In Situ Mitochondrial Ca2+ Buffering Differences of Intact Neurons and Astrocytes from Cortex and Striatum
2008; Elsevier BV; Volume: 284; Issue: 8 Linguagem: Inglês
10.1074/jbc.m807459200
ISSN1083-351X
AutoresJorge M.A. Oliveira, Jorge Gonçalves,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe striatum is particularly vulnerable to neurological disorders, such as Huntington disease. Previous studies, with nonsynaptic mitochondria isolated from cortical and striatal homogenates, suggest that striatal mitochondria are highly vulnerable to Ca2+ loads, possibly influencing striatal vulnerability. However, whether and how neuronal and glial mitochondria from cortex and striatum differ in Ca2+ vulnerability remains unknown. We test this hypothesis using a novel strategy allowing comparisons of mitochondrial Ca2+ buffering capacity in cortical and striatal neuron-astrocyte co-cultures. We provide original evidence that mitochondria not only in neurons but also in astrocytes from striatal origin exhibit a decreased Ca2+ buffering capacity when compared with cortical counterparts. The decreased mitochondrial Ca2+ buffering capacity in striatal versus cortical astrocytes does not stem from variation in mitochondrial concentration or in the rate of intracellular Ca2+ elevation, being mechanistically linked to an increased propensity to undergo cyclosporin A (CsA)-sensitive permeability transition. Indeed, 1 μm CsA selectively increased the mitochondrial Ca2+ buffering capacity of striatal astrocytes, without modifying that of neurons or cortical astrocytes. Neither thapsigargin nor FK506 modified mitochondrial Ca2+ buffering differences between cell types, excluding a predominant contribution of endoplasmic reticulum or calcineurin. These results provide additional insight into the mechanisms of striatal vulnerability, showing that the increased Ca2+ vulnerability of striatal versus cortical mitochondria resides in both intact neurons and astrocytes, thus positioning the striatum at greater risk for disturbed neuron-astrocyte interactions. Also, the selective effect of CsA over striatal astrocytes suggests that in vivo neuronal sheltering with this compound may indirectly result from astrocytic protection. The striatum is particularly vulnerable to neurological disorders, such as Huntington disease. Previous studies, with nonsynaptic mitochondria isolated from cortical and striatal homogenates, suggest that striatal mitochondria are highly vulnerable to Ca2+ loads, possibly influencing striatal vulnerability. However, whether and how neuronal and glial mitochondria from cortex and striatum differ in Ca2+ vulnerability remains unknown. We test this hypothesis using a novel strategy allowing comparisons of mitochondrial Ca2+ buffering capacity in cortical and striatal neuron-astrocyte co-cultures. We provide original evidence that mitochondria not only in neurons but also in astrocytes from striatal origin exhibit a decreased Ca2+ buffering capacity when compared with cortical counterparts. The decreased mitochondrial Ca2+ buffering capacity in striatal versus cortical astrocytes does not stem from variation in mitochondrial concentration or in the rate of intracellular Ca2+ elevation, being mechanistically linked to an increased propensity to undergo cyclosporin A (CsA)-sensitive permeability transition. Indeed, 1 μm CsA selectively increased the mitochondrial Ca2+ buffering capacity of striatal astrocytes, without modifying that of neurons or cortical astrocytes. Neither thapsigargin nor FK506 modified mitochondrial Ca2+ buffering differences between cell types, excluding a predominant contribution of endoplasmic reticulum or calcineurin. These results provide additional insight into the mechanisms of striatal vulnerability, showing that the increased Ca2+ vulnerability of striatal versus cortical mitochondria resides in both intact neurons and astrocytes, thus positioning the striatum at greater risk for disturbed neuron-astrocyte interactions. Also, the selective effect of CsA over striatal astrocytes suggests that in vivo neuronal sheltering with this compound may indirectly result from astrocytic protection. Brain regions and cell types vary in their