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• W2090693552 abstract "p75 is expressed among Purkinje cells in the adult cerebellum, but its function has remained obscure. Here we report that p75 is involved in maintaining the frequency and regularity of spontaneous firing of Purkinje cells. The overall spontaneous firing activity of Purkinje cells was increased in p75−/− mice during the phasic firing period due to a longer firing period and accompanying reduction in silence period than in the wild type. We attribute these effects to a reduction in small conductance Ca2+-activated potassium (SK) channel activity in Purkinje cells from p75−/− mice compared with the wild type littermates. The mechanism by which p75 regulates SK channel activity appears to involve its ability to activate Rac1. In organotypic cultures of cerebellar slices, brain-derived neurotrophic factor increased RacGTP levels by activating p75 but not TrkB. These results correlate with a reduction in RacGTP levels in synaptosome fractions from the p75−/− cerebellum, but not in that from the cortex of the same animals, compared with wild type littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75−/− cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels. p75 is expressed among Purkinje cells in the adult cerebellum, but its function has remained obscure. Here we report that p75 is involved in maintaining the frequency and regularity of spontaneous firing of Purkinje cells. The overall spontaneous firing activity of Purkinje cells was increased in p75−/− mice during the phasic firing period due to a longer firing period and accompanying reduction in silence period than in the wild type. We attribute these effects to a reduction in small conductance Ca2+-activated potassium (SK) channel activity in Purkinje cells from p75−/− mice compared with the wild type littermates. The mechanism by which p75 regulates SK channel activity appears to involve its ability to activate Rac1. In organotypic cultures of cerebellar slices, brain-derived neurotrophic factor increased RacGTP levels by activating p75 but not TrkB. These results correlate with a reduction in RacGTP levels in synaptosome fractions from the p75−/− cerebellum, but not in that from the cortex of the same animals, compared with wild type littermates. More importantly, we demonstrate that Rac1 modulates SK channel activity and firing patterns of Purkinje cells. Along with the finding that spine density was reduced in p75−/− cerebellum, these data suggest that p75 plays a role in maintaining normalcy of Purkinje cell firing in the cerebellum in part by activating Rac1 in synaptic compartments and modulating SK channels. p75 is widely expressed during brain development, but in the adult brain, its expression is most notable in cholinergic neurons of the septum and Purkinje cells of the cerebellum. Although the role of p75 in septal cholinergic neurons appears to be related to cell survival and degeneration (1.Chao M.V. Neurotrophins and their receptors: a convergence point for many signalling pathways.Nat. Rev. Neurosci. 2003; 4: 299-309Crossref PubMed Scopus (1725) Google Scholar), its role in Purkinje cells has remained largely undetermined. It has been reported, based on analysis of compound BDNF+/−:p75−/− mice, that p75 plays a role in dendritic development of Purkinje cells and cerebellar fissure formation, both in BDNF 3The abbreviations used are: BDNFbrain-derived neurotrophic factorGIRKG-protein coupled inwardly rectifying potassium channelVGCCvoltage-gated Ca2+ channelp-PAKphospho-PAKaCSFartificial cerebrospinal fluidTEAtetraethylammoniumAPaction potentialAHPafterhyperpolarizationTTXtetrodotoxinPnpostnatal day nSKsmall conductance Ca2+-activated K+BKlarge conductance Ca2+-activated K+.-dependent and -independent manners (2.Carter A.R. Berry E.M. Segal R.A. Regional expression of p75NTR contributes to neurotrophin regulation of cerebellar patterning.Mol. Cell Neurosci. 