Plasma Membrane Of Dendrites Research Articles

Voltage-gated calcium channels are abnormal in cultured spinal motoneurons in the G93A-SOD1 transgenic mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motoneurons. Hyperexcitability and excitotoxicity have been implicated in the early pathogenesis of ALS. Studies addressing excitotoxic motoneuron death and intracellular Ca2+ overload have mostly focused on Ca2+ influx through AMPA glutamate receptors. However, intrinsic excitability of motoneurons through voltage-gated ion channels may also have a role in the neurodegeneration. In this study we examined the function and localization of voltage-gated Ca2+ channels in cultured spinal cord motoneurons from mice expressing a mutant form of human superoxide dismutase-1 with a Gly93→Ala substitution (G93A-SOD1). Using whole-cell patch-clamp recordings, we showed that high voltage activated (HVA) Ca2+ currents are increased in G93A-SOD1 motoneurons, but low voltage activated Ca2+ currents are not affected. G93A-SOD1 motoneurons also have altered persistent Ca2+ current mediated by L-type Ca2+ channels. Quantitative single-cell RT-PCR revealed higher levels of Ca1a, Ca1b, Ca1c, and Ca1e subunit mRNA expression in G93A-SOD1 motoneurons, indicating that the increase of HVA Ca2+ currents may result from upregulation of Ca2+ channel mRNA expression in motoneurons. The localizations of the Ca1B N-type and Ca1D L-type Ca2+ channels in motoneurons were examined by immunocytochemistry and confocal microscopy. G93A-SOD1 motoneurons had increased Ca1B channels on the plasma membrane of soma and dendrites. Ca1D channels are similar on the plasma membrane of soma and lower on the plasma membrane of dendrites of G93A-SOD1 motoneurons. Our study demonstrates that voltage-gated Ca2+ channels have aberrant functions and localizations in ALS mouse motoneurons. The increased HVA Ca2+ currents and PCCa current could contribute to early pathogenesis of ALS.

Redistribution of NMDA Receptors in Estrogen-Receptor-β-Containing Paraventricular Hypothalamic Neurons following Slow-Pressor Angiotensin II Hypertension in Female Mice with Accelerated Ovarian Failure

Hypertension in male and aging female rodents is associated with glutamate-dependent plasticity in the hypothalamus, but existing models have failed to capture distinct transitional menopausal phases that could have a significant impact on the synaptic plasticity and emergent hypertension. In rodents, accelerated ovarian failure (AOF) induced by systemic injection of 4-vinylcyclohexane diepoxide mimics the estrogen fluctuations seen in human menopause including the perimenopause transition (peri-AOF) and postmenopause (post-AOF). Thus, we used the mouse AOF model to determine the impact of slow-pressor angiotensin II (AngII) administration on blood pressure and on the subcellular distribution of obligatory N-methyl-<smlcap>D</smlcap>-aspartate (NMDA) receptor GluN1 subunits in the paraventricular hypothalamic nucleus (PVN), a key estrogen-responsive cardiovascular regulatory area. Estrogen-sensitive neuronal profiles were identified in mice expressing enhanced green fluorescent protein under the promoter for estrogen receptor (ER) β, a major ER in the PVN. Slow-pressor AngII increased arterial blood pressure in mice at peri- and post-AOF time points. In control oil-injected (nonhypertensive) mice, AngII decreased the total number of GluN1 in ERβ-containing PVN dendrites. In contrast, AngII resulted in a reapportionment of GluN1 from the cytoplasm to the plasma membrane of ERβ-containing PVN dendrites in peri-AOF mice. Moreover, in post-AOF mice, AngII increased total GluN1, dendritic size and radical production in ERβ-containing neurons. These results indicate that unique patterns of hypothalamic glutamate receptor plasticity and dendritic structure accompany the elevated blood pressure in peri- and post-AOF time points. Our findings suggest the possibility that distinct neurobiological processes are associated with the increased blood pressure during perimenopausal and postmenopausal periods.

