Reserve Pool Research Articles

Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca2+ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.

A Bcl-xL–Drp1 complex regulates synaptic vesicle membrane dynamics during endocytosis

Following exocytosis, the rate of recovery of neurotransmitter release is determined by vesicle retrieval from the plasma membrane and by recruitment of vesicles from reserve pools within the synapse, the latter of which is dependent on mitochondrial ATP. The Bcl-2 family protein Bcl-xL, in addition to its role in cell death, regulates neurotransmitter release and recovery in part by increasing ATP availability from mitochondria. We now find, however, that, Bcl-xL directly regulates endocytotic vesicle retrieval in hippocampal neurons through protein/protein interaction with components of the clathrin complex. Our evidence suggests that, during synaptic stimulation, Bcl-xL translocates to clathrin-coated pits in a calmodulin-dependent manner and forms a complex of proteins with the GTPase Drp1, Mff and clathrin. Depletion of Drp1 produces misformed endocytotic vesicles. Mutagenesis studies suggest that formation of the Bcl-xL-Drp1 complex is necessary for the enhanced rate of vesicle endocytosis produced by Bcl-xL, thus providing a mechanism for presynaptic plasticity.

Insights on altered mitochondrial function and dynamics in the pathogenesis of neurodegeneration

In neurons, mitochondria are enriched to provide energy and calcium buffering required for synaptic transmission. Additionally, mitochondria localize to the synapse, where they are critical for the mobilization of reserve pool vesicles and for neurotransmitter release. Previously, functional defects in mitochondria were considered to be downstream effects of neurodegenerative diseases. However, more recent findings suggest mitochondria may serve as key mediators in the onset and progression of some types of neurodegeneration. In this review, we explore the possible roles of altered mitochondrial function and dynamics in the pathogenesis of neurodegenerative disorders, with a particular focus on Alzheimer’s disease (AD) and Parkinson’s disease (PD), which have highlighted the important role of mitochondria in neurodegeneration. While inheritable diseases like Charcot-Marie-Tooth disease type 2A are concretely linked to gene mutations affecting mitochondrial function, the cause of mitochondrial dysfunction in primarily sporadic diseases such as AD and PD is less clear. Neuronal death in PD is associated with defects in mitochondrial function and dynamics arising from mutations in proteins affecting these processes, including α-synuclein, DJ-1, LRRK2, Parkin and Pink1. In the case of AD, however, the connection between mitochondria and the onset of neurodegeneration has been less clear. Recent findings, however, have implicated altered function of ER-mitochondria contact sites and amyloid beta- and/or tau-induced defects in mitochondrial function and dynamics in the pathogenesis of AD, suggesting that mitochondrial defects may act as key mediators in the pathogenesis of AD as well. With recent findings at hand, it may be postulated that defects in mitochondrial processes comprise key events in the onset of neurodegeneration.

BDNF-Dependent Recycling Facilitates TrkB Translocation to Postsynaptic Density during LTP via a Rab11-Dependent Pathway

Brain-derived neurotrophic factor (BDNF) plays an important role in the activity-dependent regulation of synaptic structure and function via tropomyosin related kinase B (TrkB) receptor activation. However, whether BDNF could regulate TrkB levels at synapse during long-term potentiation (LTP) is still unknown. We show in cultured rat hippocampal neurons that chemical LTP (cLTP) stimuli selectively promote endocytic recycling of BDNF-dependent full-length TrkB (TrkB-FL) receptors, but not isoform T1 (TrkB.T1) receptors, via a Rab11-dependent pathway. Moreover, neuronal-activity-enhanced TrkB-FL recycling could facilitate receptor translocation to postsynaptic density and enhance BDNF-induced extracellular signal-regulated kinase and phosphatidylinositol 3-kinase activation and rat hippocampal neuron survival. Finally, we found that cLTP could stimulate the switch of Rab11 from an inactive to an active form and that GTP-bound Rab11 could enhance the interaction between TrkB-FL and PSD-95. Therefore, the recycling endosome could serve as a reserve pool to supply TrkB-FL receptors for LTP maintenance. These findings provide a mechanistic link between Rab11-dependent endocytic recycling and TrkB modulation of synaptic plasticity.