2003; 22: 1-13Crossref PubMed Scopus (36) Google Scholar). However, neither the functional consequences of such structural changes nor the mechanisms by which p75 elicits such changes are known. brain-derived neurotrophic factor G-protein coupled inwardly rectifying potassium channel voltage-gated Ca2+ channel phospho-PAK artificial cerebrospinal fluid tetraethylammonium action potential afterhyperpolarization tetrodotoxin postnatal day n small conductance Ca2+-activated K+ large conductance Ca2+-activated K+. We have previously reported that p75 activates Rac1 in response to neurotrophins in oligodendrocytes and Schwann cells (3.Harrington A.W. Kim J.Y. Yoon S.O. Activation of Rac GTPase by p75 is Necessary for c-Jun N-terminal kinase-mediated apoptosis.J. Neurosci. 2002; 22: 156-166Crossref PubMed Google Scholar, 4.Tep C. Kim M.L. Opincariu L.I. Limpert A.S. Chan J.R. Appel B. Carter B.D. Yoon S.O. BDNF induces polarized signaling of small GTPase (Rac1) at the onset of Schwann cell myelination through partitioning-defective 3 (Par3).J. Biol. Chem. 2012; 287: 1600-1608Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). As one of the small GTPases, Rac1 plays a central role in spine formation during development and in the adult, thereby regulating synaptic activity in a wide variety of neurons (5.de Curtis I. Functions of Rac GTPases during neuronal development.Dev. Neurosci. 2008; 30: 47-58Crossref PubMed Scopus (69) Google Scholar). As such, regulating its activity via its guanidine nucleotide exchange factors (6.Xie Z. Photowala H. Cahill M.E. Srivastava D.P. Woolfrey K.M. Shum C.Y. Huganir R.L. Penzes P. Coordination of synaptic adhesion with dendritic spine remodeling by AF-6 and kalirin-7.J. Neurosci. 2008; 28: 6079-6091Crossref PubMed Scopus (82) Google Scholar, 7.Tolias K.F. Bikoff J.B. Burette A. Paradis S. Harrar D. Tavazoie S. Weinberg R.J. Greenberg M.E. The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines.Neuron. 2005; 45: 525-538Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 8.Tolias K.F. Bikoff J.B. Kane C.G. Tolias C.S. Hu L. Greenberg M.E. The Rac1 guanine nucleotide exchange factor Tiam1 mediates EphB receptor-dependent dendritic spine development.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7265-7270Crossref PubMed Scopus (166) Google Scholar) and RacGTPase-activating proteins (9.Buttery P. Beg A.A. Chih B. Broder A. Mason C.A. Scheiffele P. The diacylglycerol-binding protein α1-chimaerin regulates dendritic morphology.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 1924-1929Crossref PubMed Scopus (56) Google Scholar, 10.Van de Ven T.J. VanDongen H.M. VanDongen A.M. The nonkinase phorbol ester receptor α1-chimerin binds the NMDA receptor NR2A subunit and regulates dendritic spine density.J. Neurosci. 2005; 25: 9488-9496Crossref PubMed Scopus (58) Google Scholar, 11.Oh D. Han S. Seo J. Lee J.R. Choi J. Groffen J. Kim K. Cho Y.S. Choi H.S. Shin H. Woo J. Won H. Park S.K. Kim S.Y. Jo J. Whitcomb D.J. Cho K. Kim H. Bae Y.C. Heisterkamp N. Choi S.Y. Kim E. Regulation of synaptic Rac1 activity, long-term potentiation maintenance, and learning and memory by BCR and ABR Rac GTPase-activating proteins.J. Neurosci. 2010; 30: 14134-14144Crossref PubMed Scopus (84) Google Scholar) was also known to affect not only spine morphology but also synaptic function. Interestingly, the effect of RacGTP on synaptic function is not merely a consequence of spine morphology regulation. Rac1 signaling has been shown to affect ion channels, such as Kir2.1 (12.Boyer S.B. Slesinger P.A. Jones S.V. Regulation of Kir2.1 channels by the Rho-GTPase, Rac1.J. Cell Physiol. 2009; 218: 385-393Crossref PubMed Scopus (22) Google Scholar), GABAA receptors (13.Meyer D.K. Olenik C. Hofmann F. Barth H. Leemhuis J. Brünig I. Aktories K. Nörenberg W. Regulation of somatodendritic GABAA receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Rac1.J. Neurosci. 2000; 20: 6743-6751Crossref PubMed Google Scholar), voltage-gated Ca2+ channels (VGCCs) (14.Wilk-Blaszczak M.A. Singer W.D. Quill T. Miller B. Frost J.A. Sternweis P.C. Belardetti F. The monomeric G-proteins Rac1 and/or Cdc42 are required for the inhibition of voltage-dependent calcium current by bradykinin.J. Neurosci. 1997; 17: 4094-4100Crossref PubMed Google Scholar), and GIRK channels (15.Coulson E.J. May L.M. Osborne S.L. Reid K. Underwood C.K. Meunier F.A. Bartlett P.F. Sah P. p75 neurotrophin receptor mediates neuronal cell death by activating GIRK channels through phosphatidylinositol 4,5-bisphosphate.J. Neurosci. 