Anatomical evidence for a non-synaptic influence of the K+-dependent Na+/Ca2+-exchanger, NCKX2, on hippocampal plasticity

The K+-dependent Na+/Ca2-exchanger (NCKX) family is encoded by five related genes, of which NCKX2 (solute carrier family 24, member 2) is the most abundant member present in the brain. Nckx2 knockout mice display profound loss of hippocampal long-term potentiation, and selective deficits in motor learning and spatial working memory. However, the molecular mechanisms underlying these changes have not been established. Thus, the overall goal of this project was to identify the exact subcellular localization of NCKX2 in the hippocampus, as an important step toward understanding the physiological role for NCKX2 in neuronal plasticity. To achieve this goal, we used dual immunofluorescent confocal microscopy and immunoelectron microscopy. Our data demonstrate that the majority of NCKX2 is co-localized with the dendritic marker, microtubule associated protein 2. A smaller fraction is co-localized with the presynaptic marker, synapsin 1, and the smallest amount is co-localized with the glutamatergic spine marker, N-methyl-d-aspartate receptor 1. The data from immunoelectron microscopy are consistent with the observations from dual immunofluorescence, and show that the highest fraction of NCKX2 is located on the plasma membrane of small oblique dendrites, particularly in CA1 neurons of the stratum radiatum. In the molecular layer, a greater fraction of NCKX2 is associated with axon terminals and, in addition, a fraction of NCKX2 is found not associated with the plasma membrane but located in the cytoplasm. These studies describe for the first time the exact location of NCKX2 in the hippocampus of adult mice and suggest that the function of NCKX2 in neuronal plasticity in hippocampal CA1 neurons may be mediated by its kinetic effect on the local Ca2+ concentration that influences dendritic integration. At other hippocampal locations NCKX2 has a somewhat different spatial distribution, consistent with published reports of NCKX2 expression in other brain regions, suggesting that NCKX2 contributes to Ca2+ homeostasis in distinct ways in different brain neurons.

NMDA Receptor Plasticity in the Hypothalamic Paraventricular Nucleus Contributes to the Elevated Blood Pressure Produced by Angiotensin II.

Hypertension induced by angiotensin II (Ang II) is associated with glutamate-dependent dysregulation of the hypothalamic paraventricular nucleus (PVN). Many forms of glutamate-dependent plasticity are mediated by NMDA receptor GluN1 subunit expression and the distribution of functional receptor to the plasma membrane of dendrites. Here, we use a combined ultrastructural and functional analysis to examine the relationship between PVN NMDA receptors and the blood pressure increase induced by chronic infusion of a low dose of Ang II. We report that the increase in blood pressure produced by a 2 week administration of a subpressor dose of Ang II results in an elevation in plasma membrane GluN1 in dendrites of PVN neurons in adult male mice. The functional implications of these observations are further demonstrated by the finding that GluN1 deletion in PVN neurons attenuated the Ang II-induced increases in blood pressure. These results indicate that NMDA receptor plasticity in PVN neurons significantly contributes to the elevated blood pressure mediated by Ang II.

Pharmacological inhibition of cation-chloride cotransporters for neurological diseases.

