Macroautophagy can press a brake on presynaptic neurotransmission

The mechanistic target of rapamycin (MTOR) has been implicated in regulating synaptic plasticity and neurodegeneration, but MTOR’s role in modulating presynaptic function through autophagy is unexplored. We studied presynaptic function in ventral dopamine neurons, a system from which neurotransmitter release can be measured directly by cyclic voltammetry. We generated mutant mice that were specifically deficient for macroautophagy in dopaminergic neurons by deleting the Atg7 gene in cells that express the dopamine uptake transporter. Dopamine axonal profiles in the mutant dorsal striatum were ~one third larger in the mutant mice, released ~50% more stimulus-evoked dopamine release, and exhibited more rapid presynaptic recovery than controls. Rapamycin reduced dopamine neuron axon profile size by ~30% in control mice, but had no effect on macroautophagy deficient axons. Acute rapamycin decreased dopaminergic synaptic vesicle density by ~25% and inhibited evoked dopamine release by ~25% in control mice, but not in the Atg7 deficient mutants. Thus, both basal and induced macroautophagy can provide a brake on presynaptic activity in vivo, perhaps by regulating the turnover of synaptic vesicles, and further regulates terminal volume and the kinetics of transmitter release.     

Macroautophagy can press a brake on presynaptic neurotransmission

The mechanistic target of rapamycin (MTOR) has been implicated in regulating synaptic plasticity and neurodegeneration, but MTOR’s role in modulating presynaptic function through autophagy is unexplored. We studied presynaptic function in ventral dopamine neurons, a system from which neurotransmitter release can be measured directly by cyclic voltammetry. We generated mutant mice that were specifically deficient for macroautophagy in dopaminergic neurons by deleting the Atg7 gene in cells that express the dopamine uptake transporter. Dopamine axonal profiles in the mutant dorsal striatum were ~one third larger in the mutant mice, released ~50% more stimulus-evoked dopamine release, and exhibited more rapid presynaptic recovery than controls. Rapamycin reduced dopamine neuron axon profile size by ~30% in control mice, but had no effect on macroautophagy deficient axons. Acute rapamycin decreased dopaminergic synaptic vesicle density by ~25% and inhibited evoked dopamine release by ~25% in control mice, but not in the Atg7 deficient mutants. Thus, both basal and induced macroautophagy can provide a brake on presynaptic activity in vivo, perhaps by regulating the turnover of synaptic vesicles, and further regulates terminal volume and the kinetics of transmitter release.     

Impaired dopamine release and synaptic plasticity in the striatum of Parkin−/− mice

Parkin is the most common causative gene of juvenile and early-onset familial Parkinson's diseases and is thought to function as an E3 ubiquitin ligase in the ubiquitin-proteasome system. However, it remains unclear how loss of Parkin protein causes dopaminergic dysfunction and nigral neurodegeneration. To investigate the pathogenic mechanism underlying these mutations, we used parkin −/− mice to study its physiological function in the nigrostriatal circuit. Amperometric recordings showed decreases in evoked dopamine release in acute striatal slices of parkin −/− mice and reductions in the total catecholamine release and quantal size in dissociated chromaffin cells derived from parkin −/− mice. Intracellular recordings of striatal medium spiny neurons revealed impairments of long-term depression and long-term potentiation in parkin −/− mice, whereas long-term potentiation was normal in the Schaeffer collateral pathway of the hippocampus. Levels of dopamine receptors and dopamine transporters were normal in the parkin −/− striatum. These results indicate that Parkin is involved in the regulation of evoked dopamine release and striatal synaptic plasticity in the nigrostriatal pathway, and suggest that impairment in evoked dopamine release may represent a common pathophysiological change in recessive parkinsonism.     

