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.

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.

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.

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.

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.

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.

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.

Coexistence of glutamatergic spine synapses and shaft synapses in substantia nigra dopamine neurons.

Dopamine neurons of the substantia nigra have long been believed to have multiple aspiny dendrites which receive many glutamatergic synaptic inputs from several regions of the brain. But, here, using high-resolution two-photon confocal microscopy in the mouse brain slices, we found a substantial number of common dendritic spines in the nigral dopamine neurons including thin, mushroom, and stubby types of spines. However, the number of dendritic spines of the dopamine neurons was approximately five times lower than that of CA1 pyramidal neurons. Immunostaining and morphological analysis revealed that glutamatergic shaft synapses were present two times more than spine synapses. Using local two-photon glutamate uncaging techniques, we confirmed that shaft synapses and spine synapses had both AMPA and NMDA receptors, but the AMPA/NMDA current ratios differed. The evoked postsynaptic potentials of spine synapses showed lower amplitudes but longer half-widths than those of shaft synapses. Therefore, we provide the first evidence that the midbrain dopamine neurons have two morphologically and functionally distinct types of glutamatergic synapses, spine synapses and shaft synapses, on the same dendrite. This peculiar organization could be a new basis for unraveling many physiological and pathological functions of the midbrain dopamine neurons.

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.

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)).

O-GlcNAcylation promotes non-amyloidogenic processing of amyloid-ß protein precursor via inhibition of endocytosis from the plasma membrane.

Amyloid-ß protein precursor (AßPP) is transported to the plasma membrane, where it is sequentially cleaved by α-secretase and γ-secretase. This is called non-amyloidogenic pathway since it precludes the production of amyloid-ß (Aß), the main culprit of Alzheimer's disease (AD). Alternatively, once AßPP undergoes clathrin-dependent endocytosis, it can be sequentially cleaved by ß-secretase and γ-secretase at endosomes, producing Aß (amyloidogenic pathway). ß-N-acetylglucosamine (GlcNAc) can be attached to serine/threonine residues of the target proteins. This novel type of O-linked glycosylation is called O-GlcNAcylation mediated by O-GlcNAc transferase (OGT). The removal of GlcNAc is mediated by O-GlcNAcase (OGN). Recently, it is shown that O-GlcNAcylation of AßPP increases the non-amyloidogenic pathway. To investigate the regulatory role for O-GlcNAcylation in AßPP processing, we first tested the effects of inhibitor for OGN, PUGNAc, on AßPP metabolism in HeLa cells stably transfected with Swedish mutant form of AßPP. Increasing O-GlcNAcylated AßPP level increased α-secretase product while decreased ß-secretase products. We found that PUGNAc increased the trafficking rate of AßPP from the trans-Golgi network to the plasma membrane, and selectively decreased the endocytosis rate of AßPP. These events may contribute to the increased AßPP level in the plasma membrane by PUGNAc. Inhibiting clathrin-dependent endocytosis prevented the effect of PUGNAc on Aß, suggesting that the effect of PUGNAc was mainly mediated by decreasing AßPP endocytosis. These results strongly indicate that O-GlcNAcylation promotes the plasma membrane localization of AßPP, which enhances the non-amyloidogenic processing of AßPP. Thus, O-GlcNAcylation of AßPP can be a potential therapeutic strategy for AD.

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.

Homeostatic regulation mechanism of spontaneous firing determines glutamate responsiveness in the midbrain dopamine neurons.

