Somatodendritic organization of pacemaker activity in midbrain dopamine neurons.

The slow and regular pacemaking activity of midbrain dopamine (DA) neurons requires proper spatial organization of the excitable elements between the soma and dendritic compartments, but the somatodendritic organization is not clear. Here, we show that the dynamic interaction between the soma and multiple proximal dendritic compartments (PDCs) generates the slow pacemaking activity in DA neurons. In multipolar DA neurons, spontaneous action potentials (sAPs) consistently originate from the axon-bearing dendrite. However, when the axon initial segment was disabled, sAPs emerge randomly from various primary PDCs, indicating that multiple PDCs drive pacemaking. Ca2+ measurements and local stimulation/perturbation experiments suggest that the soma serves as a stably-oscillating inertial compartment, while multiple PDCs exhibit stochastic fluctuations and high excitability. Despite the stochastic and excitable nature of PDCs, their activities are balanced by the large centrally-connected inertial soma, resulting in the slow synchronized pacemaking rhythm. Furthermore, our electrophysiological experiments indicate that the soma and PDCs, with distinct characteristics, play different roles in glutamate- induced burst-pause firing patterns. Excitable PDCs mediate excitatory burst responses to glutamate, while the large inertial soma determines inhibitory pause responses to glutamate. Therefore, we could conclude that this somatodendritic organization serves as a common foundation for both pacemaker activity and evoked firing patterns in midbrain DA neurons.

Proximal dendritic localization of NALCN channels underlies tonic and burst firing in nigral dopaminergic neurons.

In multipolar nigral dopamine (DA) neurons, the highly excitable proximal dendritic compartments (PDCs) and two Na+ -permeable leak channels, TRPC3 and NALCN, play a key role in pacemaking. However, the causal link between them is unknown. Here we report that the proximal dendritic localization of NALCN underlies pacemaking and burst firing in DA neurons. Our morphological analysis of nigral DA neurons reveals that TRPC3 is ubiquitously expressed in the whole somatodendritic compartment, but NALCN is localized within the PDCs. Blocking either TRPC3 or NALCN channels abolished pacemaking. However, only blocking NALCN, not TRPC3, degraded burst discharges. Furthermore, local glutamate uncaging readily induced burst discharges within the PDCs, compared with other parts of the neuron, and NALCN channel inhibition dissipated burst generation, indicating the importance of NALCN to the high excitability of PDCs. Therefore, we conclude that PDCs serve as a common base for tonic and burst firing in nigral DA neurons. KEY POINTS: Midbrain dopamine (DA) neurons are slow pacemakers that can generate tonic and burst firings, and the highly excitable proximal dendritic compartments (PDCs) and two Na+ -permeable leak channels, TRPC3 and NALCN, play a key role in pacemaking. We find that slow tonic firing depends on the basal activity of both the NALCN and TRPC3 channels, but that burst firing does not require TRPC3 channels but relies only on NALCN channels. We find that TRPC3 is ubiquitously expressed in the entire somatodendritic compartment, but that NALCN exists only within the PDCs in nigral DA neurons. We show that NALCN channel localization confers high excitability on PDCs and is essential for burst generation in nigral DA neurons. These results suggest that PDCs serve as a common base for tonic and burst firing in nigral DA neurons.

Regional difference in spontaneous firing inhibition by GABAA and GABAB receptors in nigral dopamine neurons.

GABAergic control over dopamine (DA) neurons in the substantia nigra is crucial for determining firing rates and patterns. Although GABA activates both GABAA and GABAB receptors distributed throughout the somatodendritic tree, it is currently unclear how regional GABA receptors in the soma and dendritic compartments regulate spontaneous firing. Therefore, the objective of this study was to determine actions of regional GABA receptors on spontaneous firing in acutely dissociated DA neurons from the rat using patch-clamp and local GABA-uncaging techniques. Agonists and antagonists experiments showed that activation of either GABAA receptors or GABAB receptors in DA neurons is enough to completely abolish spontaneous firing. Local GABA-uncaging along the somatodendritic tree revealed that activation of regional GABA receptors limited within the soma, proximal, or distal dendritic region, can completely suppress spontaneous firing. However, activation of either GABAA or GABAB receptor equally suppressed spontaneous firing in the soma, whereas GABAB receptor inhibited spontaneous firing more strongly than GABAA receptor in the proximal and distal dendrites. These regional differences of GABA signals between the soma and dendritic compartments could contribute to our understanding of many diverse and complex actions of GABA in midbrain DA neurons.