Most Vesicles in a Central Nerve Terminal Participate in Recycling

Studies over the last decade using FM dyes to label vesicles at many terminals, including the calyx-type nerve terminal, led to a well accepted "principle" that only a small fraction of vesicles (∼5-20%) participate in recycling under physiological conditions. This principle imposes a large challenge in maintaining synaptic transmission during repetitive firing, because the small recycling pool may limit the number of available vesicles for release and nerve terminals would have to distinguish the recycling pool from the reserve pool and keep reserve pool vesicles from being used. By recording the presynaptic capacitance changes and the postsynaptic EPSC at rat calyx of Held synapses in the absence or presence of transmitter glutamate in nerve terminals, we developed a new method to count functional recycling vesicles. We found that essentially all vesicles in calyces participated in recycling, challenging the small-recycling-pool principle established by FM dye labeling. Nerve terminals may use all available vesicles to maximize their ability in maintaining synaptic transmission during repetitive firing.

Rapid feedback regulation of synaptic efficacy during high-frequency activity at theDrosophilalarval neuromuscular junction

High-frequency firing of neurons depresses transmitter release at many synapses. At the glutamatergic synapse of the Drosophila larval neuromuscular junction, we find that presynaptic depression is modulated by postsynaptic ionotropic glutamate receptor (iGluR) activity. Although basal release at low frequency was insensitive to postsynaptic iGluR activity, recovery from depression elicited by high-frequency presynaptic trains decreased with partial block of native iGluRs. Moreover, recovery from depression increased with optical activation of the light-gated mammalian iGluR6 (LiGluR) expressed postsynaptically. The enhancement of recovery from depression occurred within 2 min of optical activation of LiGluR and persisted for minutes after optical deactivation. This effect depended on cAMP-dependent presynaptic recruitment of vesicles from the reserve pool. Our findings reveal a unique dimension to postsynaptic iGluR activity: fast retrograde signaling that preserves transmission efficacy during high-frequency presynaptic firing.

Amphetamine elicits opposing actions on readily releasable and reserve pools for dopamine.

Amphetamine, a highly addictive drug with therapeutic efficacy, exerts paradoxical effects on the fundamental communication modes employed by dopamine neurons in modulating behavior. While amphetamine elevates tonic dopamine signaling by depleting vesicular stores and driving non-exocytotic release through reverse transport, this psychostimulant also activates phasic dopamine signaling by up-regulating vesicular dopamine release. We hypothesized that these seemingly incongruent effects arise from amphetamine depleting the reserve pool and enhancing the readily releasable pool. This novel hypothesis was tested using in vivo voltammetry and stimulus trains of varying duration to access different vesicular stores. We show that amphetamine actions are stimulus dependent in the dorsal striatum. Specifically, amphetamine up-regulated vesicular dopamine release elicited by a short-duration train, which interrogates the readily releasable pool, but depleted release elicited by a long-duration train, which interrogates the reserve pool. These opposing actions of vesicular dopamine release were associated with concurrent increases in tonic and phasic dopamine responses. A link between vesicular depletion and tonic signaling was supported by results obtained for amphetamine in the ventral striatum and cocaine in both striatal sub-regions, which demonstrated augmented vesicular release and phasic signals only. We submit that amphetamine differentially targeting dopamine stores reconciles the paradoxical activation of tonic and phasic dopamine signaling. Overall, these results further highlight the unique and region-distinct cellular mechanisms of amphetamine and may have important implications for its addictive and therapeutic properties.