susceptibility to neurological disorders. The striatum is particularly vulnerable to both acute (e.g. hypoglycemia, hypoxia, and global ischemia) and chronic (e.g. Huntington disease (HD) 2The abbreviations used are: HD, Huntington disease; CsA, cyclosporin A; CypD, cyclophilin D; DIV, days in vitro; ER, endoplasmic reticulum; FCCP, carbonylcyanide-p-(trifluoromethoxyphenyl) hydrazone; FK506, tacrolimus; GFAP, glial fibrillary acidic protein; MK-801, (5R,10S)-(+)-5-methyl-10,11-dihydro[a,d]cyclohepten-5,10-imine hydrogen maleate; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; PMPI, plasma membrane potential indicator; ROI, region of interest; TMRM+, tetramethylrhodamine methyl ester; ANOVA, analysis of variance.2The abbreviations used are: HD, Huntington disease; CsA, cyclosporin A; CypD, cyclophilin D; DIV, days in vitro; ER, endoplasmic reticulum; FCCP, carbonylcyanide-p-(trifluoromethoxyphenyl) hydrazone; FK506, tacrolimus; GFAP, glial fibrillary acidic protein; MK-801, (5R,10S)-(+)-5-methyl-10,11-dihydro[a,d]cyclohepten-5,10-imine hydrogen maleate; NMDA, N-methyl-d-aspartate; NMDAR, NMDA receptor; PMPI, plasma membrane potential indicator; ROI, region of interest; TMRM+, tetramethylrhodamine methyl ester; ANOVA, analysis of variance.) disorders. Excitotoxicity and altered energy metabolism have been proposed as important determinants of striatal pathogenesis, leading to toxic increases in [Ca2+]i (1Calabresi P. Centonze D. Bernardi G. Neurology. 2000; 55: 1249-1255Crossref PubMed Scopus (99) Google Scholar). Studies with mitochondria isolated from brain homogenates raised the hypothesis that striatal mitochondria are more vulnerable to Ca2+ loads than cortical mitochondria, possibly contributing to selective striatal vulnerability in pathologies such as HD (2Brustovetsky N. Brustovetsky T. Purl K.J. Capano M. Crompton M. Dubinsky J.M. J. Neurosci. 2003; 23: 4858-4867Crossref PubMed Google Scholar). However, because isolated mitochondria are deprived from its physiological cellular context, and brain homogenates contain mitochondria from both neurons and glia, two fundamental questions remain unanswered: first, whether the differences in Ca2+ vulnerability, observed with isolated mitochondria, also occur in situ (i.e. when mitochondria are in their physiological environment inside intact cells) and, second, whether mitochondrial vulnerability to Ca2+ loads differs among neurons and glia from cortex and striatum.Striatal selectivity in HD is noteworthy, given that the disease-causing protein (mutant huntingtin) is widely expressed throughout the brain and not enriched in the striatum (3Li S.H. Schilling G. Young III, W.S. Li X.J. Margolis R.L. Stine O.C. Wagster M.V. Abbott M.H. Franz M.L. Ranen N.G. Folstein S.E. Hedreen J. Ross C.A. Neuron. 1993; 11: 985-993Abstract Full Text PDF PubMed Scopus (269) Google Scholar). Although HD may also affect nonstriatal regions, namely the cortex (4Rosas H.D. Salat D.H. Lee S.Y. Zaleta A.K. Pappu V. Fischl B. Greve D. Hevelone N. Hersch S.M. Brain. 2008; 131: 1057-1068Crossref PubMed Scopus (351) Google Scholar), typical striatal atrophy and neuronal loss greatly exceed those seen in nonstriatal regions, particularly when HD is not superimposed with Alzheimer's disease or age-related volumetric loss (5Vonsattel J.P. DiFiglia M. J. Neuropathol. Exp. Neurol. 1998; 57: 369-384Crossref PubMed Scopus (1202) Google Scholar). This suggests that vulnerability factors inherent to striatal neurons shape the selective pattern of HD neurodegeneration. One likely factor is the enrichment in N-methyl-d-aspartate (NMDA) receptors containing NR2B subunits, allowing prolonged Ca2+ influx following excitotoxic stimuli (6Cowan C.M. Raymond L.A. Curr. Top. Dev. Biol. 2006; 75: 25-71Crossref PubMed Scopus (129) Google Scholar), but other vulnerability factors may assist. Mitochondria have an essential role in neuronal survival following excitotoxic [Ca2+]i elevations (7Nicholls D.G. Budd S.L. Physiol. Rev. 2000; 80: 315-360Crossref PubMed Scopus (1035) Google Scholar), and mitochondria-dependent Ca2+ handling is impaired in striatal neurons from HD mice (8Oliveira J.M. Chen S. Almeida S. Riley R. Goncalves J. Oliveira C.R. Hayden M.R. Nicholls D.G. Ellerby L.M. Rego A.C. J. Neurosci. 