γ-Aminobutyric acid (GABA) is a major neurotransmitter and plays important roles in both the developing and mature central nervous system (CNS). One way that GABA can act is by binding to fast, ionotropic GABAA receptors in neurons. The binding of GABA to GABAA receptors causes a conformational change that opens ion channels, allowing the passage of Cl– ions. In mature adults, intracellular Cl– concentration [Cl–]i) is low, and the opening of these ion channels triggers influx of Cl– ions, causing hyperpolarization and neuronal inhibition (Blaesse, 2009). However, in the developing nervous system, [Cl–]i is high, and the binding of GABA to GABAA receptors induces a depolarizing excitatory response (Figure 1). This leads to the stimulation of voltage-dependent Ca2+ channels, which is important for proper neuronal proliferation and differentiation in their circuitry development. Gradual changes in [Cl–]i during development, known as the “GABA shift”, determine the strength and polarity of GABA-mediated activity. The precise regulation of [Cl–]i is determined by two major cation-chloride cotransporters (CCCs), the inwardly directed Na-K-2Cl cotransporter (NKCC1) and the Cl– extruding K-Cl cotransporter (KCC2) (Blaesse, 2009). NKCC1 is expressed in a wide range of neurons and glia, but especially in embryonic ventricular zones, which suggests an important role in neuronal proliferation. KCC2 is located in plasma membrane of both somata and dendrites of neurons in various brain regions, including cortical pyramidal neurons, thalamic relay cells, and auditory brainstem neurons (Blaesse, 2009). In embryonic and early postnatal life, neurons show high expression of NKCC1and low expression of KCC2 (Figure 1). NKCC1 functions in Cl– influx and raises the [Cl–]i during the early development of the CNS, which enables GABAA receptor-mediated neuronal depolarization and excitation. NKCC1 expression rapidly decreases during the first year of life to levels common in an adult, while KCC2 expression rises (Dzhala et al., 2005). KCC2 functions in Cl– efflux and lowering [Cl–]i, which leads to the dominance of GABAA receptor-mediated hyperpolarization and inhibition response in the maturing brain (Figure 1).

The dendritic SNARE fusion machinery involved in AMPARs insertion during long-term potentiation.

Sorting endosomes carry α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) from their maturation sites to their final destination at the dendritic plasma membrane through both constitutive and regulated exocytosis. Insertion of functional AMPARs into the postsynaptic membrane is essential for maintaining fast excitatory synaptic transmission and plasticity. Despite this crucial role in neuronal function, the machinery mediating the fusion of AMPAR-containing endosomes in dendrites has been largely understudied in comparison to presynaptic vesicle exocytosis. Increasing evidence suggests that similarly to neurotransmitter release, AMPARs insertion relies on the formation of a SNARE complex (soluble NSF-attachment protein receptor), whose composition in dendrites has just begun to be elucidated. This review analyzes recent findings of the fusion machinery involved in regulated AMPARs insertion and discusses how dendritic exocytosis and AMPARs lateral diffusion may work together to support synaptic plasticity.

Going Forward with Retromer

The retromer complex is well known to mediate retrograde transport from endosomes to the Golgi. In a recent issue of Neuron, Choy et al. (2014) identify a function for retromer in supporting fast, local delivery of neurotransmitter receptors from endosomes to the dendritic plasma membrane.

β-III spectrin underpins ankyrin R function in Purkinje cell dendritic trees: protein complex critical for sodium channel activity is impaired by SCA5-associated mutations.

Beta III spectrin is present throughout the elaborate dendritic tree of cerebellar Purkinje cells and is required for normal neuronal morphology and cell survival. Spinocerebellar ataxia type 5 (SCA5) and spectrin associated autosomal recessive cerebellar ataxia type 1 are human neurodegenerative diseases involving progressive gait ataxia and cerebellar atrophy. Both disorders appear to result from loss of β-III spectrin function. Further elucidation of β-III spectrin function is therefore needed to understand disease mechanisms and identify potential therapeutic options. Here, we report that β-III spectrin is essential for the recruitment and maintenance of ankyrin R at the plasma membrane of Purkinje cell dendrites. Two SCA5-associated mutations of β-III spectrin both reduce ankyrin R levels at the cell membrane. Moreover, a wild-type β-III spectrin/ankyrin-R complex increases sodium channel levels and activity in cell culture, whereas mutant β-III spectrin complexes fail to enhance sodium currents. This suggests impaired ability to form stable complexes between the adaptor protein ankyrin R and its interacting partners in the Purkinje cell dendritic tree is a key mechanism by which mutant forms of β-III spectrin cause ataxia, initially by Purkinje cell dysfunction and exacerbated by subsequent cell death.

