Dendrites Of Hippocampal Pyramidal Neurons Research Articles

Distinct Modulation of I h by Synaptic Potentiation in Excitatory and Inhibitory Neurons.

Selective modifications in the expression or function of dendritic ion channels regulate the propagation of synaptic inputs and determine the intrinsic excitability of a neuron. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open upon membrane hyperpolarization and conduct a depolarizing inward current (I h). HCN channels are enriched in the dendrites of hippocampal pyramidal neurons where they regulate the integration of synaptic inputs. Synaptic plasticity can bidirectionally modify dendritic HCN channels in excitatory neurons depending on the strength of synaptic potentiation. In inhibitory neurons, however, the dendritic expression and modulation of HCN channels are largely unknown. In this study, we systematically compared the modulation of I h by synaptic potentiation in hippocampal CA1 pyramidal neurons and stratum radiatum (sRad) interneurons in mouse organotypic cultures. I h properties were similar in inhibitory and excitatory neurons and contributed to resting membrane potential and action potential firing. We found that in sRad interneurons, HCN channels were downregulated after synaptic plasticity, irrespective of the strength of synaptic potentiation. This suggests differential regulation of I h in excitatory and inhibitory neurons, possibly signifying their distinct role in network activity.

Longitudinal characterization of behavioral, morphological and transcriptomic changes in a tauopathy mouse model.

Neurodegenerative disorders, such as Alzheimer's disease (AD), have the gradual onset of neurobiological changes preceding clinical diagnosis by decades. To elucidate how brain dysfunction proceeds in neurodegenerative disorders, we performed longitudinal characterization of behavioral, morphological, and transcriptomic changes in a tauopathy mouse model, P301S transgenic mice. P301S mice exhibited cognitive deficits as early as 3 months old, and deficits in social preference and social cognition at 5-6 months. They had a significant decrease of arborization in basal dendrites of hippocampal pyramidal neurons from 3 months and apical dendrites of PFC pyramidal neurons at 9 months. Transcriptomic analysis of genome-wide changes revealed the enrichment of synaptic gene upregulation at 3 months of age, while most of these synaptic genes were downregulated in PFC and hippocampus of P301S mice at 9 months. These time-dependent changes in gene expression may lead to progressive alterations of neuronal structure and function, resulting in the manifestation of behavioral symptoms in tauopathies.

Anti-Müllerian Hormone Regulation of Synaptic Transmission in the Hippocampus Requires MAPK Signaling and Kv4.2 Potassium Channel Activity.

Anti-Müllerian hormone (AMH) is a paracrine factor generated peripherally by the gonads to regulate gonadal function in adult mammals. We recently reported that AMH and AMH-specific receptor Anti-Müllerian hormone receptor 2 (AMHR2) are expressed in the hippocampus, and exogenous AMH protein rapidly increased synaptic transmission and long-term synaptic plasticity at the CA3-CA1 synapses. Here we examined the cell-specific expression of AMHR2 and the cellular mechanism of rapid boosting effect of AMH on synaptic transmission in mouse hippocampus. Immunofluorescence staining showed that AMHR2 was specifically expressed in the soma and dendrites of hippocampal pyramidal neurons, but not glial cells. Electrophysiological recordings on acute hippocampal slices showed that AMH did not affect AMPAR-mediated or N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic currents at the CA3-CA1 synapses. The small-conductance Ca2+-activated K+ channel (SK2) and A-type K+ channel (Kv4.2) contribute to shaping excitatory postsynaptic potentials (EPSPs) at the CA3-CA1 synapses. Bath application of apamin to block SK2 did not alter AMH effect on increasing EPSPs, whereas blocking Kv4.2 channel with 4-aminopyridine, or chelating internal Ca2+ with BAPTA occluded the action of AMH on boosting EPSPs. Kv4.2 activity is regulated by p38 mitogen-activated kinase (MAPK). Blocking p38 MAPK with SB203580 occluded the effect of AMH on increasing EPSPs. These results show that Kv4.2 channel contributes to the rapid action of AMH on boosting synaptic transmission in a Ca2+- and p38 MAPK-dependent manner. Our findings provide functional evidence that AMH enhances synaptic transmission through Kv4.2 channel in the hippocampus, suggesting a possible role of Kv4.2 channel in AMH-regulated neuronal process underlying learning and memory.

A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+ Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.

