Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions¹.At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods^1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology⁴, cellular and molecular biology^5-8, biochemistry⁵, imaging and microscopy^4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses¹¹, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously^4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x10⁶ cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.Download video file.^{(75M, mov)}     

Alkalinization-Induced Changes in Intracellular Calcium in Rat Spinal Cord Neurons

It is well-known that pH changes can influence a lot of cellular processes. In this work, we have specifically studied the influence of alkalinization, which can be developed in spinal cord neurons during hyperventilation (respiratory alkalosis) and chronic renal failure (metabolic alkalosis) on calcium homeostasis. Application of Tyrode solution with increased pH (pH = 8.8) to secondary sensory neurons isolated from rat spinal dorsal horn induced elevation of intracellular free calcium concentration in the cytosol ([Ca2+]i) if applied after membrane depolarization. Repetitive application of alkaline solution led to disappearance of such elevations. Depletion of endoplasmic reticulum (ER) calcium stores by 30 mM caffeine almost completely blocked the effect of elevated extracellular pH. If caffeine-induced [Ca2+]i transients were evoked during alkalinization, their amplitudes were decreased by 41%. Preapplication of 500 nM ionomycin resulted in disappearance of alkalinization-induced [Ca2+]i transients, whereas prolonged applications (for 20 min) of 200 nM thapsigargin, a blocker of Ca2+ ATPase of the endoplasmic reticulum, resulted in disappearance of the rapid phase of the [Ca2+]i transients induced by alkalinization. Preapplication of the mitochondrial protonophore CCCP (10 microM) also induced changes in the alkalinization-induced calcium response--it lost its peak and was transformed into an irregular wave terminating in several seconds. The data obtained indicate that alkalinization induces an increase of [Ca2+]i level in the investigated neurons via a combined action of both intracellular Ca2+-accumulating structures--the endoplasmic reticulum and mitochondria. This suggestion was supported by morphological data that both structures in these neurons are tightly connected and may interact during release of accumulated calcium ions.     

Polysaccharides from Wolfberry Antagonizes Glutamate Excitotoxicity in Rat Cortical Neurons

Glutamate excitotoxicity is involved in many neurodegenerative diseases including Alzheimer’s disease (AD). Attenuation of glutamate toxicity is one of the therapeutic strategies for AD. Wolfberry (Lycium barbarum) is a common ingredient in oriental cuisines. A number of studies suggest that wolfberry has anti-aging properties. In recent years, there is a trend of using dried Wolfberry as food supplement and health product in UK and North America. Previously, we have demonstrated that a fraction of polysaccharide from Wolfberry (LBA) provided remarkable neuroprotective effects against beta-amyloid peptide-induced cytotoxicity in primary cultures of rat cortical neurons. To investigate whether LBA can protect neurons from other pathological factors such as glutamate found in Alzheimer brain, we examined whether it can prevent neurotoxicity elicited by glutamate in primary cultured neurons. The glutamate-induced cell death as detected by lactate dehydrogenase assay and caspase-3-like activity assay was significantly reduced by LBA at concentrations ranging from 10 to 500 μg/ml. Protective effects of LBA were comparable to memantine, a non-competitive NMDA receptor antagonist. LBA provided neuroprotection even 1 h after exposure to glutamate. In addition to glutamate, LBA attenuated N-methyl-d-aspartate (NMDA)-induced neuronal damage. To further explore whether LBA might function as antioxidant, we used hydrogen peroxide (H₂O₂) as oxidative stress inducer in this study. LBA could not attenuate the toxicity of H₂O₂. Furthermore, LBA did not attenuate glutamate-induced oxidation by using NBT assay. Western blot analysis indicated that glutamate-induced phosphorylation of c-jun N-terminal kinase (JNK) was reduced by treatment with LBA. Taken together, LBA exerted significant neuroprotective effects on cultured cortical neurons exposed to glutamate.     