2006; 26: 11174-11186Crossref PubMed Scopus (116) Google Scholar). HD may in fact be aggravating inherent mitochondrial vulnerability in the striatum, which may reside in either neurons or glia. Indeed, glia may also contribute for non-cell-autonomous neurodegeneration in HD (9Lobsiger C.S. Cleveland D.W. Nat. Neurosci. 2007; 10: 1355-1360Crossref PubMed Scopus (364) Google Scholar) or ischemic injuries (10Bambrick L. Kristian T. Fiskum G. Neurochem. Res. 2004; 29: 601-608Crossref PubMed Scopus (90) Google Scholar, 11Dienel G.A. Hertz L. Glia. 2005; 50: 362-388Crossref PubMed Scopus (130) Google Scholar), thus stressing the importance of identifying vulnerability factors in both neurons and glia.In the present study, we test the hypothesis that mitochondrial vulnerability to Ca2+ loads differs among intact neurons and astrocytes from cortex and striatum. We prepared primary neuron-astrocyte co-cultures from rat cortex and striatum, whose cellular composition and functional differentiation were confirmed by immunolabeling and functional imaging studies, respectively. Here we report for the first time not only the development but also the application of an experimental strategy allowing comparisons of in situ mitochondrial Ca2+ buffering capacity in the preserved physiological context of intact brain cells. To allow balanced comparisons, similarly to standard procedures with isolated mitochondria experiments, we have introduced calibrations for differences in mitochondrial concentration and rate of intracellular Ca2+ elevation in intact cells.Results in the present study provide additional insight into the mechanisms of striatal vulnerability, showing for the first time that the increased vulnerability of striatal mitochondria to Ca2+ loads is present in both intact neurons and astrocytes when compared with their cortical counterparts. Interestingly, our finding of a lower mitochondrial Ca2+ buffering capacity in intact striatal versus cortical astrocytes, mechanistically linked to increased cyclosporin A-dependent permeability transition, positions the striatum at higher risk for disturbed interactions between two synaptic partners, neurons and astrocytes.EXPERIMENTAL PROCEDURESMaterials—Fura-4F acetoxymethyl ester, tetramethylrhodamine methyl ester (TMRM+), and 4-bromocalcimycin were from Invitrogen. Plasma membrane potential indicator (PMPI) (Component A from the R-8042 membrane potential assay kit) was from Molecular Devices Corp. (Sunnyvale, CA) (12Nicholls D.G. J. Biol. Chem. 2006; 281: 14864-14874Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Culture media and supplements were from Invitrogen unless otherwise stated. NMDA, (5R,10S)-(+)-5-methyl-10,11-dihydro[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), ifenprodil, myxothiazol, oligomycin B, rotenone, carbonylcyanide-p-(trifluoromethoxyphenyl) hydrazone (FCCP), cyclosporin A, tacrolimus (FK506), thapsigargin, and other reagents were from Sigma.Cell Culture—Primary cultures were generated from the offspring of Wistar rats (CRIFFA, Barcelona, Spain). Handling and care of animals were conducted according to the European Union guidelines for animal research (86/609/EEC; in agreement with the National Institutes of Health guidelines) and Portuguese law (Portarias 1005/92 and 1131/97). Cortical and striatal cultures were prepared and maintained as previously described for striatal cultures (8Oliveira J.M. Chen S. Almeida S. Riley R. Goncalves J. Oliveira C.R. Hayden M.R. Nicholls D.G. Ellerby L.M. Rego A.C. J. Neurosci. 2006; 26: 11174-11186Crossref PubMed Scopus (116) Google Scholar, 13Oliveira J.M. Jekabsons M.B. Chen S. Lin A. Rego A.C. Goncalves J. Ellerby L.M. Nicholls D.G. J. Neurochem. 