Stress hormone potentiates Zn(2+)-induced neurotoxicity via TRPM7 channel in dopaminergic neuron.

Zinc toxicity is one of the key factors responsible for the neuronal injuries associated with various neurological conditions. Zinc accumulation in some cells is accompanied by the increase of blood stress hormone levels, which might indicate a functional connection between stress and zinc toxicity. However, the cellular mechanism for the effect of stress on zinc toxicity is not known. Recently, it was reported that the zinc permeable transient receptor potential melastatin 7 (TRPM7) channel may represent a novel target for neurological disorders where zinc toxicity plays an important role. To investigate the effect of stress hormone on zinc-induced cell death, neuroblastoma SH-SY5Y cells were pretreated with urocortin, a corticotropin releasing factor (CRF)-related peptide. Urocortin potentiated zinc-induced cell death at µM range of extracellular zinc concentrations. It significantly increased TRPM7 channel expression, and zinc influx into cytosol. Moreover, application of TRPM7 channel blockers and RNA interference of TRPM7 channel expression attenuated the zinc-induced cell death in urocortin-pretreated cells, indicating that TRPM7 channel may serve as a zinc influx pathway. These results suggest that TRPM7 channel may play a critical role for zinc toxicity associated with stress.

Regulation of basal autophagy by transient receptor potential melastatin 7 (TRPM7) channel.

Macroautophagy (hereafter referred to as autophagy) is a catabolic process for the degradation and recycling of cellular components. Autophagy digests intracellular components, recycling material subsequently used for new protein synthesis. The Ca(2+)- and Mg(2+)-permeable transient receptor potential melastatin 7 (TRPM7) channel underlies the constitutive Ca(2+) influx in some cells. Since autophagy is regulated by cytosolic Ca(2+) level, we set out to determine whether Ca(2+) influx through the TRPM7 channel regulates basal autophagy. When TRPM7 channel expression was induced from HEK293 cells in a nutrient-rich condition, LC3-II level increased indicating the increased level of basal autophagy. The effect of TRPM7 channel on basal autophagy was via Ca(2+)/calmodulin-dependent protein kinase kinase ß, and AMP-activated protein kinase pathway. In contrast, the level of basal autophagy was decreased when the endogenous TRPM7 channel in SH-SY5Y cells was down-regulated using short hairpin RNA. Similarly, an inhibitor for TRPM7 channel decreased the level of basal autophagy. In addition, the inhibitory effect of channel inhibitor on basal autophagy was reversed by increasing extracellular Ca(2+)concentration, suggesting that Ca(2+) influx through TRPM7 channel directly links to basal autophagy. Thus, our studies demonstrate the new role of TRPM7 channel-mediated Ca(2+) entry in the regulation of basal autophagy.

Threonine 576 residue of amyloid-ß precursor protein regulates its trafficking and processing.

Deposition of amyloid-ß (Aß) in the brain is the main culprit of Alzheimer's disease (AD). Aß is derived from sequential proteolytic cleavage of amyloid-ß precursor protein (APP). Newly synthesized APP is transported from endoplasmic reticulum to the plasma membrane via trans-Golgi network (TGN) after post-translational modification including N- and O-glycosylation. APP is internalized through clathrin-dependent endocytosis from the plasma membrane to the early endosomes. In this study, we investigated the regulation of APP trafficking and processing by mutating three threonine residues known as O-glycosylation sites. We separately mutated three threonine residues of APP695 into alanines (T291A, T292A, and T576A) and expressed them in HeLa cells. Among these APP mutants, only T576A mutant showed reduced cell surface levels, indicating this residue regulates its trafficking. We also confirmed that trafficking from TGN to the plasma membrane was decreased in T576A mutant. Consistent with these observations, T576A mutant accumulated in the early endosomes, and the secreted Aß level was increased. Thus, these results indicate that threonine 576 residue of APP regulates its trafficking and processing.