High-Resolution Scanning Patch Clamp

The cardiomyocyte plasma membrane has 2 jobs: to conduct the action potential along its length and to transduce that action potential into the synchronous increase of cytosolic calcium, which triggers contraction. Ion channels mediate these events at the surface sarcolemma and along highly ordered membrane invaginations known as T-tubules. The types of channels present in the membrane can be identified with immunocytochemistry or electron microscopy, but most are only approximately assigned to specific domains. Moreover, the resulting images likely represent mixed populations of both functional and silent channels, as well as those in reserve pools waiting to be recruited under changing physiological or pathological conditions. Because many disease conditions involve the coordinate perturbation of membrane microstructure and electrical activity, there is much to be learned by probing channel function with greater spatial resolution along the cardiomyocyte. In this issue of Circulation Research , Bhargava et al1 describe a novel method for doing just that and report some surprising discoveries. Article, see p 1112 Scanning ion conductance microscopy2,3 has evolved in recent years to provide a wide range of cellular landscapes reminiscent of the 3-dimensional images of scanning electron microscopy, but in living tissue. The probe is an electrolyte-filled pipette that reports its proximity to the cell membrane as a decrease in conductance. It gets close to the membrane, within a distance equivalent to the internal diameter of the pipette (<100 nm). The probe is lifted and translated (it hops) from 1 point to another along the x and y coordinates, with peaks and valleys registered as different values in the z dimension at which the conductance drops. The result is …

Physiological separation of vesicle pools in low- and high-output nerve terminals

Physiological differences in low- (tonic like) and high-output (phasic like) synapses match many of the expected anatomical features of these terminals. However, investigation in the recruitment of synaptic vesicles from a reserve pool (RP) to a readily releasable pool (RRP) of synaptic vesicles within these types of nerve terminals has not been fully addressed. This study highlights physiological differences and differential modulation of the vesicles in a RP for maintaining synaptic output during evoked depression of the RRP. With the use of bafilomycin A1, a vacuolar ATPase blocker, recycling vesicles are blocked in refilling with transmitter. The tonic terminal is fatigue resistant due to a large RRP, whereas the phasic depresses rapidly upon continuous stimulation. These differences in rates of depression appear to be in the size and degree of utilization of the RRP of vesicles. The working model is that upon depression of the tonic terminal, serotonin (5-HT) has a large RP to act on in order to recruit vesicles to the RRP; whereas, the phasic terminal, 5-HT can recruit RP vesicles to the RRP prior to synaptic depression but not after depression. The vesicle pools are physiologically differentiated between phasic and tonic output terminals.

Fruit production in three masting tree species does not rely on stored carbon reserves

Fruiting is typically considered to massively burden the seasonal carbon budget of trees. The cost of reproduction has therefore been suggested as a proximate factor explaining observed mast-fruiting patterns. Here, we used a large-scale, continuous (13)C labeling of mature, deciduous trees in a temperate Swiss forest to investigate to what extent fruit formation in three species with masting reproduction behavior (Carpinus betulus, Fagus sylvatica, Quercus petraea) relies on the import of stored carbon reserves. Using a free-air CO2 enrichment system, we exposed trees to (13)C-depleted CO2 during 8 consecutive years. By the end of this experiment, carbon reserve pools had significantly lower δ(13)C values compared to control trees. δ(13)C analysis of new biomass during the first season after termination of the CO2 enrichment allowed us to distinguish the sources of built-in carbon (old carbon reserves vs. current assimilates). Flowers and expanding leaves carried a significant (13)C label from old carbon stores. In contrast, fruits and vegetative infructescence tissues were exclusively produced from current, unlabeled photoassimilates in all three species, including F. sylvatica, which had a strong masting season. Analyses of δ(13)C in purified starch from xylem of fruit-bearing shoots revealed a complete turn-over of starch during the season, likely due to its usage for bud break. This study is the first to directly demonstrate that fruiting is independent from old carbon reserves in masting trees, with significant implications for mechanistic models that explain mast seeding.

Regulation in Reserve

Sequestered microRNAs serve as a regulatory reserve pool to enable quiescent cells to effectively respond to mitogenic signals.