Calcium Regulation of Kv4-Kchip Ion Channel Complexes

The Kv4 family of A-type voltage-gated K+ channels play a unique role in the dendrites of hippocampal pyramidal neurons. In particular, Kv4.2 channels are key determinants of dendritic excitability and integration, spike timing-dependent plasticity, long-term potentiation, and learning. Downregulation of Kv4.2 channel expression occurs following hippocampal seizures and in epilepsy suggesting A-type currents as targets for novel therapeutics. In addition to pore forming Kv4 subunits, native hippocampal A-type currents require modulatory auxiliary subunits known as K-channel interacting proteins (KChIPs). KChIPs function to enhance Kv4 expression and activity. KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels as they are members of the EF-hand domain containing neuronal Ca2+ sensor protein family; however, it is currently unclear to what degree intracellular Ca2+ regulates Kv4.2-KChIP complexes. To assess this possibility, we expressed KChIP2 with Kv4.2 in HEK293T cells and performed whole-cell patch clamp recordings in either nominal or 10 μM Ca2+. In the presence of 10 μM Ca2+, we measured a 1.5 fold increase in Kv4.2 current density. Intriguingly, Ca2+ regulation was specific to short splice isoforms of KChIP2, KChIP2b and 2c. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. Sequence alignment of the variable N-termini of KChIP2 isoforms suggest a novel domain limits Ca2+ sensitivity of the longer KChIP2 isoforms, KChIP2a1 and KChIP2a.

Synaptic Potentiation at Basal and Apical Dendrites of Hippocampal Pyramidal Neurons Involves Activation of a Distinct Set of Extracellular and Intracellular Molecular Cues.

In the central nervous system, several forms of experience-dependent plasticity, learning and memory require the activity-dependent control of synaptic efficacy. Despite substantial progress in describing synaptic plasticity, mechanisms related to heterogeneity of synaptic functions at local circuits remain elusive. Here we studied the functional and molecular aspects of hippocampal circuit plasticity by analyzing excitatory synapses at basal and apical dendrites of mouse hippocampal pyramidal cells (CA1 region) in acute brain slices. In the past decade, activity of metalloproteinases (MMPs) has been implicated as a widespread and critical factor in plasticity mechanisms at various projections in the CNS. However, in the present study we discovered that in striking contrast to apical dendrites, synapses located within basal dendrites undergo MMP-independent synaptic potentiation. We demonstrate that synapse-specific molecular pathway allowing MMPs to rapidly upregulate function of NMDARs in stratum radiatum involved protease activated receptor 1 and intracellular kinases and GTPases activity. In contrast, MMP-independent scaling of synaptic strength in stratum oriens involved dopamine D1/D5 receptors and Src kinases. Results of this study reveal that 2 neighboring synaptic systems differ significantly in extracellular and intracellular cascades that control synaptic gain and provide long-searched transduction pathways relevant for MMP-dependent synaptic plasticity.

Low dietary soy isoflavonoids increase hippocampal spine synapse density in ovariectomized rats

High dietary intake of plant estrogens (phytoestrogens) can affect brain structure and function. The effects of phytoestrogen intake within the range of normal animal and human dietary consumption, however, remain uncertain. The aim of the present study was to determine the effects of the isoflavonoids present in a standard low phytoestrogen laboratory rat chow on spine synapse density in the stratum radiatum of area CA1 of the hippocampus.Weanling rats (22days old) were fed either standard chow (Teklad 2018), a nutritionally comparable diet without soy (Teklad 2016) or a custom diet containing Teklad 2016 supplemented with the principal soy isoflavonoids, daidzein and genistein, for 40days. Rats were ovariectomized at 54days of age. Eight days later, spine synapse density on the apical dendrites of hippocampal pyramidal neurons in the stratum radiatum of area CA1 was measured by electron microscopic stereological analysis. Animals maintained on Teklad 2016 exhibited an approximately 60% lower CA1 spine synapse density than animals consuming Teklad 2018. Replacing genistein and daidzein in Teklad 2016 returned synapse density to levels indistinguishable from those in animals on Teklad 2018.These results indicate that the isoflavonoids in a standard laboratory rat diet exert significant effects on spine synapse density in the CA1 region of the hippocampus. Since changes in spine synapse density in this region of the hippocampus have been linked to cognitive performance and mood state, these data suggest that even relatively low daily consumption of soy phytoestrogens may be sufficient to influence hippocampal function.

The ROCK Inhibitor Fasudil Prevents Chronic Restraint Stress-Induced Depressive-Like Behaviors and Dendritic Spine Loss in Rat Hippocampus.