2008; 28: 315-324Crossref PubMed Scopus (48) Google Scholar) in the nervous system. These were thought to occur either through the effect of Rac1 on actin cytoskeleton (13.Meyer D.K. Olenik C. Hofmann F. Barth H. Leemhuis J. Brünig I. Aktories K. Nörenberg W. Regulation of somatodendritic GABAA receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Rac1.J. Neurosci. 2000; 20: 6743-6751Crossref PubMed Google Scholar) or its action on promoting the synthesis of phosphatidylinositol 4,5-bisphosphate (15.Coulson E.J. May L.M. Osborne S.L. Reid K. Underwood C.K. Meunier F.A. Bartlett P.F. Sah P. p75 neurotrophin receptor mediates neuronal cell death by activating GIRK channels through phosphatidylinositol 4,5-bisphosphate.J. Neurosci. 2008; 28: 315-324Crossref PubMed Scopus (48) Google Scholar). Conversely, RacGTP levels are regulated by neuronal activity (16.Li Z. Aizenman C.D. Cline H.T. Regulation of rho GTPases by crosstalk and neuronal activity in vivo.Neuron. 2002; 33: 741-750Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 17.Sin W.C. Haas K. Ruthazer E.S. Cline H.T. Dendrite growth increased by visual activity requires NMDA receptor and Rho GTPases.Nature. 2002; 419: 475-480Crossref PubMed Scopus (367) Google Scholar), including the activation of NMDA receptors (7.Tolias K.F. Bikoff J.B. Burette A. Paradis S. Harrar D. Tavazoie S. Weinberg R.J. Greenberg M.E. The Rac1-GEF Tiam1 couples the NMDA receptor to the activity-dependent development of dendritic arbors and spines.Neuron. 2005; 45: 525-538Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar), which orchestrates with EphB receptors to regulate Rac1 function and thereby spine morphogenesis through actin remodeling (8.Tolias K.F. Bikoff J.B. Kane C.G. Tolias C.S. Hu L. Greenberg M.E. The Rac1 guanine nucleotide exchange factor Tiam1 mediates EphB receptor-dependent dendritic spine development.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 7265-7270Crossref PubMed Scopus (166) Google Scholar, 18.Penzes P. Beeser A. Chernoff J. Schiller M.R. Eipper B.A. Mains R.E. Huganir R.L. Rapid induction of dendritic spine morphogenesis by trans-synaptic ephrinB-EphB receptor activation of the Rho-GEF kalirin.Neuron. 2003; 37: 263-274Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Therefore, Rac1 can modulate neuronal activities at multiple levels during development and plasticity, including acute effects through ion channel regulation and long term consequences resulting from structural changes of dendritic arbors and spines. Indeed, dysregulation of Rac1 has been implicated in several mental disorders, such as schizophrenia (19.Hayashi-Takagi A. Takaki M. Graziane N. Seshadri S. Murdoch H. Dunlop A.J. Makino Y. Seshadri A.J. Ishizuka K. Srivastava D.P. Xie Z. Baraban J.M. Houslay M.D. Tomoda T. Brandon N.J. Kamiya A. Yan Z. Penzes P. Sawa A. Disrupted-in-Schizophrenia 1 (DISC1) regulates spines of the glutamate synapse via Rac1.Nat. Neurosci. 2010; 13: 327-332Crossref PubMed Scopus (331) Google Scholar) and fragile X syndrome (20.Chen L.Y. Rex C.S. Babayan A.H. Kramár E.A. Lynch G. Gall C.M. Lauterborn J.C. Physiological activation of synaptic Rac>PAK (p-21 activated kinase) signaling is defective in a mouse model of fragile X syndrome.J. Neurosci. 2010; 30: 10977-10984Crossref PubMed Scopus (116) Google Scholar). Here, we report that p75 also activates Rac1 in synaptic compartments of cerebellar Purkinje cells, and this activation plays a role in maintaining normal firing regularity of Purkinje neurons through, at least in part, regulation of small conductance Ca2+-activated K+ (SK) channels. Phospho-PAK, PAK, and PSD95 antibodies were from Cell Signaling; Rac1, Cdc42, and tubulin antibodies were from Santa Cruz Biotechnology, Inc.; SK2 antibody was from Alomone Labs; and anti-mouse calbindin was from Swant. The p75 knock-out mice that carried the mutation in exon 3 of the p75 gene (21.Lee K.F. Li E. Huber L.J. Landis S.C. Sharpe A.H. Chao M.V. Jaenisch R. Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system.Cell. 1992; 69: 737-749Abstract Full Text PDF PubMed Scopus (819) Google Scholar) and the wild type mice were obtained from heterozygote mating as littermates. The mice were back-crossed to C57/BL6 for 10 generations to make them congenic. The genotype was determined by PCR analyses of tail DNA according to Bentley and Lee (22.Bentley C.A. Lee K.F. p75 is important for axon growth and schwann cell migration during development.J. Neurosci. 2000; 20: 7706-7715Crossref PubMed Google Scholar). The cerebellum and cerebral cortex were homogenized with Dounce homogenizers in 0.32 m sucrose and 4 mm HEPES plus 10 mm NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mm vanadate, and 1 mm phenylmethylsulfonyl fluoride (PMSF). Postnuclear supernatants were centrifuged at 14,000 × g for 10 min to collect the pellet, which was subsequently resuspended in the homogenization buffer and overlaid on top of a sucrose step gradient (0.8, 1, and 1.2 m). The gradient was centrifuged at 82,500 × g for 2 h. The resulting fraction that was overlaid onto the interface between 1 and 1.2 m sucrose was collected and overlaid onto 0.8 m sucrose solution and centrifuged at 230,000 × g for 15 min. The pellet contained both presynaptic and postsynaptic membranes, or synaptosomes. The synaptosome pellet was resuspended in a lysis buffer containing 25 mm HEPES (pH 7.5), 150 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 10% glycerol, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm sodium orthovanadate, 25 mm NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 2 mm PMSF. The lysates were subjected to RacGTP assays using pull-down methods as described (3.Harrington A.W. Kim J.Y. Yoon S.O. Activation of Rac GTPase by p75 is Necessary for c-Jun N-terminal kinase-mediated apoptosis.J. Neurosci. 2002; 22: 156-166Crossref PubMed Google Scholar). Brains were sectioned at 30 µm in a sagittal or coronal plane using a cryostat and used for immunohistochemistry as described (4.Tep C. Kim M.L. Opincariu L.I. Limpert A.S. Chan J.R. Appel B. Carter B.D. Yoon S.O. BDNF induces polarized signaling of small GTPase (Rac1) at the onset of Schwann cell myelination through partitioning-defective 3 (Par3).J. Biol. Chem. 2012; 287: 1600-1608Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). For p-PAK staining, tissues were subjected to antigen retrieval at 50 °C for 50 min in 10 mm Tris-HCl (pH 9.0). The images were obtained using a Leica confocal microscope (model TCS SL) at the identical setting for wild type and p75 knock-out sections. Freshly dissected brains were cut at 200 µm on the sagittal plane at 4 °C using a vibratome (Leica), and cerebellar slices that contained all 10 lobules were placed inside a Millicell on the sagittal orientation as described (24.del Río J.A. Soriano E. Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration.Nat. Protoc. 2010; 5: 217-226Crossref PubMed Scopus (23) Google Scholar). BDNF was added to the underlying media at 50 ng/ml, incubated for 10 min at 37 °C, and processed for protein extraction. Mice (postnatal days 15–25) were anesthetized with halothane and sacrificed by decapitation. Sagittal slices of 300-µm thickness were prepared from the vermis of the cerebellum with a vibratome (World Precision Instruments) in ice-cold, oxygenated artificial cerebrospinal fluid (aCSF): 125 mm NaCl, 26 mm NaHCO3, 1.25 mm NaH2PO4, 2.5 mm KCl, 1 mm MgCl2, 2 mm CaCl2, and 10 mm glucose bubbled with 5% CO2 and 95% O2 (pH 7.4). Slices were recovered at 35 °C for 1 h and then maintained at room temperature (22–24 °C) in the aCSF until use. Littermate pairs of the wild type and p75−/− mice were used. The recordings from each pair of littermates were performed either on the same day or on two consecutive days under the same experimental conditions. From each animal, 20–40 Purkinje cells from the apex of the lobule VI of the cerebellum were randomly recorded. Prior to recording, the cerebellar slice was mounted in a chamber on the stage of a Nikon E600-FN upright microscope and continuously perfused (2 ml/min) with a modified aCSF (i.e. aCSF supplemented with 5 mm kynurenic acid (a broad spectrum ionotropic glutamate receptor antagonist) and 100 µm picrotoxin (a GABAA receptor blocker)). The solution was heated to 33–35 °C with an SC-20 in-line solution heater (Harvard