Mitral and Tufted Cells Are Potential Cellular Targets of Nitration in the Olfactory Bulb of Aged Mice

Olfactory sensory function declines with age; though, the underlying molecular changes that occur in the olfactory bulb (OB) are relatively unknown. An important cellular signaling molecule involved in the processing, modulation, and formation of olfactory memories is nitric oxide (NO). However, excess NO can result in the production of peroxynitrite to cause oxidative and nitrosative stress. In this study, we assessed whether changes in the expression of 3-nitrotyrosine (3-NT), a neurochemical marker of peroxynitrite and thus oxidative damage, exists in the OB of young, adult, middle-aged, and aged mice. Our results demonstrate that OB 3-NT levels increase with age in normal C57BL/6 mice. Moreover, in aged mice, 3-NT immunoreactivity was found in some blood vessels and microglia throughout the OB. Notably, large and strongly immunoreactive puncta were found in mitral and tufted cells, and these were identified as lipofuscin granules. Additionally, we found many small-labeled puncta within the glomeruli of the glomerular layer and in the external plexiform layer, and these were localized to mitochondria and discrete segments of mitral and tufted dendritic plasma membranes. These results suggest that mitral and tufted cells are potential cellular targets of nitration, along with microglia and blood vessels, in the OB during aging.

Membrane Trafficking of NADPH Oxidase p47phoxin Paraventricular Hypothalamic Neurons Parallels Local Free Radical Production in Angiotensin II Slow-Pressor Hypertension

NADPH oxidase-generated reactive oxygen species (ROS) are highly implicated in the development of angiotensin II (AngII)-dependent hypertension mediated in part through the hypothalamic paraventricular nucleus (PVN). This region contains vasopressin and non-vasopressin neurons that are responsive to cardiovascular dysregulation, but it is not known whether ROS is generated by one or both cell types in response to "slow-pressor" infusion of AngII. We addressed this question using ROS imaging and electron microscopic dual labeling for vasopressin and p47(phox), a cytoplasmic NADPH oxidase subunit requiring mobilization to membranes for the initiation of ROS production. C57BL/6 mice or vasopressin-enhanced green fluorescent protein (VP-eGFP) mice were infused systemically with saline or AngII (600 ng · kg(-1) · min(-1), s.c.) for 2 weeks, during which they slowly developed hypertension. Ultrastructural analysis of the PVN demonstrated p47(phox) immunolabeling in many glial and neuronal profiles, most of which were postsynaptic dendrites. Compared with saline, AngII recipient mice had a significant increase in p47(phox) immunolabeling on endomembranes just beneath the plasmalemmal surface (+42.1 ± 11.3%; p < 0.05) in non-vasopressin dendrites. In contrast, AngII infusion decreased p47(phox) immunolabeling on the plasma membrane (-35.5 ± 16.5%; p < 0.05) in vasopressin dendrites. Isolated non-VP-eGFP neurons from the PVN of AngII-infused mice also showed an increase in baseline ROS production not seen in VP-eGFP neurons. Our results suggest that chronic low-dose AngII may offset the homeostatic control of blood pressure by differentially affecting membrane assembly of NADPH oxidase and ROS production in vasopressin and non-vasopressin neurons located within the PVN.

Networks of Polarized Actin Filaments in the Axon Initial Segment Provide a Mechanism for Sorting Axonal and Dendritic Proteins

Trafficking of proteins specifically to the axonal or somatodendritic membrane allows neurons to establish and maintain polarized compartments with distinct morphology and function. Diverse evidence suggests that an actin-dependent vesicle filter within the axon initial segment (AIS) plays a critical role in polarized trafficking; however, no distinctive actin-based structures capable of comprising such a filter have been found within the AIS. Here, using correlative light and scanning electron microscopy, we visualized networks of actin filaments several microns wide within the AIS of cortical neurons in culture. Individual filaments within these patches are predominantly oriented with their plus ends facing toward the cell body, consistent with models of filter selectivity. Vesicles carrying dendritic proteins are much more likely to stop in regions occupied by the actin patches than in other regions, indicating that the patches likely prevent movement of dendritic proteins to the axon and thereby act as a vesicle filter.

In vivo optogenetic control of striatal and thalamic neurons in non-human primates.