Autonomous tonic firing of the midbrain dopamine neuron is essential for maintenance of ambient dopamine level in the brain, in which intracellular Ca2+ concentration ([Ca2+]c) plays a complex but pivotal role. However, little is known about Ca2+ signals by which dopamine neurons maintain an optimum spontaneous firing rate. In the midbrain dopamine neurons, we here show that spontaneous firing evoked [Ca2+]c changes in a phasic manner in the dendritic region but a tonic manner in the soma. Tonic levels of somatic [Ca2+]c strictly tallied with spontaneous firing rates. However, manipulatory raising or lowering of [Ca2+]c with caged compounds from the resting firing state proportionally suppressed or raised spontaneous firing rate, respectively, suggesting presence of the homeostatic regulation mechanism for spontaneous firing rate via tonic [Ca2+]c changes of the soma. More importantly, abolition of this homeostatic regulation mechanism significantly exaggerated the responses of tonic firings and high-frequency phasic discharges to glutamate. Therefore, we conclude that this Ca(2+)-dependent homeostatic regulation mechanism is responsible for not only maintaining optimum rate of spontaneous firing, but also proper responses to glutamate. Perturbation of this mechanism could cause dopamine neurons to be more vulnerable to glutamate and Ca2+ toxicities.

Cholesterol regulates HERG K+ channel activation by increasing phospholipase C ß1 expression.

Human ether-a-go-go-related gene (HERG) K(+) channel underlies the rapidly activating delayed rectifier K(+) conductance (IKr) during normal cardiac repolarization. Also, it may regulate excitability in many neuronal cells. Recently, we showed that enrichment of cell membrane with cholesterol inhibits HERG channels by reducing the levels of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] due to the activation of phospholipase C (PLC). In this study, we further explored the effect of cholesterol enrichment on HERG channel kinetics. When membrane cholesterol level was mildly increased in human embryonic kidney (HEK) 293 cells expressing HERG channel, the inactivation and deactivation kinetics of HERG current were not affected, but the activation rate was significantly decelerated at all voltages tested. The application of PtdIns(4,5)P2 or inhibitor for PLC prevented the effect of cholesterol enrichment, while the presence of antibody against PtdIns(4,5)P2 in pipette solution mimicked the effect of cholesterol enrichment. These results indicate that the effect of cholesterol enrichment on HERG channel is due to the depletion of PtdIns(4,5)P2. We also found that cholesterol enrichment significantly increases the expression of ß1 and ß3 isoforms of PLC (PLCß1, PLCß3) in the membrane. Since the effects of cholesterol enrichment on HERG channel were prevented by inhibiting transcription or by inhibiting PLCß1 expression, we conclude that increased PLCß1 expression leads to the deceleration of HERG channel activation rate via downregulation of PtdIns(4,5)P2. These results confirm a crosstalk between two plasma membrane-enriched lipids, cholesterol and PtdIns(4,5)P2, in the regulation of HERG channels.

Increasing Membrane Cholesterol Level Increases the Amyloidogenic Peptide by Enhancing the Expression of Phospholipase C.

Cerebral elevation of 42-residue amyloid ß-peptide (Aß42) triggers neuronal dysfunction in Alzheimer's disease (AD). Even though a number of cholesterol modulating agents have been shown to affect Aß generation, the role of cholesterol in the pathogenesis of AD is not clear yet. Recently, we have shown that increased membrane cholesterol levels downregulates phosphatidylinositol 4,5-bisphosphate (PIP2) via activation of phospholipase C (PLC). In this study, we tested whether membrane cholesterol levels may affect the Aß42 production via changing PIP2 levels. Increasing membrane cholesterol levels decreased PIP2 and increased secreted Aß42. Supplying PIP2, by using a PIP2-carrier system, blocked the effect of cholesterol on Aß42. We also found that cholesterol increased the expressions of ß1 and ß3 PLC isoforms (PLCß1, PLCß3). Silencing the expression of PLCß1 prevented the effects of cholesterol on PIP2 levels as well as on Aß42 production, suggesting that increased membrane cholesterol levels increased secreted Aß42 by downregulating PIP2 via enhancing the expression of PLCß1. Thus, cholesterol metabolism may be linked to Aß42 levels via PLCß1 expression and subsequent changes in PIP2 metabolism.

Facial soft tissue thickness database for craniofacial reconstruction in Korean adults.