How addictive drugs disrupt presynaptic dopamine neurotransmission

The fundamental principle that unites addictive drugs appears to be that each enhances synaptic dopamine by means that dissociate it from normal behavioral control, so that they act to reinforce their own acquisition. This occurs via the modulation of synaptic mechanisms that can be involved in learning, including enhanced excitation or disinhibition of dopamine neuron activity, blockade of dopamine reuptake, and altering the state of the presynaptic terminal to enhance evoked over basal transmission. Amphetamines offer an exception to such modulation in that they combine multiple effects to produce nonexocytic stimulation-independent release of neurotransmitter via reverse transport independent from normal presynaptic function. Questions about the molecular actions of?addictive drugs, prominently including the actions of alcohol and solvents, remain unresolved, but their ability to co-opt normal presynaptic functions helps to explain why treatment for addiction has been challenging.     

Coordinate Regulation of Mature Dopaminergic Axon Morphology by Macroautophagy and the PTEN Signaling Pathway

Macroautophagy is a conserved mechanism for the bulk degradation of proteins and organelles. Pathological studies have implicated defective macroautophagy in neurodegeneration, but physiological functions of macroautophagy in adult neurons remain unclear. Here we show that Atg7, an essential macroautophagy component, regulates dopaminergic axon terminal morphology. Mature Atg7-deficient midbrain dopamine (DA) neurons harbored selectively enlarged axonal terminals. This contrasted with the phenotype of DA neurons deficient in Pten – a key negative regulator of the mTOR kinase signaling pathway and neuron size – that displayed enlarged soma but unaltered axon terminals. Surprisingly, concomitant deficiency of both Atg7 and Pten led to a dramatic enhancement of axon terminal enlargement relative to Atg7 deletion alone. Similar genetic interactions between Atg7 and Pten were observed in the context of DA turnover and DA-dependent locomotor behaviors. These data suggest a model for morphological regulation of mature dopaminergic axon terminals whereby the impact of mTOR pathway is suppressed by macroautophagy.     

Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice

Recessively inherited loss-of-function mutations in the parkin , DJ-1 , or PINK1 gene are linked to familial cases of early-onset Parkinson's diseases (PD), and heterozygous mutations are associated with increased incidence of late-onset PD. We previously reported that single knockout mice lacking Parkin, DJ-1, or PINK1 exhibited no nigral degeneration, even though evoked dopamine release from nigrostriatal terminals was reduced and striatal synaptic plasticity was impaired. In this study, we tested whether inactivation of all three recessive PD genes, each of which was required for nigral neuron survival in the aging human brain, resulted in nigral degeneration during the lifespan of mice. Surprisingly, we found that triple knockout mice lacking Parkin, DJ-1, and PINK1 have normal morphology and numbers of dopaminergic and noradrenergic neurons in the substantia nigra and locus coeruleus, respectively, at the ages of 3, 16, and 24 months. Interestingly, levels of striatal dopamine in triple knockout mice were normal at 16 months of age but increased at 24 months. These results demonstrate that inactivation of all three recessive PD genes is insufficient to cause significant nigral degeneration within the lifespan of mice, suggesting that these genes may be protective rather than essential for the survival of dopaminergic neurons during the aging process. These findings also support the notion that mammalian Parkin and PINK1 may function in the same genetic pathway as in Drosophila .     

Persistent Posttetanic Depression at Cerebellar Parallel Fiber to Purkinje Cell Synapses