Apparatus). The Rac1 inhibitor, NSC23766 (Calbiochem), was diluted in the modified aCSF to 100 µm and applied through whole-chamber perfusion. Extracellular field potentials were recorded using glass electrodes with a tip size of 0.5–1 µm filled with aCSF without the synaptic blockers. The pipette tip was positioned near the initial axon segment of the Purkinje cell soma. Action potentials appeared as fast negative deflections of 100–1000 µV. Only clearly isolated single cell signals were chosen for recording and subsequent analysis. Signals were preamplified and filtered (at 3 kHz) by NPI EXT-10C and LPBF-01GX amplifier/filter modules before being digitized and sampled (at 10 kHz) by the built-in AD/DA converter of an EPC10 amplifier (HEKA Electronics Inc.). Data were analyzed using the Spike2 program (Cambridge Electronic Design). Recording pipettes were pulled from micropipette glass (A-M Systems, Inc., Carlsborg, WA) to 3–5 MΩ and filled with an intracellular solution containing 122 mm potassium gluconate, 9 mm NaCl, 1.8 mm MgCl2, 0.9 mm EGTA, 9 mm HEPES, 14 mm Tris-creatine phosphate, 4 mm Mg-ATP, and 0.3 mm Tris-GTP (pH 7.2) and placed in the modified aCSF. When needed, NSC23766 was diluted in the intracellular solution and applied into the cytoplasm of Purkinje cells through dialysis from the recording pipette. Purkinje cells in lobule VI of mouse cerebellar slices were selected for whole-cell recording using an EPC10 amplifier and the PatchMaster version 2.2.0 software while the slice was continuously perfused with the modified aCSF at 33 °C. Tight (GΩ) seal and high quality break-in were made using the ez-gSEAL pressure controller (NeoBiosystems, Inc., San Jose, CA). As soon as the whole-cell configuration was established, fast and slow capacitances were canceled, and the holding potential was set to −70 mV. Spontaneous firing was recorded after switching to current clamp mode and setting the current injection value to 0. Voltage signals were sampled at 20 kHz using a gap-free recording protocol. Cerebellar Purkinje neurons were isolated as described (69.Raman I.M. Bean B.P. Ionic currents underlying spontaneous action potentials in isolated cerebellar Purkinje neurons.J. Neurosci. 1999; 19: 1663-1674Crossref PubMed Google Scholar). In brief, the vermal layer of the cerebellum from the wild type or p75−/− mice (P13–P18) was removed and minced in an ice-cold, oxygenated dissociation solution containing 82 mm Na2SO4, 30 mm K2SO4, 5 mm MgCl2, 10 mm HEPES, 10 mm glucose, and 0.001% phenol red (pH 7.4). The minced tissue was subsequently incubated in the dissociation solution with 3 mg/ml protease XXIII (pH 7.4; Sigma) at 35 °C for 7 min while oxygen was blown over the surface of the solution. Subsequently, the tissue was washed in prewarmed, oxygenated dissociation solution containing 1 mg/ml bovine serum albumin and 1 mg/ml trypsin inhibitor and then maintained in Tyrode's solution containing 150 mm NaCl, 4 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 10 mm HEPES, and 10 mm glucose at room temperature (pH 7.4) while oxygen was blown over the surface of the fluid. Occasionally, an aliquot of the tissue suspension was withdrawn and triturated with a fire-polished Pasteur pipette to liberate individual neurons. Purkinje cells were identified by their large diameter and characteristic pearlike shape. Dendrites were rarely seen, but some dendritic membrane may have been incorporated into the isolated neurons. Cells were used between 30 min and 4 h of dissociation. Acutely dissociated Purkinje cells were allowed to settle in the recording chamber for 5 min. After washing out tissue debris with Tyrode's solution, Purkinje cells with a pearlike shape and smooth surface were selected. Recording pipettes (2–4 MΩ) were filled with an intracellular solution containing 122 mm potassium gluconate, 9 mm NaCl, 1.8 mm MgCl2, 0.9 mm EGTA, 9 mm HEPES, 14 mm Tris-creatine phosphate, 4 mm MgATP, and 0.3 mm Tris-GTP (pH 7.2). The control external solution was Tyrode's solution, which was applied to the recorded cell during the gigaseal formation. After adding 300 nm tetrodotoxin (TTX) to Tyrode's solution to block all major sodium channels, the solution was referred to as “Ca ECS”. A “Co ECS” solution was made by replacing CaCl2 in Ca ECS with 2 mm CoCl2, which reversibly blocks calcium currents through all types of calcium channels. In order to block certain potassium channels, tetraethylammonium (TEA; 1 mm) was added to Ca ECS and Co ECS to make “CaTEA ECS” and “CoTEA ECS,” respectively. Fast change of different ECS was achieved by using a pressure-driven, solenoid valve-controlled perfusion system (SmartSquirt 8, AutoMate Scientific). The open end of the perfusion probe was positioned near the cell to ensure complete coverage of the cell by the perfusate. Whole-cell recordings of isolated Purkinje cells were performed at room temperature (22–24 °C) using the EPC10 amplifier. Current signals were digitized at 10 kHz. After establishment of the whole-cell configuration, the cell was voltage-clamped at −80 mV. Fast and slow capacitances were canceled using the auto compensation function of the PatchMaster software. For isolation of potassium currents, voltage step protocols were repeated when the cell was exposed to Ca ECS, Co ECS, CaTEA ECS, and CoTEA ECS sequentially. For quantification of ionic currents of interest, recordings from the last three ECS solutions were subtracted from their corresponding control records. To evaluate SK channel activities, we adopted a voltage clamp protocol used previously to isolate SK-mediated tail currents in cerebellar Purkinje cells (25.Cingolani L.A. Gymnopoulos M. Boccaccio A. Stocker M. Pedarzani P. Developmental regulation of small-conductance Ca2+-activated K+ channel expression and function in rat Purkinje neurons.J. Neurosci. 2002; 22: 4456-4467Crossref PubMed Google Scholar). For this study, cerebellar slices prepared from a pair of wild type and p75−/− littermates were recorded on the same day. The slice was perfused with aCSF supplemented with 1 µm TTX and 1 mm TEA to block voltage-gated Na+ channels, most of the voltage-gated K+ channels, and large conductance Ca2+-activated K+ (BK) channels. Drugs (100 nm apamin or 100 µm NSC23766) were applied to the cerebellar slice through bath perfusion. Purkinje cells located in lobules V–VII were randomly picked. The patch electrode was filled with an intracellular solution containing 135 mm potassium gluconate, 10 mm KCl, 1 mm MgCl2, 2 mm Na2-ATP, 0.4 mm Na3-GTP, 10 mm HEPES (pH 7.2–7.3 with KOH). Purkinje cells were held at −50 mV before stepping to potentials ranging from −10 to +30 mV every 30 s for a period of 100–300 ms to induce unclamped Ca2+ spike complexes. At the end of the voltage step, the membrane potential was stepped back to −50 mV to allow for the generation of Ca2+-activated tail currents. Using this protocol, currents evoked during the voltage step were not fully clamped due to space clamp errors. However, the voltage control after the step back to −50 mV can be well maintained (26.Sah P. Role of calcium influx and buffering in the kinetics of Ca2+-activated K+ current in rat vagal motoneurons.J. Neurophysiol. 1992; 68: 2237-2247Crossref PubMed Scopus (47) Google Scholar, 27.Constanti A. Sim J.A. Calcium-dependent potassium conductance in guinea-pig olfactory cortex neurones in vitro.J. Physiol. 1987; 387: 173-194Crossref PubMed Scopus (134) Google Scholar). Because individual Purkinje cells responded to the same voltage step with distinct numbers and positions of Ca2+ spike complexes, which could strongly affect the size and shape of the tail currents, special care was taken to maintain the constant number and position of Ca2+ spikes before and during the drug application. In most recordings, step parameters were adjusted to allow only one Ca2+ spike complex with the peak occurring right in the middle of the step (see Fig. 3A (a), region 2, inward peak). This ensured no or little overlap between currents of the Ca2+ spike complex and the tail current and a good consistency of all tail currents recorded from the same cell. SK currents were evaluated based on either the peak tail current amplitude (for the comparison between p75+/− and p75−/− neurons) or the tail current amplitude at the time when maximal inhibition by NSC23766 was achieved. Because the size of the Ca2+ spike may potentially affect tail current amplitude, SK