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1.  Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABAA receptors in cortical pyramidal cells 
The Journal of Physiology  2012;590(Pt 7):1517-1534.
Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABAARγ277Ilox) in which the γ2 subunit of the GABAA receptor (GABAAR) was mutated to become zolpidem insensitive (γ277I) and used viral vectors to swap γ277I with wild-type, zolpidem-sensitive γ2 subunits (γ277F). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABAARγ2−/− mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 (AU1γ277F) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the AU1γ277F subunit (Cre-2A-AU1γ277F) into the neocortex of GABAARγ277Ilox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the AU1γ277F subunits in perisomatic GABAergic synapses. In line with this, miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ277I and AU1γ277F GABAARs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.
doi:10.1113/jphysiol.2012.227538
PMCID: PMC3413495  PMID: 22351636
2.  Molecular Identity of Dendritic Voltage-Gated Sodium Channels 
Science (New York, N.Y.)  2010;328(5980):906-909.
Active invasion of the dendritic tree by action potentials (APs) generated in the axon is essential for associative synaptic plasticity and neuronal ensemble formation. In cortical pyramidal cells (PCs) this AP backpropagation is supported by dendritic voltage-gated Na+ channels (Nav), whose molecular identity is unknown. Using a highly sensitive electron microscopic immunogold technique, we reveal the presence of the Nav1.6 subunit in hippocampal CA1 PC proximal and distal dendrites. Here the subunit density is lower by a factor of 35 to 80 than that found in axon initial segments. A gradual decrease in Nav1.6 density along the proximodistal axis of the dendritic tree was also detected without any labeling in dendritic spines. Our results reveal the characteristic subcellular distribution of the Nav1.6 subunit, identifying this molecule as a key substrate enabling dendritic excitability.
doi:10.1126/science.1187958
PMCID: PMC3546315  PMID: 20466935
3.  Release probability of hippocampal glutamatergic terminals scales with the size of the active zone 
Nature neuroscience  2012;15(7):988-997.
Cortical synapses display remarkable structural, molecular and functional heterogeneity. Our knowledge regarding the relationship between the ultrastructural and functional parameters is still fragmented. Here we asked how the release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active zone area. The peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated Ca2+ channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active zone area. Gold particles for Cav2.1 showed a nonrandom distribution within the active zones. Our results demonstrate that the number of several active zone proteins, including presynaptic Ca2+ channels, docked vesicles and the release probability scales linearly with the active zone area.
doi:10.1038/nn.3137
PMCID: PMC3386897  PMID: 22683683
4.  Unique somato-dendritic distribution pattern of Kv4.2 channels on hippocampal CA1 pyramidal cells 
A-type K+ current (IA) plays a critical role in controlling the excitability of pyramidal cell (PC) dendrites. In vitro dendritic patch-pipette recordings have demonstrated a prominent, 6-fold increase in IA density along the main apical dendrites of rat hippocampal CA1 PCs. In these cells, IA is mediated by Kv4.2 subunits, whose precise subcellular distribution and densities in small diameter oblique dendrites and dendritic spines are still unknown. Here we examined the densities of the Kv4.2 subunit in 13 axo-somato-dendritic compartments of CA1 PCs using a highly sensitive, high-resolution quantitative immunogold localization method (SDS-digested freeze-fracture replica-labelling). Only a ~70% increase in Kv4.2 immunogold density was observed along the proximo-distal axis of main apical dendrites in the stratum radiatum with a slight decrease in density in stratum lacunosum-moleculare. A similar pattern was detected for all dendritic compartments, including main apical dendrites, small diameter oblique dendrites and dendritic spines. The specificity of the somato-dendritic labelling was confirmed in Kv4.2−/− tissue. No specific immunolabelling for the Kv4.2 subunit was found in SNAP-25 containing presynaptic axons. Our results demonstrate a novel distribution pattern of a voltage-gated ion channel along the somato-dendritic surface of CA1 PCs and suggest that the increase in the IA along the proximo-distal axis of PC dendrites cannot be solely explained by a corresponding increase in Kv4.2 channel number.
doi:10.1111/j.1460-9568.2011.07907.x
PMCID: PMC3428895  PMID: 22098631
potassium channels; pyramidal cell; immunohistochemistry; rat; hippocampus; dendrite
5.  Generalized neuromuscular hypoplasia, reduced smooth muscle myosin and altered gut motility in the klotho model of premature aging 
Background
Gastrointestinal symptoms, particularly constipation, increase with aging, but their underlying mechanisms are poorly understood due to a lack of experimental models. Previously we established the progeric klotho mouse as a model of aging-associated anorexia and gastric dysmotility. We also detected reduced fecal output in these animals; therefore, the aim of this study was to investigate in-vivo function and cellular make-up of the small intestinal and colonic neuromuscular apparatus.