Citrobacter bitternis sp. nov. isolated from bitterns.

In this study, we reported two gram-negative bacteria that were isolated from bitterns, designated as SKKU-TP7(T) and SKKU-TP20, representing a novel species of Citrobacter. Based on the 16S rRNA gene sequences, the two strains were found to be closely related and showed the highest pairwise similarity with Citrobacter farmeri CDC 2992-81(T) (97.1-97.3 %) and other Citrobacter species. Cellular fatty acid analysis revealed that the profiles of strains SKKU-TP7(T) and SKKU-TP20 were similar to those of related species of Citrobacter. The major cellular fatty acids were C16:0 (31.5 %), summed feature 3 (C16:1 ω7c, C16:1 ω6c, 19.7 %), summed feature 8 (C18:1 ω7c, C18:1 ω6c, 11.9 %), C17:0 cyclo (10.7 %), and summed feature 2 (C12:0 aldehyde/unknown 10928, 9.5 %). Although the strains could utilize sucrose and raffinose as a carbon source, they did not produce ornithine decarboxylase and urease. The biochemical and genotypic characteristics indicate that strains SKKU-TP7(T) and SKKU-TP20 represent a novel species of Citrobacter, for which the name Citrobacter bitterns sp. nov. is proposed. The type strain is SKKU-TP7(T) (=KCTC 42139(T) = JCM 30009(T)).

Balance between the proximal dendritic compartment and the soma determines spontaneous firing rate in midbrain dopamine neurons.

Midbrain dopamine (DA) neurons are slow intrinsic pacemakers that require the elaborate composition of many ion channels in the somatodendritic compartments. Understanding the major determinants of the spontaneous firing rate (SFR) of midbrain DA neurons is important because they determine the basal DA levels in target areas, including the striatum. As spontaneous firing occurs synchronously at the soma and dendrites, the electrical coupling between the soma and dendritic compartments has been regarded as a key determinant for the SFR. However, it is not known whether this somatodendritic coupling is served by the whole dendritic compartments or only parts of them. In the rat substantia nigra pars compacta (SNc) DA neurons, we demonstrate that the balance between the proximal dendritic compartment and the soma determines the SFR. Isolated SNc DA neurons showed a wide range of soma size and a variable number of primary dendrites but preserved a quite consistent SFR. The SFR was not correlated with soma size or with the number of primary dendrites, but it was strongly correlated with the area ratios of the proximal dendritic compartments to the somatic compartment. Tetrodotoxin puff and local Ca(2+) perturbation experiments, computer simulation, and local glutamate uncaging experiments suggest the importance of the proximal dendritic compartments in pacemaker activity. These data indicate that the proximal dendritic compartments, not the whole dendritic compartments, play a key role in the somatodendritic balance that determines the SFR in DA neurons.

Cholesterol inhibits M-type K+ channels via protein kinase C-dependent phosphorylation in sympathetic neurons.

M-type (KCNQ) potassium channels play an important role in regulating the action potential firing in neurons. Here, we investigated the effect of cholesterol on M current in superior cervical ganglion (SCG) sympathetic neurons, using the patch clamp technique. M current was inhibited in a dose-dependent manner by cholesterol loading with a methyl-beta-cyclodextrin-cholesterol complex. This effect was prevented when membrane cholesterol level was restored by including empty methyl-beta-cyclodextrin in the pipette solution. Dialysis of cells with AMP-PNP instead of ATP prevented cholesterol action on M currents. Protein kinase C (PKC) inhibitor, calphostin C, abolished cholesterol-induced inhibition whereas the PKC activator, PDBu, mimicked the inhibition of M currents by cholesterol. The in vitro kinase assay showed that KCNQ2 subunits of M channel can be phosphorylated by PKC. A KCNQ2 mutant that is defective in phosphorylation by PKC failed to show current inhibition not only by PDBu but also by cholesterol. These results indicate that cholesterol-induced inhibition of M currents is mediated by PKC phosphorylation. The inhibition of M currents by PDBu and cholesterol was completely blocked by PIP(2) loading, indicating that the decrease in PIP(2)-channel interaction underlies M channel inhibition by PKC-mediated phosphorylation. We conclude that cholesterol specifically regulates M currents in SCG neurons via PKC activation.