2007; 101: 241-249Crossref PubMed Scopus (113) Google Scholar) but without cytosine arabinoside to allow the growth of astrocytes. Briefly, hemicortices (free from meninges, olfactory bulb, striatum, and hippocampus) and hemistriata were dissected from the same embryonic day 18 rats, pooled according to brain region (cortex or striatum), and processed in parallel (sister cultures). Dissociated cells (1 × 105) were plated in 12-mm round polyethyleneimine-coated glass coverslips and used in functional imaging experiments between 11 and 13 days in vitro (DIV).Immunocytochemistry—Cells were fixed with 4% paraformaldehyde for 15 min at 37 °C and permeabilized with 0.4% Triton X-100 in phosphate-buffered saline for 15 min. Blocking was performed with 3% bovine serum albumin in phosphate-buffered saline for 30 min at room temperature. Cells were then incubated with primary antibodies for the neuronal marker (mouse anti-MAP-2 (microtubule-associated protein); Sigma; 1:500) and the astrocytic marker (rabbit anti-glial fibrillary acidic protein (GFAP); Dako; 1:250) in 3% bovine serum albumin phosphate-buffered saline for 1 h at room temperature. After washing, cells were incubated with appropriate secondary antibodies conjugated to either AlexaFluor 488 or 594 (Invitrogen; 1:200) for 1 h at room temperature and shielded from light. Nuclei labeling was performed with Hoechst 33342 (Invitrogen; 2 μg/ml; 10 min at room temperature). To test for antibody selectivity, primary antibodies were omitted in control coverslips processed in parallel. No immunolabeling was observed in control coverslips, which were used for shading correction of epifluorescent images, acquired with the same equipment settings.Monitoring of Dynamic Changes in [Ca2+]i, Mitochondrial Membrane Potential (ΔΨm), and Plasma Membrane Potential (ΔΨp) in Single Cells—Functional imaging at single-cell resolution was performed with a system composed by an inverted epifluorescence microscope (Eclipse TE300, Nikon, Tokyo, Japan) equipped with 20×, 40×, and 60× air objectives, a monochromator (Polychrome II; TILL Photonics, Martinsried, Germany), a CCD camera (C6790; Hamamatsu Photonics, Hamamatsu, Japan), and a computer with analysis software (Aquacosmos 2.5; Hamamatsu Photonics). Cells were loaded with the [Ca2+]i probe Fura-4F (5 μm) for 45 min at 37 °C. For simultaneous recordings of changes in [Ca2+]i and in TMRM+ fluorescence (indicative of changes in ΔΨm), 50 nm TMRM+ (quench mode) (14Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar). was present during Fura-4F loading and throughout the experiments. Simultaneous recordings of changes in [Ca2+]i and in ΔΨp were performed in Fura-4F-loaded cells with a constant PMPI concentration present throughout the experiment. The PMPI concentration was the same as previously described (12Nicholls D.G. J. Biol. Chem. 2006; 281: 14864-14874Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), corresponding to a 1:2,000 dilution of a PMPI stock solution prepared by reconstituting one R-8042 Component A vial in 1 ml of ultrapure water. Experiments were performed at 37 °C in buffer containing 133 mm NaCl, 5 mm KCl, 1 mm Na2SO4, 0.4 mm KH2PO4, 20 mm HEPES, 1.3 mm CaCl2, and 15 mm glucose, pH 7.4. Where indicated, glucose was replaced by 2 mm 2-deoxy-d-glucose, and mitochondria were energized with 10 mm pyruvate. Fura-4F was excited at 340 and 380 nm, TMRM+ at 550 nm, and PMPI at 500 nm. Dual Fura-4F and TMRM+ emission was collected with a 73100bs dichroic and a 73101 m emitter (Chroma Technology Corp., Rockingham, VT), whereas dual Fura-4F and PMPI emission was collected with a 505-nm beam splitter and a 520-nm long pass filter. There was no significant bleed-through between Fura-4F and TMRM+ or between Fura-4F and PMPI with these excitation and emission settings. Individual cells were identified as regions of interest (ROIs) for the determination of fluorescence time courses.In situ calibration of Ca2+ responses for each individual cell was performed at the end of every experiment, according to previously detailed procedures (8Oliveira J.M. Chen S. Almeida S. Riley R. Goncalves J. Oliveira C.R. Hayden M.R. Nicholls D.G. Ellerby L.M. Rego A.C. J. Neurosci. 