Plasticity at the cerebellar parallel fiber to Purkinje cell synapse may underlie information processing and motor learning. In vivo, parallel fibers appear to fire in short high frequency bursts likely to activate sparsely distributed synapses over the Purkinje cell dendritic tree. Here, we report that short parallel fiber tetanic stimulation evokes a ∼7–15% depression which develops over 2 min and lasts for at least 20 min. In contrast to the concomitantly evoked short-term endocannabinoid-mediated depression, this persistent posttetanic depression (PTD) does not exhibit a dependency on the spatial pattern of synapse activation and is not caused by any detectable change in presynaptic calcium signaling. This persistent PTD is however associated with increased paired-pulse facilitation and coefficient of variation of synaptic responses, suggesting that its expression is presynaptic. The chelation of postsynaptic calcium prevents its induction, suggesting that post- to presynaptic (retrograde) signaling is required. We rule out endocannabinoid signaling since the inhibition of type 1 cannabinoid receptors, monoacylglycerol lipase or vanilloid receptor 1, or incubation with anandamide had no detectable effect. The persistent PTD is maximal in pre-adolescent mice, abolished by adrenergic and dopaminergic receptors block, but unaffected by adrenergic and dopaminergic agonists. Our data unveils a novel form of plasticity at parallel fiber synapses: a persistent PTD induced by physiologically relevant input patterns, age-dependent, and strongly modulated by the monoaminergic system. We further provide evidence supporting that the plasticity mechanism involves retrograde signaling and presynaptic diacylglycerol.     

Effects of mercury compounds on the spontaneous and potassium-evoked release of [3H]dopamine from mouse striatal slices

The effects of mercury compounds on the spontaneous and potassium-evoked release of [3H]dopamine from mouse striatal slices have been examined. All mercury compounds examined produced concentration-dependent increases in the spontaneous release of [3H]dopamine, with an order of potency of methylmercury greater than mercuric (Hg2+) mercury greater than p-choloromercuribenzene sulfonic acid. Methylmercury had no effect on the 25 mM potassium evoked release of [3H]dopamine in the presence of 1.3 mM calcium. However, in calcium-free conditions, methylmercury significantly increased the potassium-evoked release of [3H]dopamine. Mercuric mercury significantly reduced the 25 mM potassium evoked release of [3H]dopamine in the presence of 1.3 mM calcium, and this response was not reversible with brief washing of the tissue. In calcium-free conditions, mercuric mercury significantly elevated the evoked release of [3H]dopamine, similar to the result obtained with methylmercury. It is suggested that mercury compounds alter dopaminergic synaptic function, possibly by disrupting calcium homeostasis or calcium-dependent processes, and that methylmercury and mercuric mercury can have differential effects to alter dopaminergic neurotransmission.     

Mechanisms underlying the rapid induction and sustained expression of synaptic homeostasis

Homeostatic signaling systems are thought to interface with the mechanisms of neural plasticity to achieve stable yet flexible neural circuitry. However, the time course, molecular design, and implementation of homeostatic signaling remain poorly defined. Here we demonstrate that a homeostatic increase in presynaptic neurotransmitter release can be induced within minutes following postsynaptic glutamate receptor blockade. The rapid induction of synaptic homeostasis is independent of new protein synthesis and does not require evoked neurotransmission, indicating that a change in the efficacy of spontaneous quantal release events is sufficient to trigger the induction of synaptic homeostasis. Finally, both the rapid induction and the sustained expression of synaptic homeostasis are blocked by mutations that disrupt the pore-forming subunit of the presynaptic Ca(V)2.1 calcium channel encoded by cacophony. These data confirm the presynaptic expression of synaptic homeostasis and implicate presynaptic Ca(V)2.1 in a homeostatic retrograde signaling system.     

Presynaptic regulation of dopamine release by beta-phenylethylamine

Experiments with local perfusion of the rat neostriatum and subsequent chromatography of the perfusate have shown that addition of beta-phenylethylamine (beta-PEA) to the perfusion medium in a concentration of 10(-3) M enhanced spontaneous and inhibited the K+-induced release of 3H-dopamine preliminarily applied to the neostriatum. The stimulating effect of beta-PEA was Ca2+-dependent and was potentiated in sodium-free media. The inhibitory effect of beta-PEA on the K+-induced release of 3H-DA was abolished by haloperidol, a blocker of dopamine receptors. This fact allows one to suggest that this effect of beta-PEA is mediated by presynaptic dopamine autoreceptors. The data obtained indicate that beta-PEA can modulate the dopaminergic synaptic transmission depending on functional activity of dopaminergic neurons.     