Background:Dendritic arbor simplification and dendritic spine loss in the hippocampus, a limbic structure implicated in mood disorders, are assumed to contribute to symptoms of depression. These morphological changes imply modifications in dendritic cytoskeleton. Rho GTPases are regulators of actin dynamics through their effector Rho kinase. We have reported that chronic stress promotes depressive-like behaviors in rats along with dendritic spine loss in apical dendrites of hippocampal pyramidal neurons, changes associated with Rho kinase activation. The present study proposes that the Rho kinase inhibitor Fasudil may prevent the stress-induced behavior and dendritic spine loss.Methods:Adult male Sprague-Dawley rats were injected with saline or Fasudil (i.p., 10 mg/kg) starting 4 days prior to and maintained during the restraint stress procedure (2.5 h/d for 14 days). Nonstressed control animals were injected with saline or Fasudil for 18 days. At 24 hours after treatment, forced swimming test, Golgi-staining, and immuno-western blot were performed.Results:Fasudil prevented stress-induced immobility observed in the forced swimming test. On the other hand, Fasudil-treated control animals showed behavioral patterns similar to those of saline-treated controls. Furthermore, we observed that stress induced an increase in the phosphorylation of MYPT1 in the hippocampus, an exclusive target of Rho kinase. This change was accompanied by dendritic spine loss of apical dendrites of pyramidal hippocampal neurons. Interestingly, increased pMYPT1 levels and spine loss were both prevented by Fasudil administration.Conclusion:Our findings suggest that Fasudil may prevent the development of abnormal behavior and spine loss induced by chronic stress by blocking Rho kinase activity.

CUB and Sushi multiple domains 3 regulates dendrite development

CUB and Sushi multiple domains 3 (CSMD3) is a large protein expressed in fetal and adult brain. Recently, mutations of the CSMD3 gene were identified in schizophrenia and autism patients. However, biochemical properties and functions of the CSMD3 protein remain unknown. Here, we demonstrate that CSMD3 is an oligomeric type I transmembrane protein localized in the apical dendrites of hippocampal pyramidal neurons in the postnatal brain. In cultured hippocampal neurons, CSMD3 is expressed only after 7 days in vitro. Overexpression of CSMD3 induced dendritic branching in hippocampal neurons. Regulation of dendritic morphology by CSMD3 depended on the presence of its extracellular region, while CSMD3 intracellular region was dispensable for this activity. These results suggest that CSMD3 acts as a co-receptor of an unidentified membrane protein to regulate dendrite development. Therefore, malfunctions of CSMD3 may be one of the factors in the pathogenesis of psychiatric disorders.

Dendritic sodium spikes are required for long-term potentiation at distal synapses on hippocampal pyramidal neurons.

Dendritic integration of synaptic inputs mediates rapid neural computation as well as longer-lasting plasticity. Several channel types can mediate dendritically initiated spikes (dSpikes), which may impact information processing and storage across multiple timescales; however, the roles of different channels in the rapid vs long-term effects of dSpikes are unknown. We show here that dSpikes mediated by Nav channels (blocked by a low concentration of TTX) are required for long-term potentiation (LTP) in the distal apical dendrites of hippocampal pyramidal neurons. Furthermore, imaging, simulations, and buffering experiments all support a model whereby fast Nav channel-mediated dSpikes (Na-dSpikes) contribute to LTP induction by promoting large, transient, localized increases in intracellular calcium concentration near the calcium-conducting pores of NMDAR and L-type Cav channels. Thus, in addition to contributing to rapid neural processing, Na-dSpikes are likely to contribute to memory formation via their role in long-lasting synaptic plasticity.

Small-conductance Ca2+-activated K+ channels modulate action potential-induced Ca2+ transients in hippocampal neurons.

In hippocampal pyramidal neurons, voltage-gated Ca(2+) channels open in response to action potentials. This results in elevations in the intracellular concentration of Ca(2+) that are maximal in the proximal apical dendrites and decrease rapidly with distance from the soma. The control of these action potential-evoked Ca(2+) elevations is critical for the regulation of hippocampal neuronal activity. As part of Ca(2+) signaling microdomains, small-conductance Ca(2+)-activated K(+) (SK) channels have been shown to modulate the amplitude and duration of intracellular Ca(2+) signals by feedback regulation of synaptically activated Ca(2+) sources in small distal dendrites and dendritic spines, thus affecting synaptic plasticity in the hippocampus. In this study, we investigated the effect of the activation of SK channels on Ca(2+) transients specifically induced by action potentials in the proximal processes of hippocampal pyramidal neurons. Our results, obtained by using selective SK channel blockers and enhancers, show that SK channels act in a feedback loop, in which their activation by Ca(2+) entering mainly through L-type voltage-gated Ca(2+) channels leads to a reduction in the subsequent dendritic influx of Ca(2+). This underscores a new role of SK channels in the proximal apical dendrite of hippocampal pyramidal neurons.