Tracking carbon within the trees

Tracking carbon within the trees

Overexpression of synapsin Ia in the rat calyx of Held accelerates short-term plasticity and decreases synaptic vesicle volume and active zone area

Synapsins are synaptic vesicle (SV) proteins organizing a component of the reserve pool of vesicles at most central nervous system synapses. Alternative splicing of the three mammalian genes results in multiple isoforms that may differentially contribute to the organization and maintenance of the SV pools. To address this, we first characterized the expression pattern of synapsin isoforms in the rat calyx of Held. At postnatal day 16, synapsins Ia, Ib, IIb and IIIa were present, while IIa—known to sustain repetitive transmission in glutamatergic terminals—was not detectable. To test if the synapsin I isoforms could mediate IIa-like effect, and if this depends on the presence of the E-domain, we overexpressed either synapsin Ia or synapsin Ib in the rat calyx of Held via recombinant adeno-associated virus-mediated gene transfer. Although the size and overall structure of the perturbed calyces remained unchanged, short-term depression and recovery from depression were accelerated upon overexpression of synapsin I isoforms. Using electron microscopic three-dimensional reconstructions we found a redistribution of SV clusters proximal to the active zones (AZ) alongside with a decrease of both AZ area and SV volume. The number of SVs at individual AZs was strongly reduced. Hence, our data indicate that the amount of synapsin Ia expressed in the calyx regulates the rate and extent of short-term synaptic plasticity by affecting vesicle recruitment to the AZ. Finally, our study reveals a novel contribution of synapsin Ia to define the surface area of AZs.

LTP requires a reserve pool of glutamate receptors independent of subunit type

SummaryLong-term potentiation (LTP) of synaptic transmission is thought to be a key cellular mechanism underlying memory formation. A widely accepted model posits that LTP requires the cytoplasmic tail of the AMPA receptor subunit GluA1. To find the minimum necessary requirement of the GluA1 C-tail for LTP in CA1 hippocampal pyramidal neurons, we used a single-cell molecular replacement strategy to replace all endogenous AMPA receptors with transfected subunits. In striking contrast to the prevailing model, we found no requirement of the GluA1 C-tail for LTP. In fact, replacement with the GluA2 subunit showed normal LTP, as did an artificially expressed kainate receptor not normally found at these synapses. The only conditions under which LTP was impaired were those with dramatically decreased AMPA receptor surface expression, indicating a requirement for a reserve pool of receptors. These results demonstrate the synapse’s remarkable flexibility to potentiate with a variety of glutamate receptor subtypes, requiring a fundamental change in our thinking with regard to the core molecular events underlying synaptic plasticity.

Neuromuscular physiology and pharmacology: an update

This consists of the terminal part of the motor neurone which originates from the ventral horn of the spinal cord, losing its myelin as it nears the muscle fibre. Here, the Schwann cell (SC) anchors the nerve to the muscle membrane. SCs play an important role in the maintenance of nerve homeostasis. In addition to providing stability and secreting growth and trophic factors, they also participate in axon development and synaptic formation from the fetal state and throughout life. SCs control the number of NMJs and remove superfluous presynaptic nerve terminals, especially during re-innervation, for instance, after crush injury. The mechanism involves interaction with the immune molecules, major histocompatibility Class 1a (MHC Class 1a), present in motor neurones which mediate non-immune modulation of synaptic function and plasticity. In the absence of MHC Class 1a, NMJ organization is disturbed, and recovery of motor nerve lesions is delayed. The presynaptic terminal also contains AChRs on the surface of the nerve membrane. These are nicotinic receptors identified as neuronal nAChR (a3b2). Non-depolarizing and depolarizing neuromuscular blocking agents (NMBAs) act on these receptors, altering the mobilization of ACh: the former inhibit them and the latter stimulate them. Such mobilization involves the acquisition and synthesis of ACh, its storage in reserve vesicles, and its release by a nerve action potential. ACh is stored in two pools: in vesicles in the reserve pool which contain abundant mitochondria and microtubules to synthesize neurotransmitter; and as readily releasable vesicles. The release of ACh from the readily releasable pool on arrival of a nerve impulse results in sodium channel activation on the prejunctional nerve membrane. This in turn activates voltagedependent calcium channels (P-type fast channels) on the motor neurone causing an influx of calcium into the nerve cytoplasm that promotes further ACh release. Three proteins, synaptobrevin, syntaxin, and synaptosome-associated protein SNAP-25, are involved in the attachment of ACh vesicles to the presynaptic cell membrane. These proteins along with vesicle membrane-associated synaptotagmins cause the docking, fusion, and release (exocytosis) of neurotransmitter from the vesicles. The P-type calcium channels are in contrast to the L-type (slow channels) which are present in the heart. There is evidence that L-type calcium channels may also be present on the nerve terminals which could explain why the action of non-depolarizing NMBAs is prolonged by calcium channel blockers. By blocking the L-type channels, these drugs may prevent calcium accumulation within the prejunctional membrane and thus release of ACh from the vesicles. The P-type calcium channels are blocked by cations such as magnesium, cadmium, and manganese. By blocking calcium entry, magnesium sulphate reduces ACh release from the vesicles, resulting in a reduction in muscle tone. Antibodies to the calcium channels may develop in some forms of cancer (Eaton–Lambert syndrome in small cell lung cancer) causing muscle weakness. An increase in calcium concentration in the prejunctional cytoplasm triggers ACh release. The calcium inflow is balanced by Key points The functioning of the neuromuscular junction (NMJ) and the release of acetylcholine to stimulate the post-synaptic nicotinic receptors is dependent on the interaction of several proteins such as agrin and muscle-specific tyrosine kinase, which are responsible for the formation and maintenance of the NMJ. A new non-depolarizing agent, gantacurium, is being investigated as a replacement for succinylcholine. It is degraded in the plasma non-enzymatically by endogenous L-cysteine. The new antagonist to rocuronium, sugammadex, can reverse profound block when given in the correct dose immediately after rocuronium. Postoperative residual curarization is still common. A train-of-four ratio of .0.9 is necessary before extubation to prevent postoperative respiratory complications.