"Passive stabilization" of striatal extracellular dopamine across the lesion spectrum encompassing the presymptomatic phase of Parkinson's disease: a voltammetric study in the 6-OHDA-lesioned rat

Symptoms of Parkinson's disease do not present until the degeneration of nigrostriatal dopaminergic neurons is nearly complete. Maintenance of dopaminergic tone governing striatal efferents is postulated to preserve motor control during the presymptomatic phase, but the neuroadaptation responsible for normalization is not completely understood. In particular, the prevailing view that surviving dopaminergic neurons compensate by up-regulating release has been difficult to demonstrate directly. Here we investigate dopaminergic neurotransmission in the hemiparkinsonian rat using fast-scan cyclic voltammetry at carbon-fiber microelectrodes. Electrical stimulation was used to elicit extracellular dopamine levels mimicking the steady-state dynamics of tonic dopaminergic signaling. In agreement with microdialysis studies, evoked steady-state dopamine levels remained constant over the entire lesion spectrum (0 to approximately 85%) observed during the presymptomatic stage. Kinetic analysis of the voltammetric recordings demonstrated that evoked dopamine concentrations were normalized without plasticity of dopamine release and uptake, suggesting that the primary mechanisms controlling ambient levels of extracellular dopamine were not actively altered. In the present study, we formalize this neuroadaptation as "passive stabilization" . We further propose that passive stabilization is mediated by the simple physical principles of diffusion and steady state, is predicated on extrasynaptic transmission, and forms the basis for a new compensation model of preclinical parkinsonism.     

Dopamine transporter cell surface localization facilitated by a direct interaction with the dopamine D2 receptor

Altered synaptic dopamine levels have been implicated in several neurological/neuropsychiatric disorders, including drug addiction and schizophrenia. However, it is unclear what precipitates these changes in synaptic dopamine levels. One of the key presynaptic components involved in regulating dopaminergic tone is the dopamine transporter (DAT). Here, we report that the DAT is also regulated by the dopamine D2 receptor through a direct protein-protein interaction involving the DAT amino-terminus and the third intracellular loop of the D2 receptor. This physical coupling facilitates the recruitment of intracellular DAT to the plasma membrane and leads to enhanced dopamine reuptake. Moreover, mice injected with peptides that disrupt D2-DAT interaction exhibit decreased synaptosomal dopamine uptake and significantly increased locomotor activity, reminiscent of DAT knockout mice. Our data highlight a novel mechanism through which neurotransmitter receptors can functionally modulate neurotransmitter transporters, an interaction that can affect the synaptic neurotransmitter levels in the brain.     

Hydrogen peroxide induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells

A novel mechanism for H?O?-induced autophagic cell death in GSH-depleted RAW 264.7 cells, a murine macrophage cell line, is proposed. Under GSH-depleted conditions, H?O?-induced autophagic cell, characterized by an increased LC3-II/I ratio, a decreased level of p62 and the formation of autophagic vacuoles, was inhibited by bafilomycin A1 and by Atg5 siRNA transfection, whereas the cell death was not inhibited by zVAD-fmk, by PI3K inhibitors or by Beclin 1 siRNA transfection. In addition, H?O? treatment reduced the activity of mTOR and promoted the ubiquitination and degradation of Rheb, a key upstream activator of mTOR. Furthermore, proteasome inhibition with MG132 restored the expression of Rheb and increased mTOR activity, resulting in an increased viability of H?O?-treated cells. Collectively, these findings demonstrate that H?O? induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells.     