Augmentation of Kv4.2-encoded Currents by Accessory Dipeptidyl Peptidase 6 and 10 Subunits Reflects Selective Cell Surface Kv4.2 Protein Stabilization

Rapidly activating and inactivating somatodendritic voltage-gated K(+) (Kv) currents, I(A), play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal I(A) channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K(+) channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

Generation of computer models of dendritic signal processing and synaptic plasticity mechanisms in hippocampal nerve cells by high-resolution microscopy

Event Abstract Back to Event Generation of computer models of dendritic signal processing and synaptic plasticity mechanisms in hippocampal nerve cells by high-resolution microscopy Stefan Remy1, 2*, William Kath3, Stephen Smith4 and Nelson Spruston5 1 Deutsches Zentrum für Neurodegenerative Erkrankungen, Germany 2 University of Bonn, Germany 3 Northwestern University, United States 4 Stanford University, United States 5 HHMI Janelia Farm Research Center, United States Neuronal dendrites constitute an elaborate web of branching processes, excitatory and inhibitory synapses, neurotransmitter receptors and voltage-gated channels. How can we visualize these components, determine their spatial relationships, and reveal their functional interactions? If we can see the component parts at high resolution, we can then use this information to generate more accurate computational models of dendrites and the complex integrative functions they perform. We propose to combine two high-resolution imaging methodologies: two-photon microscopy, which provides high-resolution functional imaging in living tissue, and array tomography, which provides even higher resolution and the ability to identify the location of molecular constituents of the dendrite. These experiments will provide a view of the dendritic tree with unprecedented structural and functional detail. Using these data, we will then develop improved computational models of the dendritic tree that combine and integrate this underlying organizational complexity. A focus of the proposal is the distribution of synapses over a vast dendritic tree. Of the tens of thousands of synapses on a single CA1 pyramidal neuron, many are located electrically far from the site of action. Furthermore, there is abundant evidence that the size and properties of excitatory synapses vary according to their dendritic position. Likewise, inhibitory synapses that target dendrites are diverse in their location, molecular markers, and functional properties. Our multidisciplinary, collaborative approach will use the hippocampus as a model system for studying this neuronal complexity at the level of cells, synapses and molecules, but the results will provide working hypotheses and approaches that can be applied in future studies to other types of pyramidal neurons, such as those in the neocortex. In our view, this is an important step, because understanding the intricacies of neurons and their connections is a necessary component of understanding complex neural circuits. The two experimental approaches we will employ – two-photon uncaging and array tomography – are both relatively new and both provide sub-micron resolution regarding synaptic structure and function. The results of our experiments will be used to develop computational models, which will be used to generate and test hypotheses regarding the complex integration of synaptic inputs that occurs in the dendrites of hippocampal pyramidal neurons. Keywords: Dendrites, Synapses, array tomography, two-photon microscopy, STDP, dendritic spike Conference: Bernstein Conference 2012, Munich, Germany, 12 Sep - 14 Sep, 2012. Presentation Type: Poster Topic: Other Citation: Remy S, Kath W, Smith S and Spruston N (2012). Generation of computer models of dendritic signal processing and synaptic plasticity mechanisms in hippocampal nerve cells by high-resolution microscopy. Front. Comput. Neurosci. Conference Abstract: Bernstein Conference 2012. doi: 10.3389/conf.fncom.2012.55.00041 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: 18 Sep 2012; Published Online: 12 Sep 2012. * Correspondence: Prof. Stefan Remy, Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn, Germany, stefan.remy@ukb.uni-bonn.de 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 Stefan Remy William Kath Stephen Smith Nelson Spruston Google Stefan Remy William Kath Stephen Smith Nelson Spruston Google Scholar Stefan Remy William Kath Stephen Smith Nelson Spruston PubMed Stefan Remy William Kath Stephen Smith Nelson Spruston 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.