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta.

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain.

TRPC3 and NALCN channels drive pacemaking in substantia nigra dopaminergic neurons.

Midbrain dopamine (DA) neurons are slow pacemakers that maintain extracellular DA levels. During the interspike intervals, subthreshold slow depolarization underlies autonomous pacemaking and determines its rate. However, the ion channels that determine slow depolarization are unknown. Here we show that TRPC3 and NALCN channels together form sustained inward currents responsible for the slow depolarization of nigral DA neurons. Specific TRPC3 channel blockade completely blocked DA neuron pacemaking, but the pacemaking activity in TRPC3 knock-out (KO) mice was perfectly normal, suggesting the presence of compensating ion channels. Blocking NALCN channels abolished pacemaking in both TRPC3 KO and wild-type mice. The NALCN current and mRNA and protein expression are increased in TRPC3 KO mice, indicating that NALCN compensates for TRPC3 currents. In normal conditions, TRPC3 and NALCN contribute equally to slow depolarization. Therefore, we conclude that TRPC3 and NALCN are two major leak channels that drive robust pacemaking in nigral DA neurons.

Cholesterol modulates ion channels via down-regulation of phosphatidylinositol 4,5-bisphosphate.

Ubiquitously expressed Mg(2+)-inhibitory cation (MIC) channels are permeable to Ca2+ and Mg2+ and are essential for cell viability. When membrane cholesterol level was increased by pre-incubating cells with a water-soluble form of cholesterol, the endogenous MIC current in HEK293 cells was negatively regulated. The application of phosphatidylinositol 4,5-bisphosphate (PIP2) recovered MIC current from cholesterol effect. As PIP2 is the direct modulator for MIC channels, high cholesterol content may cause down-regulation of PIP2. To test this possibility, we examined the effect of cholesterol on two exogenously expressed PIP2-sensitive K+ channels: human Ether-a-go-go related gene (HERG) and KCNQ. Enrichment with cholesterol inhibited HERG currents, while inclusion of PIP2 in the pipette solution blocked the cholesterol effect. KCNQ channel was also inhibited by cholesterol. The effects of cholesterol on these channels were blocked by pre-incubating cells with inhibitors for phospholipase C, which may indicate that cholesterol enrichment induces the depletion of PIP2 via phospholipase C activation. Lipid analysis showed that cholesterol enrichment reduced gamma-(32)P incorporation into PIP2 by approximately 35%. Our results suggest that cholesterol may modulate ion channels by changing the levels of PIP2. Thus, an important cross-talk exists among two plasma membrane-enriched lipids, cholesterol and PIP2.

N-benzhydryl quinuclidine compounds are a potent and Src kinase-independent inhibitor of NALCN channels.

BACKGROUND AND PURPOSE: NALCN is a Na+ leak, GPCR-activated channel that regulates the resting membrane potential and neuronal excitability. Despite numerous possible roles for NALCN in both normal physiology and disease processes, lack of specific blockers hampers further investigation. EXPERIMENTAL APPROACH: The effect of N-benzhydryl quinuclidine compounds on NALCN channels was demonstrated using whole-cell patch-clamp recordings in HEK293T cells overexpressing NALCN and acutely isolated nigral dopaminergic neurons that express NALCN endogenously. Src kinase activity was measured using a Src kinase assay kit, and voltage and current-clamp recordings from nigral dopaminergic neurons were used to measure NALCN currents and membrane potentials. KEY RESULTS: N-benzhydryl quinuclidine compounds inhibited NALCN channels without affecting TRPC channels, another important route for Na+ leak. In HEK293T cells overexpressing NALCN, N-benzhydryl quinuclidine compounds potently suppressed muscarinic M3 receptor-activated NALCN currents. Structure-function relationship studies suggest that the quinuclidine ring with a benzhydryl group imparts the ability to inhibit NALCN currents regardless of Src family kinases. Moreover, N-benzhydryl quinuclidine compounds inhibited not only GPCR-activated NALCN currents but also background Na+ leak currents and hyperpolarized the membrane potential in native midbrain dopaminergic neurons that express NALCN endogenously. CONCLUSION AND IMPLICATIONS: These findings suggest that N-benzhydryl quinuclidine compounds have a pharmacological potential to directly inhibit NALCN channels and could be a useful tool to investigate functions of NALCN channels.