Effects of Cannabinoids on Dopamine Release in the Corpus Striatum and the Nucleus Accumbens In Vitro

Cannabinoid receptors are widely distributed in the nuclei of the extrapyramidal motor and mesolimbic reward systems; their exact functions are, however, not known. The aim of the present study was to characterize the effects of cannabinoids on the electrically evoked release of endogenous dopamine in the corpus striatum and the nucleus accumbens. In rat brain slices dopamine release elicited by single electrical pulses was determined by fast cyclic voltammetry. Dopamine release was markedly inhibited by the OP2 opioid receptor agonist U-50488 and the D2/D3 dopamine receptor agonist quinpirole, indicating that our method is suitable for studying presynaptic modulation of dopamine release. In contrast, the CB1/CB2 cannabinoid receptor agonists WIN55212-2 (10(-6) M) and CP55940 (10(-6)-10(-5) M) and the CB1 cannabinoid receptor antagonist SR141716A (10(-6) M) had no effect on the electrically evoked dopamine release in the corpus striatum and the nucleus accumbens. The lack of a presynaptic effect on terminals of nigrostriatal and mesolimbic dopaminergic neurons is in accord with the anatomical distribution of cannabinoid receptors: The perikarya of these neurons in the substantia nigra and the ventral tegmental area do not synthesize mRNA, and hence protein, for CB1 and CB2 cannabinoid receptors. It is therefore unlikely that presynaptic modulation of dopamine release in the corpus striatum and the nucleus accumbens plays a role in the extrapyramidal motor and rewarding effects of cannabinoids.     

MPTP modulates hippocampal synaptic transmission and activity-dependent synaptic plasticity via dopamine receptors

Parkinson's disease (PD)-like symptoms and cognitive deficits are inducible by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Since cognitive abilities, including memory formations rely also on hippocampus, we set out to clarify the effects of MPTP on hippocampal physiology. We show that bath-application of MPTP (25?μM) to acute hippocampal slices enhanced AMPA receptor-mediated field excitatory postsynaptic potentials (AMPAr-fEPSPs) transiently, whereas N-methyl-D-aspartate (NMDA) receptor-mediated fEPSPs (NMDAr-fEPSPs) were facilitated persistently. The MPTP-mediated transient AMPAr-fEPSP facilitation was antagonized by the dopamine D2-like receptor antagonists, eticlopride (1?μM) and sulpiride (1 and 40?μM). In contrast, the persistent enhancement of NMDAr-fEPSPs was prevented by the dopamine D1-like receptor antagonist SCH23390 (10?μM). In addition, we show that MPTP decreased paired-pulse facilitation of fEPSPs and mEPSCs frequency. Regarding activity-dependent synaptic plasticity, 25?μM MPTP transformed short-term potentiation (STP) into a long-term potentiation (LTP) and caused a slow onset potentiation of a non-tetanized synaptic input after induction of LTP in a second synaptic input. This heterosynaptic slow onset potentiation required activation of dopamine D1-like and NMDA-receptors. We conclude that acute MPTP application affects basal synaptic transmission by modulation of presynaptic vesicle release and facilitates NMDAr-fEPSPs as well as activity-dependent homo- and heterosynaptic plasticity under participation of dopamine receptors.     

Extrasynaptic release of dopamine in a retinal neuron: activity dependence and transmitter modulation

Extrasynaptic release of dopamine is well documented, but its relation to the physiological activity of the neuron is unclear. Here we show that in absence of presynaptic active zones, solitary cell bodies of retinal dopaminergic neurons release by exocytosis packets of approximately 40,000 molecules of dopamine at irregular intervals and low frequency. The release is triggered by the action potentials that the neurons generate in a rhythmic fashion upon removal of all synaptic influences and therefore depends upon the electrical events at the neuronal surface. Furthermore, it is stimulated by kainate and abolished by GABA and quinpirole, an agonist at the D(2) dopamine receptor. Since the somatic receptors for these ligands are extrasynaptic, we suggest that the composition of the extracellular fluid directly modulates extrasynaptic release.     

Preserved morphology and physiology of excitatory synapses in profilin1-deficient mice

Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity.     