Synapsins Contribute to the Dynamic Spatial Organization of Synaptic Vesicles in an Activity-Dependent Manner

The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III(-/-) triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity.

The regulation and packaging of synaptic vesicles as related to recruitment within glutamatergic synapses

The reserve pool (RP) and readily releasable pool (RRP) of synaptic vesicles within presynaptic nerve terminals, at crayfish and larval Drosophila neuromuscular junctions (NMJs), were examined for physiological differentiation into distinctly separate functional groups. These NMJs are glutamatergic and produce graded excitatory postsynaptic potentials (EPSPs). The packaging of glutamate was perturbed by blocking the vesicular glutamate transporter (VGlut) with bafilomycin A1. Various frequencies of motor nerve stimulation, exposure time, and concentration of bafilomycin A1 were examined. The low-output tonic opener NMJs in crayfish exposed to 4μM bafilomycin A1 and 20-Hz continuous stimulation decreased the EPSP amplitude to 50% in ∼30min with controls lasting 3h. After activity and bafilomycin A1-induced synaptic depression, the EPSPs were rapidly revitalized by serotonin (5-HT, 1μM) in the crayfish preparations. The 5-HT action can be blocked with a PLC inhibitor. We postulate 5-HT recruits unused vesicles from the RP. The perception is the RRP is selectively activated during rapid electrical stimulation (20Hz) sparing the RP. When stimulation frequency is high (40Hz) the RP is recruited to the RRP and dampens subsequent recruitment with 5-HT. The higher output synapses of the larval Drosophila NMJ when stimulated at 1Hz or 5Hz and exposed to 4μM of bafilomycin A1 showed a depression rate of 50% within ∼10min with controls lasting ∼40min. After low frequency depression and/or exposure to bafilomycin A1 a burst of higher frequency (10Hz) can recruit vesicles from the RP to the RRP.