Assessment of dosimetric leaf gap correction factor in Mobius3D commissioning affected by couch top.

This study assesses the dosimetric leaf gap (DLG) correction factor in Mobius3D commissioning affected by a couch top platform and calculates the optimal DLG value according to the point dose difference function. DLG optimizations were performed for 3 LINAC machines and a total of 30 patient volumetric modulated arc therapy plans (i.e., 10 plans per each LINAC). Point dose calculations were performed using an automatic dose calculation system in Mobius3D as well as Mobis3D calculation using a Mobius Verification Phantom (MVP)-based quality assurance plan with a carbon fiber couch top. Subsequently, the results were compared with measurement data. The averaged point dose measured for the MVP with a couch top decreased by approximately 2% relative to that without the couch top. The average of the optimal DLG factors increased by 1.153 mm due to the couch top effect for a dose decrease of 2% at the measured point. In the procedure of Mobius beam commissioning, users should adjust the DLG correction factor using a specific phantom (including MVP) with a couch top structure. If the DLG optimization were performed by using MVP automatic dose calculation system, the factor should be increased by approximately 1.2 mm per 2% dose difference considering user's couch top effect.

Localized Ca2+ uncaging reveals polarized distribution of Ca2+-sensitive Ca2+ release sites: mechanism of unidirectional Ca2+ waves.

Ca2+-induced Ca2+ release (CICR) plays an important role in the generation of cytosolic Ca2+ signals in many cell types. However, it is inherently difficult to distinguish experimentally between the contributions of messenger-induced Ca2+ release and CICR. We have directly tested the CICR sensitivity of different regions of intact pancreatic acinar cells using local uncaging of caged Ca2+. In the apical region, local uncaging of Ca2+ was able to trigger a CICR wave, which propagated toward the base. CICR could not be triggered in the basal region, despite the known presence of ryanodine receptors. The triggering of CICR from the apical region was inhibited by a pharmacological block of ryanodine or inositol trisphosphate receptors, indicating that global signals require coordinated Ca2+ release. Subthreshold agonist stimulation increased the probability of triggering CICR by apical uncaging, and uncaging-induced CICR could activate long-lasting Ca2+ oscillations. However, with subthreshold stimulation, CICR could still not be initiated in the basal region. CICR is the major process responsible for global Ca2+ transients, and intracellular variations in sensitivity to CICR predetermine the activation pattern of Ca2+ waves.

Elevated cellular cholesterol in Familial Alzheimer's presenilin 1 mutation is associated with lipid raft localization of ß-amyloid precursor protein.

Familial Alzheimer's disease (FAD)-associated presenilin 1 (PS1) serves as a catalytic subunit of γ-secretase complex, which mediates the proteolytic liberation of ß-amyloid (Aß) from ß-amyloid precursor protein (APP). In addition to its proteolytic role, PS1 is involved in non-proteolytic functions such as protein trafficking and ion channel regulation. Furthermore, postmortem AD brains as well as AD patients showed dysregulation of cholesterol metabolism. Since cholesterol has been implicated in regulating Aß production, we investigated whether the FAD PS1-associated cholesterol elevation could influence APP processing. We found that in CHO cells stably expressing FAD-associated PS1 ΔE9, total cholesterol levels are elevated compared to cells expressing wild-type PS1. We also found that localization of APP in cholesterol-enriched lipid rafts is substantially increased in the mutant cells. Reducing the cholesterol levels by either methyl-ß-cyclodextrin or an inhibitor of CYP51, an enzyme mediating the elevated cholesterol in PS1 ΔE9-expressing cells, significantly reduced lipid raft-associated APP. In contrast, exogenous cholesterol increased lipid raft-associated APP. These data suggest that in the FAD PS1 ΔE9 cells, the elevated cellular cholesterol level contributes to the altered APP processing by increasing APP localized in lipid rafts.

The endoplasmic reticulum as an integrator of multiple dendritic events.