The Ig cell adhesion molecule Basigin controls compartmentalization and vesicle release at Drosophila melanogaster synapses

Synapses can undergo rapid changes in size as well as in their vesicle release function during both plasticity processes and development. This fundamental property of neuronal cells requires the coordinated rearrangement of synaptic membranes and their associated cytoskeleton, yet remarkably little is known of how this coupling is achieved. In a GFP exon-trap screen, we identified Drosophila melanogaster Basigin (Bsg) as an immunoglobulin domain-containing transmembrane protein accumulating at periactive zones of neuromuscular junctions. Bsg is required pre- and postsynaptically to restrict synaptic bouton size, its juxtamembrane cytoplasmic residues being important for that function. Bsg controls different aspects of synaptic structure, including distribution of synaptic vesicles and organization of the presynaptic cortical actin cytoskeleton. Strikingly, bsg function is also required specifically within the presynaptic terminal to inhibit nonsynchronized evoked vesicle release. We thus propose that Bsg is part of a transsynaptic complex regulating synaptic compartmentalization and strength, and coordinating plasma membrane and cortical organization.     

Evidence for plasticity of the dopaminergic system in Parkinsonism

A series of compensatory mechanisms within the dopaminergic system have been shown to maintain clinical function in the presence of dopamine loss. Experimental evidence for increased presynaptic dopamine turnover owing to increased dopamine synthesis, release, and reduced reuptake exists. Direct evidence that these mechanisms maintain extracellular dopamine levels is provided by intracerebral microdialysis techniques. Postsynaptic denervation supersensitivity clearly occurs with D2 dopamine receptors, although this is less evident with D1 receptors. Similarly, mechanisms of plasticity have been shown to be relevant in human postmortem and Positron Emission Tomographic studies of patients with Parkinson's disease. However, although presynaptic increases in dopamine turnover are well documented, postsynaptic D1 and D2 receptor changes have been more difficult to establish, mainly because of methodological difficulties. D2, but not D1, receptor increases have been documented in drug naive Parkinsonian patients with PET techniques. In transplantation of adrenal gland to striatum in animal models and patients with Parkinsonism where clinical improvement occurs, plasticity of host response may be as important as plasticity of the graft. Although some elements of the compensatory mechanism of dopamine plasticity may be deleterious, such as dyskinesias owing to dopamine receptor supersensitivity, the overall effect of delay and minimization of the clinical expression of disease is advantageous. An even greater understanding of the mechanisms involved may assist in developing future therapeutic strategies.     

Effect of Dopaminergic D1 Receptors on Plasticity Is Dependent of Serotoninergic 5-HT1A Receptors in L5-Pyramidal Neurons of the Prefrontal Cortex

Major depression and schizophrenia are associated with dysfunctions of serotoninergic and dopaminergic systems mainly in the prefrontal cortex (PFC). Both serotonin and dopamine are known to modulate synaptic plasticity. 5-HT_1A receptors (5-HT_1ARs) and dopaminergic type D1 receptors are highly represented on dendritic spines of layer 5 pyramidal neurons (L5PyNs) in PFC. How these receptors interact to tune plasticity is poorly understood. Here we show that D1-like receptors (D1Rs) activation requires functional 5HT_1ARs to facilitate LTP induction at the expense of LTD. Using 129/Sv and 5-HT_1AR-KO mice, we recorded post-synaptic currents evoked by electrical stimulation in layer 2/3 after activation or inhibition of D1Rs. High frequency stimulation resulted in the induction of LTP, LTD or no plasticity. The D1 agonist markedly enhanced the NMDA current in 129/Sv mice and the percentage of L5PyNs displaying LTP was enhanced whereas LTD was reduced. In 5-HT_1AR-KO mice, the D1 agonist failed to increase the NMDA current and orientated the plasticity towards L5PyNs displaying LTD, thus revealing a prominent role of 5-HT_1ARs in dopamine-induced modulation of plasticity. Our data suggest that in pathological situation where 5-HT_1ARs expression varies, dopaminergic treatment used for its ability to increase LTP could turn to be less and less effective.