Proteomic Analysis of Primary Cultured Rat Cortical Neurons in Chemical Ischemia

To elucidate the molecular events involved in early ischemic neuronal death, we performed two-dimensional proteome profiling of primary cultures of rat cortical neurons following chemical ischemia induced by the administration of sodium azide under glucose-free conditions. Using a lactic dehydrogenase assay and Western blot analysis of dephosporylation of the voltage-gated potassium channel Kv2.1, we determined duration of chemical ischemia of 2 h to be the relevant time-point for early ischemic neuronal death. Sixty-one proteins were differentially expressed, and 26 different proteins were identified by MALDI-TOF with Mascot database searching. The proteome data indicated that chemical ischemia altered the expression of 20 proteins that are involved in stress response/chaperone, brain development, cytoskeletal/structural proteins, metabolic enzymes, and calcium ion homeostasis. Western blotting and immunocytochemical studies of the 6-most functionally significant proteins showed that, in the ischemia-treated group, the expression of glucose-related protein 78, heat shock protein 90 alpha, and α-enolase was significantly increased, while the expression of inositol triphosphate receptor 1 and ATP synthase beta subunit was decreased. In addition, the expression of dihydropyrimidinase-like 3 showed a truncated pattern in the ischemia group. The changes in the expression of these proteins might be significant indicators of early ischemic neuronal death.     

Corticotropin-Peptide Regulation of Intracellular Cyclic AMP Production in Cortical Neurons in Primary Culture

Previous studies have provided evidence for adrenocorticotropic hormone (ACTH) effects on a wide variety of behaviors. However, the precise sites of action and the mechanisms by which these effects may be mediated have yet to be clearly elucidated. Although ACTH was shown to augment cyclic AMP levels in glial cells isolated from whole brain, other studies found little or no effect of ACTH peptides on cyclic nucleotide metabolism in slices of cerebral cortex or homogenates of whole brain. In the present study, our objective was to determine whether ACTH peptides regulate intracellular cyclic AMP levels in neurons of the cerebral cortex in primary culture. ACTH peptides stimulated cyclic AMP synthesis up to threefold in a dose-dependent manner; stimulation was complete within 5-10 min of exposure to agonists. Neurohormone efficacy was augmented by 0.1 microM forskolin (which was virtually ineffective alone); potency was unaffected. The order of potency (EC50) for increasing intracellular cyclic AMP levels was as follows: ACTH (1-24), ACTH (1-17) (10 nM) greater than alpha-melanocyte stimulating hormone, beta-melanocyte stimulating hormone (alpha-MSH, beta-MSH) (100 nM) greater than ACTH (1-10) (1 microM) greater than ACTH (4-10) (5 microM). The hexapeptide ACTH (4-9) as well as ACTH (11-24) were inactive at concentrations as high as 10 microM. Other neuropeptides derived from proopiocortin, such as beta-endorphin and Met- and Leu-enkephalin were without effect on basal or hormonally stimulated cyclic AMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quinolinic Acid Stimulates Somatostatin Gene Expression in Cultured Rat Cortical Neurons

Striatal atrophy in Huntington's disease (HD) is characterized by selective preservation of a subclass of neurons colocalizing NADPH-diaphorase (NADPH-d), somatostatin (SS), and neuropeptide Y (NPY), which have been reported to show three- to fivefold increases in SS-like immunoreactivity (SSLI) and NPY content. Since HD brain is capable of producing excessive quantities of the excitotoxin quinolinic acid (Quin), an N-methyl-D-aspartate (NMDA) receptor agonist, and since experimental Quin lesions show neuronal loss with sparing of NADPH-d/SS/NPY neurons, it has been suggested that Quin may be important in the pathogenesis of HD. In the present study we determined whether Quin stimulates SS gene function in cultured cortical cells known to be rich in NADPH-d/SS/NPY neurons. Cultures of dispersed fetal rat cortical cells were exposed to Quin (1 and 10 mM) with or without (-)-2-amino-5-phosphonovaleric acid (APV; 0.5 mM), an NMDA receptor antagonist, NMDA (0.2 and 0.5 mM), and glutamate (Glu; 0.5 mM). Medium and cellular SSLI was determined by radioimmunoassay and SS mRNA by Northern analysis with a cRNA probe. Quin induced significant (p less than 0.01) 1.6- and 2.5-4 fold increases in SSLI and SS mRNA accumulation, respectively, which were abolished by APV. Release of SSLI into the culture medium was stimulated two- to fivefold by Quin over a 2- to 20-h period. The increase in SS mRNA produced by Quin was time and dose dependent. A similar dose-dependent increase in SS mRNA comparable with that observed with Quin was induced by NMDA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.     