One hundred Korean adults (50 men, 50 women) were scanned in the upright position using a cone-beam CT (CBCT) scanner. The soft tissue (ST) thicknesses were measured at 31 landmarks, 10 midline and 21 bilateral landmark sites, and the means and standard deviations were obtained for male and female subjects. While 18 of 31 landmarks showed sex differences, the majority showed higher values for male subjects with the exception of a few landmark sites corresponding to the zygoma area, which showed smaller values in men than in women. The mandibular area showed greater differences between the right and left sides. Overall, the ST thickness measurements obtained in this study can be used as a database for the forensic craniofacial reconstruction of Korean adult faces.

Modulation of transient receptor potential melastatin related 7 channel by presenilins.

Presenilins (PS1 and PS2) are multifunctional proteins involved in a diverse array of molecular and cellular functions, including proteolysis, development, neurogenesis, synaptic plasticity, ion channel regulation and phospholipid metabolism. Mutations in presenilin genes are responsible for the majority of Familial Alzheimer disease (FAD). Consequently, FAD-associated mutations in genes encoding PS1 or PS2 lead to several key cellular phenotypes, including alterations in proteolysis of ß-amyloid precursor protein (APP) and Ca(2+) entry. The mechanism underlying presenilin (PS)-mediated modulation of Ca(2+) entry remains to be determined. Our previous studies showed that the PS-dependent down-regulation of phosphatidylinositol-4,5-bisphosphate (PIP2) is attributable to the observed Ca(2+) deficits. In this study, we attempted to identify the ion channel that is subject to the PIP2 and PS-dependent modulation. We found that Ca(2+) or Zn(2+) entry via the transient receptor potential melastatin 7 (TRPM7) channel was attenuated by the presence of FAD-associated PS1 mutants, such as ΔE9 and L286V. TRPM7 has been implicated in Mg(2+) homeostasis and embryonic development. The intracellular delivery of PIP2 restored TRPM7-mediated Ca(2+) influx, indicating that the observed deficits in Ca(2+) entry are due to downregulation of PIP2. Conversely, PS1 and PS2 deficiency, previously shown to upregulate PIP2 levels, potentiated TRPM7-mediated Ca(2+) influx. PS-dependent changes in Ca(2+) influx could be neutralized by a TRPM7 channel blocker. Collectively, these results indicate that TRPM7 may underlie the Ca(2+) entry deficits observed in FAD-associated PS mutants and suggest that the normal function of PS involves regulation of TRPM7 through a PIP2-dependent mechanism.

Functional organization of dendritic Ca2+ signals in midbrain dopamine neurons.

Dendritic Ca2+ plays an important role not only in synaptic integration and synaptic plasticity, but also in dendritic excitability in midbrain dopamine neurons. However, the functional organization of dendritic Ca2+ signals in the dopamine neurons remains largely unknown. We therefore investigated dendritic Ca2+ signals by measuring glutamate-induced Ca2+ increases along the dendrites of acutely isolated midbrain dopamine neurons. Maximal doses of glutamate induced a [Ca2+]c rise with similar amplitudes in proximal and distal dendritic regions of a dopamine neuron. Glutamate receptors contributed incrementally to the [Ca2+]c rise according to their distance from the soma, with a reciprocal decrement in the contribution of voltage-operated Ca2+ channels (VOCCs). The contribution of AMPA and NMDA receptors increased with dendritic length, but that of metabotropic glutamate receptors decreased. At low doses of glutamate at which spontaneous firing was sustained, the [Ca2+]c rise was higher in the distal than the proximal regions of a dendrite, possibly due to the increased spontaneous firing rate. These results indicate that functional organization of Ca2+ signals in the dendrites of dopamine neurons requires different combination of VOCCs and glutamate receptors according to dendritic length, and that regional Ca2+ rises in dendrites respond differently to applied glutamate concentration.