2006; 26: 11174-11186Crossref PubMed Scopus (116) Google Scholar). Briefly, maximal 340/380 ratios (Rmax) were determined with 20 μm 4-bromocalcimycin with 1.3 mm extracellular Ca2+, and minimal 340/380 ratios (Rmin) were determined in Ca2+-free buffer with 5 mm EGTA. This Ca2+-free buffer contained neither TMRM+ nor PMPI, and thus the subsequent decays in their respective fluorescence should not be interpreted as changes in ΔΨm or ΔΨp. In experiments addressing mitochondrial Ca2+ buffering capacity, maximal mitochondrial depolarization (ΔΨm collapse) was confirmed/evoked before [Ca2+]i calibration by adding protonophore (1 μm FCCP), with ATP synthase reversal prevented by oligomycin (3 μm). Further details on the analysis of cellular responses are provided below.Image Processing and Data Analysis—Analysis of [Ca2+]i responses was performed as previously detailed (8Oliveira J.M. Chen S. Almeida S. Riley R. Goncalves J. Oliveira C.R. Hayden M.R. Nicholls D.G. Ellerby L.M. Rego A.C. J. Neurosci. 2006; 26: 11174-11186Crossref PubMed Scopus (116) Google Scholar). Briefly, background-corrected 340/380 Fura-4F ratios were normalized to the Rmax (100 units) and Rmin (0 units) determined for each single cell during in situ calibration. The left yy axes of Ca2+ graphs are scaled with these normalized values, where 50 units should correspond to the Ca2+ probe Kd, regardless of its absolute value inside the different cells. The right yy axis is an approximate log [Ca2+]i scale adjusted based on the dynamic range of Fura-4F and Kd determinations (15Haugland R.P. Handbook of Fluorescent Probes and Research Products. Molecular Probes, Inc., Eugene, OR2002: 776-781Google Scholar, 16Wokosin D.L. Loughrey C.M. Smith G.L. Biophys. J. 2004; 86: 1726-1738Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar).Changes in TMRM+ and PMPI fluorescence in single cells are expressed as arbitrary fluorescence units. Individual signals are corrected for epifluorescence shading, using images acquired in the same experimental xx-yy-zz plane, without TMRM+ (during Rmin determination at the end of the experiment) and in the absence of PMPI (before starting the experiment, in PMPI-free buffer). Exposure times for image acquisition were kept constant in all experiments, with signal saturation and underexposure being avoided merely by adjusting camera gain before starting the experiments. Mathematical correction for different gains was introduced so that all fluorescent signals are compared under the same circumstances.Conversions to mV of neuronal PMPI fluorescence (Figs. 1, B and D (ii), and 2, A (ii)) were performed in a manner similar to that described previously (12Nicholls D.G. J. Biol. Chem. 2006; 281: 14864-14874Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) but with in situ calibration for each individual neuron. Briefly, extracellular Na+ was replaced equimolarly by K+ in a stepwise fashion, up to 42 mm extracellular K+ in the assay buffer. ΔΨp values (in mV) were derived from the Goldman-Hodgkin-Katz equation, assuming conductances for K+, Na+, and Cl- of 100:1:100. PMPI fluorescence enhancements evoked by K+ in each neuron were plotted in a function of ΔΨp and fit monoexponentially (correlation coefficients, r2 > 0.990) to generate individual calibration curves to derive approximate ΔΨp changes in mV.FIGURE 2Cultured cortical and striatal neurons are morphologically and functionally distinct. A, simultaneous monitoring of changes in [Ca2+]i (i) and ΔΨp (ii) in cortical and striatal neurons. NMDA (10 μm + 1 μm glycine), ifenprodil (IFEN; 2 μm), and MK-801 (5 μm) were added where indicated. Matched color lines in i and ii represent measurements performed in the same representative neuron (three are shown for each type). Time bar, 2 min. B, three-dimensional morphofunctional clustering of cortical (black circles) and striatal (white circles) neurons using the three parameters in xx, yy, and zz. Data are from a total of 46 (23 cortical and 23 striatal) neurons from single representative experiments with sister cultures. The solid lines converge to the respective centroid, and the vertical red dashed line divides neurons according to the blinded "two-step cluster analysis."View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cell size was determined from the ROI area (μm2) using micrometer-calibrated images. Neuronal areas reflect only the soma, due to impossibility of tracing the entire neuronal processes. Area conversion into approximate somatic diameter (e.g. Fig. 2B) was performed by a circular approximation using Equation 1,d=2×SQRT(a/π)(Eq. 1) where d represents the diameter, SQRT is the square