Dendrites are integrating elements that receive numerous subsets of heterogeneous synaptic inputs, which generate temporally and spatially distinct changes in membrane potential and intracellular Ca2+ levels in local domains. The ubiquitously distributed endoplasmic reticulum (ER) in dendrites is luminally connected to the bulk ER in the soma, constituting a huge interconnected intracellular network that allows rapid Ca2+ diffusion and equilibration. The ER is an excitable organelle that can elicit or terminate cytosolic Ca2+ signals in local or global domains. The absolute level or changes in the Ca2+ concentration in the ER lumen are also very important for the synthesis and maturation of proteins, regulation of gene expression, mitochondrial functions, neuronal excitability, and synaptic plasticity. Through the connected lumen of the ER, information from multiple dendritic events in neurons appears to be delivered into the bulk ER in the soma. Therefore, the ER network in neurons is emerging as a conveyor and integrator of signals. In this article, we will discuss the various roles of the ER and the functional and structural organization of the ER network in neurons.

Voltage-operated Ca2+ channels regulate dopamine release from somata of dopamine neurons in the substantia nigra pars compacta.

Dopamine (DA) neurons release DA not only from axon terminals at the striatum, but from their somata and dendrites at the substantia nigra pars compacta (SNc). Released DA may auto-regulate further DA release or modulate non-DA cells. However, the actual mechanism of somatodendritic DA release, especially the Ca(2+) dependency of the process, remains controversial. In this study, we used amperometry to monitor DA release from somata of acutely isolated rat DA neurons. We found that DA neurons spontaneously released DA in the resting state. Removal of extracellular Ca(2+) and application of blockers for voltage-operated Ca(2+) channels (VOCCs) suppressed the frequency of secretion events. Activation of VOCCs by stimulation with K(+)-rich saline increased the frequency of secretion events, which were also sensitive to blockers for L- and T-type Ca(2+) channels. These results suggest that Ca(2+) influx through VOCCs regulates DA release from somata of DA neurons.

Involvement of corticotropin-releasing factor receptor 2 beta in differentiation of dopaminergic MN9D cells.

Corticotropin releasing factor (CRF) mediates various responses to stress through CRF receptors 1 and 2. CRF receptor 2 has two forms, 2alpha and 2beta each of which appears to have distinct roles. Here we used dopaminergic neuron-derived MN9D cells to investigate the function of CRF receptor 2 in dopamine neurons. We found that n-butyrate, a histone deacetylase inhibitor, induced MN9D cell differentiation and increased gene expression of all CRF receptors. CRF receptor 2beta was minimally expressed in MN9D cells; however, its expression dramatically increased during differentiation. CRF receptor 2beta expression levels appeared to correlate with neurite outgrowth, suggesting CRF receptor 2beta involvement in neuronal differentiation. To validate this statement, we made a CRF receptor 2beta-overexpressing MN9D/CRFR2 beta stable cell line. This cell line showed robust neurite outgrowth and GAP43 overexpression, together with MEK and ERK activation, suggesting MN9D cell neuronal differentiation. From these results, we conclude that CRF receptor 2beta plays an important role in MN9D cell differentiation by activating the MEK/ERK signaling pathway.

Regional interaction of endoplasmic reticulum Ca2+ signals between soma and dendrites through rapid luminal Ca2+ diffusion.

The endoplasmic reticulum (ER) Ca2+ store plays a key role in integration and conveyance of Ca2+ signals in highly polarized neurons. The interconnected ER network in neurons generates Ca2+ signals in local domains, but the regional interaction is unclear. Here, we show that continuous or repetitive applications of caffeine produced robust Ca2+ release from the ER Ca2+ store in dendritic areas without severe store depletion, but that similar stimuli applied to soma caused rapid store depletion in acutely isolated midbrain dopamine neurons. Partial emptying of the ER Ca2+ store within a dendrite caused a similar level of store depletion in unstimulated dendrites, as well as in soma. Photobleaching and local stimulation experiments revealed that Ca2+ and the dye trapped within the ER diffused rapidly from the soma to dendrites up to 90 microm, which we could resolve, suggesting that the ER network acts as a functional tunnel for rapid Ca2+ transport. These data imply that the ER in soma acts as a Ca2+ reservoir supplying Ca2+ to the dendritic store, and that the dendritic store, hence, is able to respond to Ca2+-mobilizing input signals endurably.