Remodeling of Lipid Vesicles into Cylindrical Micelles by α-Synuclein in an Extended α-Helical Conformation

α-Synuclein (αS) is a protein with multiple conformations and interactions. Natively unfolded in solution, αS accumulates as amyloid in neurological tissue in Parkinson disease and interacts with membranes under both physiological and pathological conditions. Here, we used cryoelectron microscopy in conjunction with electron paramagnetic resonance (EPR) and other techniques to characterize the ability of αS to remodel vesicles. At molar ratios of 1:5 to 1:40 for protein/lipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol), large spherical vesicles are converted into cylindrical micelles ~50 Å in diameter. Other lipids of the same charge (negative) exhibit generally similar behavior, although bilayer tubes of 150-500 Å in width are also produced, depending on the lipid acyl chains. At higher protein/lipid ratios, discoid particles, 70-100 Å across, are formed. EPR data show that, on cylindrical micelles, αS adopts an extended amphipathic α-helical conformation, with its long axis aligned with the tube axis. The observed geometrical relationship between αS and the micelle suggests that the wedging of its long α-helix into the outer leaflet of a membrane may cause curvature and an anisotropic partition of lipids, leading to tube formation.

Presenilin conditional double knockout mice exhibit decreases in drebrin a at hippocampal CA1 synapses.

Presenilin conditional double knockout (PScDKO) mice have been used as animal models to study the development of Alzheimer's disease (AD) phenotypes. Studies to date indicate that these animals exhibit memory dysfunction and decreased synaptic plasticity before the onset of neurodegeneration. Therefore, the current study sought to examine how the loss of presenilin expression leads to these defects. Drebrin A, a neuron-specific actin-binding protein, has been shown to play an important role in the activity-dependent redistribution of the NMDA type of glutamate receptors at the synapse which, in turn, is a critical step for enabling synaptic plasticity. It is hypothesized that defects in the activity dependent redistribution of NMDA receptors in PScDKO mice may be due to loss of drebrin A. In this study, electron microscopic immunocytochemistry (EM-ICC) was used to quantify and locate drebrin A in the CA1 field of the hippocampus of PScDKO mice. The high resolution of EM-ICC allowed for differentiation between drebrin A at the synapse and at nonsynaptic sites, the latter of which would reflect the protein's role in regulating the reserve or degradative pool of NMDA receptors. The results here demonstrate that loss of function of presenilin in mice leads to a decrease in immunoreactivity for drebrin A at both synaptic (54% decrease, P < 0.01) and nonsynaptic areas (40% decrease, P < 0.01) and overall (44% decrease, P < 0.01). The reduction of drebrin A in both synaptic and nonsynaptic locations of the spine may implicate impairment in glutamate receptor trafficking to and from the postsynaptic plasma membrane. In addition, because of reduced drebrin A at nonsynaptic sites, the regulation of the reserve and degradative pools of glutamate receptors may also be impaired, leading to further synaptic dysfunction. Therefore, this study provides evidence to the theory that drebrin A has a causal role in compromising activity-dependent glutamate receptor trafficking in PScDKO mice.

Attenuated response to methamphetamine sensitization and deficits in motor learning and memory after selective deletion of β-catenin in dopamine neurons

In the present study, we analyzed mice with a targeted deletion of β-catenin in DA neurons (DA-βcat KO mice) to address the functional significance of this molecule in the shaping of synaptic responses associated with motor learning and following exposure to drugs of abuse. Relative to controls, DA-βcat KO mice showed significant deficits in their ability to form long-term memories and displayed reduced expression of methamphetamine-induced behavioral sensitization after subsequent challenge doses with this drug, suggesting that motor learning and drug-induced learning plasticity are altered in these mice. Morphological analyses showed no changes in the number or distribution of tyrosine hydroxylase-labeled neurons in the ventral midbrain. While electrochemical measurements in the striatum determined no changes in acute DA release and uptake, a small but significant decrease in DA release was detected in mutant animals after prolonged repetitive stimulation, suggesting a possible deficit in the DA neurotransmitter vesicle reserve pool. However, electron microscopy analyses did not reveal significant differences in the content of synaptic vesicles per terminal, and striatal DA levels were unchanged in DA-βcat KO animals. In contrast, striatal mRNA levels for several markers known to regulate synaptic plasticity and DA neurotransmission were altered in DA-βcat KO mice. This study demonstrates that ablation of β-catenin in DA neurons leads to alterations of motor and reward-associated memories and to adaptations of the DA neurotransmitter system and suggests that β-catenin signaling in DA neurons is required to facilitate the synaptic remodeling underlying the consolidation of long-term memories.