root, a is the area, and π is the circular constant.Mitochondrial concentration in single cells was estimated from the analysis of the average TMRM+ fluorescence in each ROI (for a representative measurement, see Fig. 3A (ii), solid line, γ - δ). The difference between resting TMRM+ fluorescence (start of experiment) and nonmitochondrial fluorescence (TMRM+ signal after FCCP, prior to Rmax determination; Figs. 3 and 4 (ii), solid lines) was assumed to reflect mitochondrial concentration. Our assumption is based on evidence that when the amount of TMRM+ stacked in the mitochondria surpasses the quench threshold, autoquenching occurs and fluorescence becomes invariant with matrix TMRM+ concentration (14Ward M.W. Rego A.C. Frenguelli B.G. Nicholls D.G. J. Neurosci. 2000; 20: 7208-7219Crossref PubMed Google Scholar); TMRM+ quenching in the present study is evidenced by the dequenching spikes in Fig. 4, A–D, ii, solid lines). Therefore, the resting cellular TMRM+ fluorescence in our experiments is primarily governed by the amount of mitochondria per cell volume (mitochondrial concentration) with superimposed nonmitochondrial fluorescence. This nonmitochondrial fluorescence reflects TMRM+ in extracellular buffer in the cytosol and non-specifically bound to cellular elements. Hence, subtracting the residual TMRM+ signal following full ΔΨm collapse (as confirmed with FCCP in the presence of oligomycin) should eliminate the vast majority of nonmitochondrial fluorescence, thus yielding an estimate of mitochondrial concentration. We further substantiate this procedure by providing both medium and high magnification images (supplemental material) of TMRM+-loaded neurons and astrocytes used in functional experiments. Collectively, the images show that the mitochondrial distribution is sparser in astrocytes that in neurons, visually showing a lower mitochondrial concentration. We also provide fluorescence intensity profiles across representative cells as well as time lapse experiments illustrating how ΔΨm collapse with oligomycin plus FCCP influences TMRM+ distribution and fluorescence intensity values in mitochondria-rich and mitochondria-free cellular regions.FIGURE 3In situ monitoring of mitochondrial Ca2+ buffering capacity in neurons and astrocytes from cortex and striatum. A–D, changes in [Ca2+]i (i) and in TMRM+ (ii; solid lines) or PMPI (ii; dashed lines) fluorescence in arbitrary units (A.F.U.). The solid or dashed line in i and ii depicts measurements performed simultaneously in the same single cell. Representative cells with identical [Ca2+]i kinetics were chosen (i) in order to allow comparison of changes in TMRM+ and PMPI fluorescence (ii). Assay buffer contained 10 mm pyruvate, and glucose was replaced by 2 mm 2-deoxy-d-glucose before the addition of oligomycin (3 μm; O). FCCP addition (1 μm; F) confirms full ΔΨm collapse. R and r, Rmax and Rmin determinations, respectively. A (i), β - α, duration of the [Ca2+]i plateau (i.e. apparent mitochondrial Ca2+ buffering). A (ii), γ - δ, mitochondrial concentration. Up to 50 random single cells of each type were analyzed per experiment; only a few are shown for clarity, being representative of n = 7 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4Intracellular Ca2+ dynamics in neurons and astrocytes from cortex and striatum under conditions where mitochondria are prevented from buffering [Ca2+]i. A–D, changes in [Ca2+]i (i) and in TMRM+ or PMPI (ii; solid or dashed lines, respectively) fluorescence in arbitrary units (A.F.U.). The solid or dashed line in i and ii depicts measurements performed simultaneously in the same single cell. i, representative cells with identical [Ca2+]i kinetics to allow comparisons in ii. Oligomycin (O; 3 μm) and myxothiazol (M; 2 μm) were added where indicated. FCCP (F; 1 μm) addition and in situ determination of Rmax and Rmin (R and r, respectively) were performed at the indicated times for cells depicted by solid lines and slightly earlier for those shown by dashed lines (A and B, 10 min earlier; C and D, 5 min earlier). This time variation results from the need to anticipate and avoid excessive Fura-4F leakage to allow accurate [Ca2+]i calibration. Assay buffer contained 