Three-dimensional imaging and photostimulation by remote-focusing and holographic light patterning

Access to three-dimensional structures in the brain is fundamental to probe signal processing at multiple levels, from integration of synaptic inputs to network activity mapping. Here, we present an optical method for independent three-dimensional photoactivation and imaging by combination of digital holography with remote-focusing. We experimentally demonstrate compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We apply this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging. By bringing extended portions of tilted dendrites simultaneously in-focus, we monitor the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na(+) channels.

DPP6 Establishes the A-Type K+ Current Gradient Critical for the Regulation of Dendritic Excitability in CA1 Hippocampal Neurons

Subthreshold-activating A-type K(+) currents are essential for the proper functioning of the brain, where they act to delay excitation and regulate firing frequency. In CA1 hippocampal pyramidal neuron dendrites, the density of A-type K(+) current increases with distance from the soma, playing an important role in synaptic integration and plasticity. The mechanism underlying this gradient has, however, remained elusive. Here, dendritic recordings from mice lacking the Kv4 transmembrane auxiliary subunit DPP6 revealed that this protein is critical for generating the A-current gradient. Loss of DPP6 led to a decrease in A-type current, specifically in distal dendrites. Decreased current density was accompanied by a depolarizing shift in the voltage dependence of channel activation. Together these changes resulted in hyperexcitable dendrites with enhanced dendritic AP back-propagation, calcium electrogenesis, and induction of synaptic long-term potentiation. Despite enhanced dendritic excitability, firing behavior evoked by somatic current injection was mainly unaffected in DPP6-KO recordings, indicating compartmentalized regulation of neuronal excitability.

Time-lapse imaging of disease progression in deep brain areas using fluorescence microendoscopy

The combination of intravital microscopy and animal models of disease has propelled studies of disease mechanisms and treatments. However, many disorders afflict tissues inaccessible to light microscopy in live subjects. Here we introduce cellular-level time-lapse imaging deep within the live mammalian brain by one- and two-photon fluorescence microendoscopy over multiple weeks. Bilateral imaging sites allowed longitudinal comparisons within individual subjects, including of normal and diseased tissues. Using this approach we tracked CA1 hippocampal pyramidal neuron dendrites in adult mice, revealing these dendrites' extreme stability (>8,000 day mean lifetime) and rare examples of their structural alterations. To illustrate disease studies, we tracked deep lying gliomas by observing tumor growth, visualizing three-dimensional vasculature structure, and determining microcirculatory speeds. Average erythrocyte speeds in gliomas declined markedly as the disease advanced, notwithstanding significant increases in capillary diameters. Time-lapse microendoscopy will be applicable to studies of numerous disorders, including neurovascular, neurological, cancerous, and trauma-induced conditions.

MRNA for the EAAC1 subtype of glutamate transporter is present in neuronal dendrites in vitro and dramatically increases in vivo after a seizure

The neuronal Na+-dependent glutamate transporter, excitatory amino acid carrier 1 (EAAC1, also called EAAT3), has been implicated in the control of synaptic spillover of glutamate, synaptic plasticity, and the import of cysteine for neuronal synthesis of glutathione. EAAC1 protein is observed in both perisynaptic regions of the synapse and in neuronal cell bodies. Although amino acid residues in the carboxyl terminal tail have been implicated in the dendritic targeting of EAAC1 protein, it is not known if mRNA for EAAC1 may also be targeted to dendrites. Sorting of mRNA to specific cellular domains provides a mechanism by which signals can rapidly increase translation in a local environment; this form of regulated translation has been linked to diverse biological phenomena ranging from establishment of polarity during embryogenesis to synapse development and synaptic plasticity. In the present study, EAAC1 mRNA sequences were amplified from dendritic samples that were mechanically harvested from low-density hippocampal neuronal cultures. In parallel analyses, mRNA for histone deacetylase 2 (HDAC-2) and glial fibrillary acidic protein (GFAP) was not detected, suggesting that these samples are not contaminated with cell body or glial mRNAs. EAAC1 mRNA also co-localized with Map2a (a marker of dendrites) but not Tau1 (a marker of axons) in hippocampal neuronal cultures by in situ hybridization. In control rats, EAAC1 mRNA was observed in soma and proximal dendrites of hippocampal pyramidal neurons. Following pilocarpine- or kainate-induced seizures, EAAC1 mRNA was present in CA1 pyramidal cell dendrites up to 200μm from the soma. These studies provide the first evidence that EAAC1 mRNA localizes to dendrites and suggest that dendritic targeting of EAAC1 mRNA is increased by seizure activity and may be regulated by neuronal activity/depolarization.