Methods
Klotho expression was studied by RT-PCR and immunohistochemistry. Motility was assessed by dye transit and bead expulsion. Smooth muscle and neuron-specific gene expression was studied by Western immunoblotting. Interstitial cells of Cajal (ICC) and precursors were analyzed by flow cytometry, confocal microscopy and 3-dimensional reconstruction. HuC/D+ myenteric neurons were enumerated by fluorescent microscopy.
Key Results
Klotho protein was detected in neurons, smooth muscle cells and some ICC classes. Small intestinal transit was slower but whole-gut transit of klotho mice was accelerated due to faster colonic transit and shorter intestinal lengths, apparent only after weaning. Fecal water content remained normal despite reduced output. Smooth muscle myosin expression was reduced. ICC, ICC precursors, as well as nitrergic and cholinergic neurons maintained their normal proportions in the shorter intestines.
Conclusions & Inferences
Progeric klotho mice express less contractile proteins and develop generalized intestinal neuromuscular hypoplasia mainly arising from stunted post-weaning growth. Since reduced fecal output in these mice occurs in the presence of accelerated colonic and whole-gut transit, it likely reflects reduced food intake rather than intestinal dysmotility.
doi:10.1111/j.1365-2982.2011.01730.x
PMCID: PMC3149585  PMID: 21605285
Aging; gastrointestinal motility; smooth muscle; interstitial cells of Cajal; enteric neurons
6.  Kitlow stem cells cause resistance to Kit/Pdgfra inhibitors in murine gastrointestinal stromal tumors 
Gastroenterology  2010;139(3):942-952.
Background & Aims
Gastrointestinal stromal tumors (GIST) are related to interstitial cells of Cajal (ICC) and often contain activating Kit or Pdgfra mutations. Inhibitors of Kit/Pdgfra signaling such as imatinib mesylate have increased progression-free survival in metastatic GIST but are not curative. In mouse models we investigated whether Kitlow adult ICC progenitors could represent an inherently Kit/Pdgfra inhibitor-resistant reservoir for GIST.
Methods
KitlowCd44+Cd34+ cells were isolated and characterized after serial clonal re-derivation. Tumorigenic potential of spontaneously transformed cells was investigated in nude mice. The KitlowCd44+Cd34+ cells responsiveness to Kit activation and blockade was studied by enumerating them in KitK641E mice (a GIST model), in mice with defective Kit signaling, and pharmacologically.
Results
Single isolated KitlowCd44+Cd34+ cells were clonogenic and capable of self-renewal and differentiation into ICC. In nude mice, spontaneously transformed cells formed malignant tumors expressing GIST markers. The KitlowCd44+Cd34+ cells were resistant to in vitro Kit blockade, including by imatinib, and occurred in normal numbers in mice with reduced Kit signaling. In KitK641E mice, the mutant ICC stem cells were grossly hyperplastic but remained imatinib-resistant. In contrast, the cancer-initiating cell-targeting drug salinomycin blocked the proliferation of KitlowCd44+Cd34+ cells and increased their sensitivity to imatinib.
Conclusions
KitlowCd44+Cd34+ progenitors are true stem cells for normal and hyperplastic ICC and give rise to GIST. Resistance to Kit/Pdgfra inhibitors is inherent in GIST and is due to the native ICC stem cells lack of dependence on Kit for survival, which is maintained after the acquisition of oncogenic Kit mutation. Cancer stem cell drugs may target these cells.
doi:10.1053/j.gastro.2010.05.083
PMCID: PMC2933938  PMID: 20621681
Cancer-initiating cell; imatinib; neoplasm
7.  Specificity of immunoreactions 
doi:10.1523/JNEUROSCI.2494-08.2008
PMCID: PMC2629537  PMID: 18784286
immunohistochemistry; antibodies; ion channels; receptors; specificity
8.  Cell-Type-Dependent Molecular Composition of the Axon Initial Segment 
The exact site of initiation and shape of action potentials vary among different neuronal types. The reason for this variability is largely unknown, but the subunit composition, density and distribution of voltage-gated sodium (Nav) and potassium (Kv) channels within the axon initial segment (AIS) are likely to play a key role. Here, we asked how heterogeneous are the density and distribution of Nav and Kv channels within the AISs of a variety of excitatory and inhibitory neurons. Most of the studied cell types expressed a high density of Nav1.6, Kv1.1, and Kv1.2 subunits in their AIS, but the Nav1.1 subunit could only be detected in GABAergic interneurons. A proximo-distal gradient in the density of these subunits was observed within the AIS of certain nerve cells but not in others. For example, a gradual increase of the Nav1.6 subunit was observed in cortical layer 2/3 and hippocampal CA1 pyramidal cell (PC) AISs, whereas its density was rather uniform in layer 5 PC AISs. The Nav1.1 subunit was distributed evenly along the AIS of short-axon cells of the main olfactory bulb but was restricted to the proximal part of the AIS in cortical and cerebellar interneurons. Our results reveal a cell type-dependent expression of sodium and potassium channel subunits with varying densities along the proximo-distal axis of the AISs. This precise arrangement is likely to contribute to the diversity of firing properties observed among central neurons.
doi:10.1523/JNEUROSCI.4833-08.2008
PMCID: PMC2628579  PMID: 19118165
voltage-gated ion channels; immunohistochemistry; axon initial segment; cortex; hippocampus; cerebellum; olfactory bulb
9.  Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABAA receptors in cortical pyramidal cells 
The Journal of Physiology  2012;590(7):1517-1534.
Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABAARγ277Ilox) in which the γ2 subunit of the GABAA receptor (GABAAR) was mutated to become zolpidem insensitive (γ277I) and used viral vectors to swap γ277I with wild-type, zolpidem-sensitive γ2 subunits (γ277F). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABAARγ2−/− mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 (AU1γ277F) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the AU1γ277F subunit (Cre-2A-AU1γ277F) into the neocortex of GABAARγ277Ilox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the AU1γ277F subunits in perisomatic GABAergic synapses. In line with this, miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ277I and AU1γ277F GABAARs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it does not require any extrinsic protein that might endow some unforeseen alterations of the genetically modified cells. In addition, this virus-based approach opens up the possibility of modifying multiple cell types in distinct brain regions and performing alternative recombination-based intersectional genetic manipulations.
doi:10.1113/jphysiol.2012.227538
PMCID: PMC3413495  PMID: 22351636
10.  Unique somato-dendritic distribution pattern of Kv4.2 channels on hippocampal CA1 pyramidal cells 
A-type K+ current (IA) plays a critical role in controlling the excitability of pyramidal cell (PC) dendrites. In vitro dendritic patch-pipette recordings have demonstrated a prominent, sixfold increase in IA density along the main apical dendrites of rat hippocampal CA1 PCs. In these cells, IA is mediated by Kv4.2 subunits, whose precise subcellular distribution and densities in small-diameter oblique dendrites and dendritic spines are still unknown. Here we examined the densities of the Kv4.2 subunit in 13 axo-somato-dendritic compartments of CA1 PCs using a highly sensitive, high-resolution quantitative immunogold localization method (sodium dodecyl sulphate-digested freeze-fracture replica-labelling). Only an approximately 70% increase in Kv4.2 immunogold density was observed along the proximo-distal axis of main apical dendrites in the stratum radiatum with a slight decrease in density in stratum lacunosum-moleculare. A similar pattern was detected for all dendritic compartments, including main apical dendrites, small-diameter oblique dendrites and dendritic spines. The specificity of the somato-dendritic labelling was confirmed in Kv4.2−/− tissue. No specific immunolabelling for the Kv4.2 subunit was found in SNAP-25-containing presynaptic axons. Our results demonstrate a novel distribution pattern of a voltage-gated ion channel along the somato-dendritic surface of CA1 PCs, and suggest that the increase in the IA along the proximo-distal axis of PC dendrites cannot be solely explained by a corresponding increase in Kv4.2 channel number.
doi:10.1111/j.1460-9568.2011.07907.x
PMCID: PMC3428895  PMID: 22098631
dendrite; hippocampus; immunohistochemistry; potassium channels; pyramidal cell; rat
11.  Distinct axo-somato-dendritic distributions of three potassium channels in CA1 hippocampal pyramidal cells 
The European Journal of Neuroscience  2014;39(11):1771-1783.
Potassium channels comprise the most diverse family of ion channels and play critical roles in a large variety of physiological and pathological processes. In addition to their molecular diversity, variations in their distributions and densities on the axo-somato-dendritic surface of neurons are key parameters in determining their functional impact. Despite extensive electrophysiological and anatomical investigations, the exact location and densities of most K+ channels in small subcellular compartments are still unknown. Here we aimed at providing a quantitative surface map of two delayed-rectifier (Kv1.1 and Kv2.1) and one G-protein-gated inwardly rectifying (Kir3.2) K+ channel subunits on hippocampal CA1 pyramidal cells (PCs). Freeze-fracture replica immunogold labelling was employed to determine the relative densities of these K+ channel subunits in 18 axo-somato-dendritic compartments. Significant densities of the Kv1.1 subunit were detected on axon initial segments (AISs) and axon terminals, with an approximately eight-fold lower density in the latter compartment. The Kv2.1 subunit was found in somatic, proximal dendritic and AIS plasma membranes at approximately the same densities. This subunit has a non-uniform plasma membrane distribution; Kv2.1 clusters are frequently adjacent to, but never overlap with, GABAergic synapses. A quasi-linear increase in the Kir3.2 subunit density along the dendrites of PCs was detected, showing no significant difference between apical dendritic shafts, oblique dendrites or dendritic spines at the same distance from the soma. Our results demonstrate that each subunit has a unique cell-surface distribution pattern, and predict their differential involvement in synaptic integration and output generation at distinct subcellular compartments.
doi:10.1111/ejn.12526
PMCID: PMC4150533  PMID: 24606584
confocal microscopy; electron microscopy; immunohistochemistry; ion channels

Results 1-11 (11)