10 mm pyruvate, and glucose was replaced by 2 mm 2-deoxy-d-glucose before the addition of oligomycin (3 μm). A (i), Kexp, the rate of [Ca2+]i elevation. Up to 50 random single cells of each type were analyzed per experiment; only a few are shown for clarity, being representative of n = 5 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The rate of [Ca2+]i elevation was estimated by curve-fitting (with nonlinear regression) the normalized Fura-4F 340/380 ratios in experiments such as in Fig. 4, where the combination of oligomycin and a respiratory chain inhibitor (myxothiazol) collapsed ΔΨm, preventing mitochondrial Ca2+ uptake (17Nicholls D.G. Cell Calcium. 2005; 38: 311-317Crossref PubMed Scopus (273) Google Scholar). Best fit values, with r2 > 0.990, were obtained with Equation 2,y=IF(χlt;x0),THENy=b,ELSEy=b+(t - b)×(1 - e-Kexp×(x-x0)))(Eq. 2) where y represents normalized [Ca2+]i at time x, x0 is the first time point, b is basal [Ca2+]i, t is the top part of the curve (constrained to be below the 100 value achieved during Rmax), e is Neper's number, and Kexp is the exponential constant used to compare the rate of [Ca2+]i elevation among cells (e.g. Fig. 4A (i), Kexp arrow). It should be noted that myxothiazol, albeit at higher concentrations, was previously shown to inhibit Ca2+ influx through store-operated channels (18Makowska A. Zablocki K. Duszynski J. Eur. J. Biochem. 2000; 267: 877-884Crossref PubMed Scopus (53) Google Scholar). However, 3 μm oligomycin (specifically oligomycin B, which has a 0.5 μm K50 for the inhibition of store-operated Ca2+ influx (19Cho J.H. Balasubramanyam M. Chernaya G. Gardner J.P. Aviv A. Reeves J.P. Dargis P.G. Christian E.P. Biochem. J. 1997; 324: 971-980Crossref PubMed Scopus (29) Google Scholar)) was present in our experiments either with or without myxothiazol, thus preventing differences in store-operated Ca2+ influx between these experiments.Image processing was performed with Aquacosmos 2.5 (Hamamatsu Photonics) and with ImageJ (National Institutes of Health; available on the World Wide Web). Calculations on numerical data derived from ROIs were automated in Excel spreadsheets (Microsoft Corp., Redmond, WA). Values throughout are presented as mean ± S.E. unless otherwise stated. Nonlinear regressions, curve fitting, and t tests were performed using GraphPad Prism version 4.0 (San Diego, CA). Other statistical analyses were performed with Statistical Package for the Social Sciences version 15.0 for Windows (SPSS Inc., Chicago, IL). Type I error probability was set at 0.05 for all analyses.RESULTSCharacterization of Neurons and Astrocytes in Cortical and Striatal Cultures—Immunolabeling revealed that cortical and striatal cultures were predominantly composed of neurons and astrocytes (over 95% of all nuclei stained with Hoechst 33342 were also positive for either the neuronal marker, MAP-2, or the astrocytic marker, GFAP) (supplemental Fig. 1). At 11 DIV, cells were functionally differentiated. Neurons responded to K+ or NMDA receptor (NMDAR) activation with [Ca2+]i elevation and plasma membrane depolarization in a concentration-dependent manner (Fig. 1, A and C, and solid lines in B and D). In contrast, astrocytes did not respond to NMDA (up to 100 μm), despite exhibiting changes in plasma membrane polarization upon K+ challenging. Also, no changes in astrocytic [Ca2+]i were detected during either K+ or NMDA challenging, despite clear responses to Ca2+-ionophore during [Ca2+]i calibration (Rmax; Fig. 1, A and C, and dashed lines in B and D). These clear functional differences ensured cellular identification, particularly in experiments with PMPI (e.g. Fig. 1, A and C (ii), bottom left; Fig. 3, A and B (ii), dashed lines).To appraise differences between cortical and striatal neurons, we integrated three parameters (somatic diameter, NMDA pEC50, and proportion of functional NMDAR-2B) in a cluster analysis with n = 23 random neurons of each type. The average somatic diameter was larger for cortical neurons (17.4 ± 0.4 versus 12.9 ± 0.2 μm; p < 0.001). Despite similar NMDA pEC50 (5.48 ± 0.05 versus 5.36 ± 0.05, cortical and striatal neurons, respectively; p = 0.087), the contribution of the NMD
Referência(s)