大鼠生后发育过程中听皮层神经元特征频率的可塑性

应用常规电生理学技术,以神经元的特征频率和频率调谐曲线为指标,分别在生后2、3、4、5、6和8周龄SD大鼠上,研究生后发育过程中,听皮层神经元特征频率的可塑性.结果表明,在给予条件刺激频率和神经元特征频率相差1.0kHz范围内,条件刺激都可诱导各年龄组神经元特征频率向频率调谐曲线的低频端、高频端或调谐曲线的两端相应的偏移.特征频率偏移的概率与年龄相关.随着年龄的增长,特征频率偏移的比例下降,而不偏移的比例则上升.随着年龄增长,那些Q10-dB值大和频率调谐曲线对称指数大于零的神经元,特征频率偏移到频率调谐曲线高频端的比例增加更为明显(P<0.01).诱导特征频率完全偏移的时程和特征频率恢复的时程也与动物的年龄相关,随着年龄增长,诱导和恢复时程都明显延长(P<0.05).结果提示,大鼠听皮层神经元特征频率的可塑性与生后年龄相关,为深入研究中枢神经元功能活动可塑性的机制提供了重要实验资料.     

Suppression of the Nicotinic Acetylcholine Response in Rat Superior Cervical Ganglionic Neurons by Steroids

Abstract : The effects of various types of steroids on the nicotinic acetylcholine (ACh) receptor (nAChR)-mediated responses were investigated in superior cervical ganglionic neurons acutely dissociated from rats using nystatin perforated patch recording. ACh induced a peak followed by a gradual decrease in the inward current at a holding potential of -40 mV. Nicotine, but not muscarine, mimicked ACh. Hydrocortisone at a concentration of > 10^-6 M reversibly suppressed both the peak and steady-state nicotine-induced currents ( I _nic) in a noncompetitive manner. The inhibition of I _nic by hydrocortisone did not show any voltage dependency and persisted in the presence of either cyclic AMP modulators, forskolin and 3-isobutyl-1-methylxanthine, or a protein kinase A inhibitor, N -[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89). β-Estradiol, androsterone, aldosterone, and 17α-estradiol mimicked hydrocortisone in its inhibitory action on ACh-induced currents ( I _ACh). The potency for the inhibitory actions on I _Ach was as follows : androsterne > β-estradiol > hydrocortisone ≥ aldosterone =17α-estradiol. Cholesterol had no effect on the I _ACh. In conclusion, the structural characteristics of steroid are thus considered to be necessary to block nicotinic I _ACh in rat superior cervical ganglionic cells, whereas the cholesterol side chain might disturb the inhibitory action of the steroid skeleton on nAChRs.     

Modulation of Synapsin I Gene Expression in Rat Cortical Neurons by Extracellular Matrix

1. Neuronal differentiation depends on crosstalk between genetic program and environmental cues. In this study we tried to dissect this complex interplay by culturing neurons from fetal rat brain cortices in a chemically defined, neuron-specific, medium and on different substrata, either artificial (poly-D-lysine) or natural.2. Among the extracellular matrix compounds used in this study, two (collagen I and fibronectin) allowed only a weak attachment of cortical neurons to the substratum, while the others (collagen IV, laminin, and basal lamina from Engelbreth-Holm-Swarm sarcoma) allowed both firm attachment and moderate to extensive neurite outgrowth from neuronal cell bodies.3. By using synapsin I gene expression as a parameter of neuronal differentiation, we found that neurite outgrowth and neuronal differentiation are not linearly linked. Synapsin I gene expression, in fact, was maximal in neurons cultured on laminin, while the fastest neuritic outgrowth was recorded in cultures on poly-D-lysine.4. The data presented in this paper are consistent with the hypothesis that the extracellular matrix plays an active role in modulating the differentiative program of neurons.     

The Desglycinyl Metabolite of Remacemide Hydrochloride Is Neuroprotective in Cultured Rat Cortical Neurons