tail current was normalized to the integral of the Ca2+ spike complex from the same recording and then used for paired comparison between p75+/− and p75−/− cells (for Fig. 3C (c)) and for paired comparison before and during the NSC23766 treatment for the same cell (for Fig. 6B (b)).FIGURE 6SK channel activity is regulated by Rac1 in Purkinje cells. A, representative traces of SK-mediated tail currents in a WT Purkinje cell before (black) and during (gray) bath application of 100 µm NSC23766. The same protocol was used as in Fig. 3A (a). The arrow indicates the point of maximal inhibition, from where the inhibitory effect of NSC23766 was measured for data analysis. The inset shows Ca2+ spike complexes from the same records. Scale bars, 30 ms, 20 pA; 50 ms, 300 pA for inset. B, individual (open circles) and summary data (filled circles) for integral currents of Ca2+ spike complexes (B (a)) and amplitudes of SK-mediated tail currents at the point indicated in A normalized to the integral currents of Ca2+ spike complexes (B (b)) before and during the treatment with NSC23766. **, p < 0.01, paired t test (n = 14).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data were processed in Microsoft Excel 2010, Origin version 8.0 (OriginLab), and Statistica version 7.1 (StatSoft) programs. Data are presented as means ± S.E. Statistical analysis was performed using Student's t test and analysis of variance. p < 0.05 was defined as statistically significant. Mice were perfused with 2% paraformaldehyde, 2% glutaldehyde in 0.1 m sodium cacodylate buffer (pH 7.2) prior to dissecting the brain (n = 3 for each genotype). The brain was first cut in a sagittal plane, and 1 × 1-mm square areas were cut out from the lobule VI/VII. The tissues were rinsed in 0.1 m sodium cacodylate buffer and placed in 1% osmium, 0.1 m sodium cacodylate for 1–1.5 h at room temperature. The tissues were stained en bloc for 1 h in 2% uranyl acetate and embedded in Spurr resin following dehydration procedures. Sections were cut at 80 nm using a Reichert Ultracut E ultramicrotome and collected on 300-mesh grids. Sections were stained in 2% uranyl acetate and Reynolds lead citrate before observation in an FEI Technai G2 Spirit transmission electron microscope at 60 kV (Ohio State University Campus Microscopy and Imaging Facility). For quantification of spine head areas, the electron photomicrographs were taken at ×15,750 magnification, and the spine length and spine head area were calculated using ImageJ software in a blinded manner. For statistical analyses, Student's t test was used. Freshly dissected brains were stained for Golgi analysis using the Rapid GolgiStainTM kit as directed by the manufacturer (FD Neurotechnologies Inc.) and sectioned in the sagittal plane at 200 µm. Five Purkinje neurons from each of lobes IV, V, VI, and VII of each cerebellum per set of p75+/+ and p75−/− mice (n = 3) were then analyzed in a blinded manner using the Neurolucida® 7 image analysis system (MBF Biosciences), and the spine density was determined. For each Purkinje neuron, at least five different dendritic secondary and tertiary branches were randomly selected for spine analysis at ×100 magnification to obtain the number of spines per given length of the dendritic branch. For statistical analyses, Student's t test was used. Rodent cerebellar Purkinje cells are known to fire spontaneously in the absence of any afferent inputs at a relatively constant rate (tonic firing) or intermittently with pauses of variable lengths that separate the firing periods (phasic firing). During phasic firing, there can also be a short duration of bursting, which typically precedes long pauses or silence periods, giving rise to the so-called trimodal firing pattern of Purkinje neurons (28.Womack M. Khodakhah K. Active contribution of dendrites to the tonic and trimodal patterns of activity in cerebellar Purki" @default.
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• W2090693552 date "2014-11-01" @default.
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• W2090693552 title "p75 Regulates Purkinje Cell Firing by Modulating SK Channel Activity through Rac1" @default.
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