Downregulation of Dendritic HCN Channel Gating in Epilepsy Is Mediated by Altered Phosphorylation Signaling

The onset of spontaneous seizures in the pilocarpine model of epilepsy causes a hyperpolarized shift in the voltage-dependent activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated current (Ih) in CA1 hippocampal pyramidal neuron dendrites, contributing to neuronal hyperexcitability and possibly to epileptogenesis. However, the specific mechanisms by which spontaneous seizures cause downregulation of HCN channel gating are yet unknown. We asked whether the seizure-dependent downregulation of HCN channel gating was due to altered phosphorylation signaling mediated by the phosphatase calcineurin (CaN) or the kinase p38 mitogen-activated protein kinase (p38 MAPK). We first found that CaN inhibition upregulated HCN channel gating and reduced neuronal excitability under normal conditions, showing that CaN is a strong modulator of HCN channels. We then found that an in vitro model of seizures (1 h in 0 Mg2+ and 50 microM bicuculline at 35-37 degrees C) reproduced the HCN channel gating change seen in vivo. Pharmacological inhibition of CaN or activation of p38 MAPK partially reversed the in vitro seizure-induced hyperpolarized shift in HCN channel gating, and the shift was fully reversed by the combination of CaN inhibition and p38 MAPK activation. We then demonstrated enhanced CaN activity as well as reduced p38 MAPK activity in vivo in the CA1 hippocampal area of chronically epileptic animals. Pharmacological reversal of these phosphorylation changes restored HCN channel gating downregulation and neuronal hyperexcitability in epileptic tissue to control levels. Together, these results suggest that alteration of two different phosphorylation pathways in epilepsy contributes to the downregulation of HCN channel gating, which consequently produces neuronal hyperexcitability and thus may be a target for novel antiepileptic therapies.

Retrograde BMP Signaling Controls Synaptic Growth at the NMJ by Regulating Trio Expression in Motor Neurons

Retrograde signaling is essential for coordinating the growth of synaptic structures; however, it is not clear how it can lead to modulation of cytoskeletal dynamics and structural changes at presynaptic terminals. We show that loss of retrograde bone morphogenic protein (BMP) signaling at the Drosophila larval neuromuscular junction (NMJ) leads to a significant reduction in levels of Rac GEF Trio and a diminution of transcription at the trio locus. We further find that Trio is required in motor neurons for normal structural growth. Finally, we show that transgenic expression of Trio in motor neurons can partially restore NMJ defects in larvae mutant for BMP signaling. Based on our findings, we propose a model in which a retrograde BMP signal from the muscle modulates GTPase activity through transcriptional regulation of Rac GEF trio, thereby regulating the homeostasis of synaptic growth at the NMJ.

Alternatively Spliced Isoforms of TRIP8b Differentially Control h Channel Trafficking and Function

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) are the molecular basis for the current, I(h), which contributes crucially to intrinsic neuronal excitability. The subcellular localization and biophysical properties of h channels govern their function, but the mechanisms controlling these characteristics, and especially the potential role of auxiliary subunits or other binding proteins, remain unclear. We focused on TRIP8b, an h channel-interacting protein that colocalizes with HCN1 in cortical and hippocampal pyramidal neuron dendrites, and found that it exists in multiple alternative splice variants with distinct effects on h channel trafficking and function. The developmentally regulated splice variants of TRIP8b all shared dual, C terminus-located interaction sites with HCN1. When coexpressed with HCN1 in heterologous cells individual TRIP8b isoforms similarly modulated gating of I(h), causing a hyperpolarizing shift in voltage dependence of channel activation, but differentially upregulated or downregulated I(h) current density and HCN1 surface expression. In hippocampal neurons, coexpression of TRIP8b isoforms with HCN1 produced isoform-specific changes of HCN1 localization. Interestingly, the TRIP8b isoforms most abundant in the brain are those predicted to enhance h channel surface expression. Indeed, shRNA knockdown of TRIP8b in hippocampal neurons significantly reduced native I(h). Thus, although TRIP8b exists in multiple splice isoforms, our data suggest that the predominant role of this protein in brain is to promote h channel surface expression and enhance I(h). Because I(h) expression is altered in models of several diseases, including temporal lobe epilepsy, TRIP8b may play a role in both normal neuronal function and in aberrant neuronal excitability associated with neurological disease.