Electrical and pharmacological stimulation methods are commonly used to study neuronal brain circuits in vivo, but are problematic, because electrical stimulation has limited specificity, while pharmacological activation has low temporal resolution. A recently developed alternative to these methods is the use of optogenetic techniques, based on the expression of light sensitive channel proteins in neurons. While optogenetics have been applied in in vitro preparations and in in vivo studies in rodents, their use to study brain function in nonhuman primates has been limited to the cerebral cortex. Here, we characterize the effects of channelrhodopsin-2 (ChR2) transfection in subcortical areas, i.e., the putamen, the external globus pallidus (GPe) and the ventrolateral thalamus (VL) of rhesus monkeys. Lentiviral vectors containing the ChR2 sequence under control of the elongation factor 1α promoter (pLenti-EF1α -hChR2(H134R)-eYFP-WPRE, titer 109 particles/ml) were deposited in GPe, putamen and VL. Four weeks later, a probe combining a conventional electrode and an optic fiber was introduced in the previously injected brain areas. We found light-evoked responses in 31.5% and 32.7% of all recorded neurons in the striatum and thalamus, respectively, but only in 2.5% of recorded GPe neurons. As expected, most responses were time-locked increases in firing, but decreases or mixed responses were also seen, presumably via ChR2-mediated activation of local inhibitory connections. Light and electron microscopic analyses revealed robust expression of ChR2 on the plasma membrane of cell somas, dendrites, spines and terminals in the striatum and VL. This study demonstrates that optogenetic experiments targeting the striatum and basal ganglia-related thalamic nuclei can be successfully achieved in monkeys. Our results indicate important differences of the type and magnitude of responses in each structure. Experimental conditions such as the vector used, the number and rate of injections, or the light stimulation conditions have to be optimized for each structure studied.

Methamphetamine-Evoked Depression of GABAB Receptor Signaling in GABA Neurons of the VTA

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

Neuronal P2X2 Receptors Are Mobile ATP Sensors That Explore the Plasma Membrane When Activated

ATP-gated ionotropic P2X2 receptors are widely expressed in neurons. Although the electrophysiological properties of P2X2 receptors have been extensively studied, little is known about the plasma membrane lateral mobility of P2X2 receptors or whether receptor mobility is regulated by ATP. Here we used single-molecule imaging with simultaneous whole-cell voltage-clamp recordings to track quantum dot-labeled P2X2 receptors in the dendrites of rat hippocampal neurons to explore P2X2 receptor mobility and its regulation. We find that plasma membrane P2X2 receptor lateral mobility in dendrites is heterogeneous but mostly Brownian in nature, consisting of mobile and slowly mobile receptor pools. Moreover, lateral mobility is P2X2 subunit and cell specific, is increased in an activation-dependent manner, and is regulated by cytosolic VILIP1, a calcium binding protein. Our data provide the first direct measures of P2X receptor mobility and show that P2X2 receptors are mobile ATP sensors, sampling more of the dendritic plasma membrane in response to ATP.

Trafficking of AMPA Receptors at Plasma Membranes of Hippocampal Neurons

The number of AMPA receptors at synapses depends on receptor cycling. Because receptors diffuse rapidly in plasma membranes, their exocytosis and endocytosis need not occur near synapses. Here, pre-embedding immunogold electron microscopy is applied to dissociated rat hippocampal cultures to provide sensitive, high-resolution snapshots of the distribution of surface AMPA receptors in spines, dendrites, and cell bodies that will be informative about trafficking of AMPA receptors. The density of the label for GluR2 varies, but is consistent throughout cell body and dendrites in each individual neuron, except at postsynaptic densities (PSDs), where it is typically higher. Glutamate receptor 2 (GluR2) labels at PSDs significantly increase after synaptic activation by glycine treatment and increase further upon depolarization by high K(+). Islands of densely packed labels have consistent size and density but vary in frequency under different experimental conditions. These patches of label, which occur on plasma membranes of cell bodies and dendrites but not near PSDs, are taken to be the aftermath of exocytosis of AMPA receptors. A subpopulation of clathrin-coated pits in cell bodies and dendrites label for GluR2, and the number and amount of label in individual pits increase after NMDA treatment. Coated pits near synapses typically lack GluR2 label under basal conditions, but ∼40% of peri-PSD pits label for GluR2 after NMDA treatment. Thus, exocytosis and endocytosis of AMPA receptors occur mainly at extrasynaptic locations on cell bodies and dendrites. Receptors are not preferentially exocytosed near PSDs, but may be removed via endocytosis at peri-PSD locations after activation of NMDA receptors.

