Nestler
  • Icahn School of Medicine at Mount Sinai
Raymond J Dolan
  • University College London
Eliezer Masliah
  • University of California, San Diego
Trevor W Robbins
  • University of Cambridge
Ronald S Duman
  • Yale University