Cellular and subcellular localization of the neuron-specific plasma membrane calcium ATPase PMCA1a in the rat brain.

Regulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca(2+) extrusion from the cell, the plasma membrane calcium-dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a "housekeeping" function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites, and spines. Thus, rather than serving a general housekeeping function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca(2+) transients.

Cellular and subcellular localization of the neuron‐specific plasma membrane calcium ATPase PMCA1a in the rat brain

AbstractRegulation of intracellular calcium is crucial both for proper neuronal function and survival. By coupling ATP hydrolysis with Ca2+ extrusion from the cell, the plasma membrane calcium‐dependent ATPases (PMCAs) play an essential role in controlling intracellular calcium levels in neurons. In contrast to PMCA2 and PMCA3, which are expressed in significant levels only in the brain and a few other tissues, PMCA1 is ubiquitously distributed, and is thus widely believed to play a “housekeeping” function in mammalian cells. Whereas the PMCA1b splice variant is predominant in most tissues, an alternative variant, PMCA1a, is the major form of PMCA1 in the adult brain. Here, we use immunohistochemistry to analyze the cellular and subcellular distribution of PMCA1a in the brain. We show that PMCA1a is not ubiquitously expressed, but rather is confined to neurons, where it concentrates in the plasma membrane of somata, dendrites, and spines. Thus, rather than serving a general housekeeping function, our data suggest that PMCA1a is a calcium pump specialized for neurons, where it may contribute to the modulation of somatic and dendritic Ca2+ transients. J. Comp. Neurol. 518:3169–3183, 2010. © 2010 Wiley‐Liss, Inc.

Polarized targeting of DNER into dendritic plasma membrane in hippocampal neurons depends on endocytosis

The targeting of membrane proteins into axons and dendrites is of critical importance for directional signal transmission within specific neural circuits. Many dendritic proteins have been shown to reach the somatodendritic membrane based on selective sorting and transport of carrier vesicles. Using rat hippocampal neurons in culture, we investigated the trafficking pathways of Delta/Notch-like EGF-related receptor (DNER), a transmembrane Notch ligand which is specifically expressed in CNS dendrites. Mutations in the cytoplasmic domain of DNER that abolished somatodendritic localization also increased its surface expression. Furthermore, inhibition of endocytosis resulted in disruption of the somatodendritic localization of DNER, indicating that the somatodendritic targeting of DNER is dependent on endocytosis. The DNER cytoplasmic domain binds to a clathrin adaptor protein complex-2 via a proximal tyrosine motif and a 40 amino acid stretch in the mid-domain, but not by the C-terminal tail. Molecular and pharmacological inhibition revealed that the surface expression of DNER is regulated by clathrin-dependent and -independent endocytosis. In contrast, the somatodendritic targeting of DNER is predominantly regulated by clathrin- and adaptor protein complex-2-independent endocytosis via the C-terminal tail of DNER. Our data suggest that clathrin-independent endocytosis is critical for the polarized targeting of somatodendritic proteins.

Molecular identity of dendritic voltage-gated sodium channels in CA1 pyramidal cells

Event Abstract Back to Event Molecular identity of dendritic voltage-gated sodium channels in CA1 pyramidal cells Andrea Lorincz1* and Zoltán Nusser1 1 Hungarian Academy of Sciences, Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Hungary High density of voltage-gated sodium (Nav) channels in axon initial segments (AIS) of pyramidal cells (PC) ensures low threshold for the initiation of action potential (AP). Nav channels-mediated currents were found in the somato-dendritic domain of PCs, where they contribute to dendritic spikes and backpropagating APs. However, the molecular identity of somato-dendritic Nav channels is unknown, most likely owing to the limited sensitivity of the applied methods. Here we used immunofluorescent reactions and a highly sensitive electron microscopic SDS-digested freeze-fracture replica-labeling (SDS-FRL) technique to identify the Nav subunit content of the axo-somato-dendritic plasma membranes of CA1 pyramidal cells (PCs). Modified immunofluorescent reactions with a pan-Nav antibody demonstrated the presence of Nav channels in the axo-somato-dendritic compartments of CA1 PCs, but the method failed to detect Nav1.1, Nav1.2 or Nav1.6 subunits on the somato-dendritic surface of CA1 PCs. Using SDS-FRL, high densities of gold particles for the Nav1.6 subunit were detected in Nodes of Ranvier and AISs of CA1 PCs. We found a graded increase in the density of gold particles along the proximo-distal axis of AISs and the gold particles consistently avoided GABAergic axo-axonic synapses. Somatic and dendritic plasma membranes also contained gold particles for the Nav1.6 subunit at low, but significant densities. However, we could not detect gold particles in dendritic spines. Quantitative analysis revealed that AISs contained ~40-fold higher density of gold particles than somatic and proximal dendritic plasma membranes. A distance dependent decrease in the density of gold particles was also observed along the proximo-distal axis of the dendrites. Our results demonstrate for the first time the somato-dendritic presence of the Nav1.6 subunit and reveal quantitative differences in the amount of the Nav1.6 subunit between different subcellular compartments of CA1 PCs. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Cellular neuroscience Citation: Lorincz A and Nusser Z (2010). Molecular identity of dendritic voltage-gated sodium channels in CA1 pyramidal cells. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00078 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 Apr 2010; Published Online: 21 Apr 2010. * Correspondence: Andrea Lorincz, Hungarian Academy of Sciences, Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, Budapest, Hungary, lorincz@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Andrea Lorincz Zoltán Nusser Google Andrea Lorincz Zoltán Nusser Google Scholar Andrea Lorincz Zoltán Nusser PubMed Andrea Lorincz Zoltán Nusser Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Subcellular distribution of low‐voltage activated T‐type Ca2+ channel subunits (Cav3.1 and Cav3.3) in reticular thalamic neurons of the cat

Low-voltage-activated (LVA) Ca(2+) channels play a critical role in the generation of burst firing in the thalamus. Recently, three LVA Ca(2+) channel isoforms (Ca(v)3.1, Ca(v)3.2, Ca(v)3.3) have been identified in the reticular thalamic nucleus (RE). Previous electrophysiological and modelling studies have suggested that kinetically different T-type channels might be expressed in a compartmentalized manner in RE cells. However, their precise subcellular distribution has not been fully elucidated. Using light and electron microscopic (EM) immunocytochemistry, we investigated the subcellular expression pattern of Ca(v)3.1 and Ca(v)3.3 channel subunits in RE neurons of the cat. Fluorescent and peroxidase labelling demonstrated the presence of Ca(v)3.1 channel predominantly on the somata and proximal dendrites and Ca(v)3.3 channels on cell bodies. Quantitative immunogold localization disclosed that Ca(v)3.1 and Ca(v)3.3 isoforms showed 5.8- and 8.7-fold higher density, respectively, in the cytoplasm compared with somatic plasma membrane. Density of Ca(v)3.1 isoform in the somatic plasma membrane was 2.21-fold higher compared with Ca(v)3.3 subunit. In the dendritic plasma membrane, Ca(v)3.1 channel isoform was expressed throughout the entire dendritic tree. In contrast, Ca(v)3.3 isoform was absent from large-caliber, presumably proximal dendritic segments. Quantitative comparison showed that the relative density of immunogold particles compared with dendritic surface was 8.9- and 14.8-fold higher for Ca(v)3.1 and Ca(v)3.3, respectively, in small-diameter dendrites than in large proximal dendritic segments or somata. Our results demonstrate a higher density of low-threshold Ca(2+) channels in distal dendrites and provide further evidence of the role of RE neuron dendrites in the generation of prolonged, low-threshold spike bursts.