Abstract: The neuroprotective actions of remacemide and its anticonvulsant metabolite 1,2-diphenyl-2-propylamine monohydrochloride (desglycinylremacemide; DGR), a low-affinity NMDA receptor antagonist, were investigated using primary rat cortical neuronal cultures. Exposure of cortical cultures to NMDA (100 µ M ) for 15 min killed 85% of the neurons during the next 24 h. This neurotoxicity was blocked in a concentration-dependent manner by adding DGR (5–20 µ M ), but not its remacemide precursor (10–100 µ M ), to the cultures during the time of NMDA exposure. This suggests that the neuroprotective, as well as the anticonvulsant, activity of remacemide is mediated by DGR. Neuroprotective concentrations of DGR also inhibited two of the principal acute effects of NMDA. DGR (5–20 µ M ) prevented the loss of membrane-associated protein kinase C (PKC) activity that developed by 4 h after transient exposure to 100 µ M NMDA and reduced the NMDA-triggered increases in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by up to 70%. By contrast, remacemide (50 and 100 µ M ) did not prevent the NMDA-induced loss of PKC activity or reduce the [Ca²⁺]_i responses. These data suggest that DGR protection against NMDA-mediated toxicity in cultured cortical neurons is associated with a reduction of NMDA-triggered [Ca²⁺]_i surges and a prevention of the loss of membrane-associated PKC activity. In addition, the inhibition of NMDA-triggered [Ca²⁺]_i responses by DGR was qualitatively different from the inhibition of these responses by the high-affinity NMDA-receptor antagonists MK-801 and phencyclidine. This may be a consequence of DGR's lower affinity for the NMDA receptor.     

Inhibitory Effects of Carvone Isomers on the GABAA Receptor in Primary Cultures of Rat Cortical Neurons

Carvone is a natural terpene which can be purified as R‐(?) or S‐(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA_A receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA_A by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA‐induced stimulation of [³H]flunitrazepam binding, suggesting their interaction with the GABA_A receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R‐(?)‐carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA_A receptor at its noncompetitive blocker site. Chirality 26:368–372, 2014. © 2014 Wiley Periodicals, Inc.     

Subthalamic Nucleus High-Frequency Stimulation Restores Altered Electrophysiological Properties of Cortical Neurons in Parkinsonian Rat

Electrophysiological recordings performed in parkinsonian patients and animal models have confirmed the occurrence of alterations in firing rate and pattern of basal ganglia neurons, but the outcome of these changes in thalamo-cortical networks remains unclear. Using rats rendered parkinsonian, we investigated, at a cellular level in vivo, the electrophysiological changes induced in the pyramidal cells of the motor cortex by the dopaminergic transmission interruption and further characterized the impact of high-frequency electrical stimulation of the subthalamic nucleus, a procedure alleviating parkinsonian symptoms. We provided evidence that a lesion restricted to the substantia nigra pars compacta resulted in a marked increase in the mean firing rate and bursting pattern of pyramidal neurons of the motor cortex. These alterations were underlain by changes of the electrical membranes properties of pyramidal cells including depolarized resting membrane potential and increased input resistance. The modifications induced by the dopaminergic loss were more pronounced in cortico-striatal than in cortico-subthalamic neurons. Furthermore, subthalamic nucleus high-frequency stimulation applied at parameters alleviating parkinsonian signs regularized the firing pattern of pyramidal cells and restored their electrical membrane properties.     

Effects of Estradiol and IGF-1 on the Sodium Calcium Exchanger in Rat Cultured Cortical Neurons

The Na⁺/Ca²⁺ exchanger (NCX) is an important bidirectional transporter of calcium in neurons and has been shown to be involved in neuroprotection. Calcium can activate a number of cascades that can result in apoptosis and cell death, and NCX is a key factor in regulating the cytoplasmic concentration of this ion. 17-β-estradiol and insulin-like growth factor 1 (IGF-1) are known neuroprotective hormones with interacting mechanisms and effects on intracellular calcium; however, their relationship with the NCX has not been explored. In this article, the effects of these two hormones on neuronal NCX were tested using the whole-cell patch clamp technique on rat primary culture neurons. Both 17-β-estradiol and IGF-1 produced an increase in the NCX-mediated inward current and a decrease in the NCX-mediated outward current. However, the IGF-1 effect was lower than that of 17-β-estradiol, and the effect of both agents together was greater than the sum of each agent alone. Neither of the agents affected the pattern of regulation by extracellular or intrapipette calcium. Inhibitors of the IGF-1 and 17-β-estradiol receptors and inhibitors of the main signaling pathways failed to change the observed effects, indicating that these actions were not mediated by the classical receptors of these hormones. These effects on the NCX could be a mechanism explaining the neuroprotective actions of 17-β-estradiol and IGF-1, and these findings could help researchers to understand the role of the NCX in neuroprotection.     

Sensitivity of Rat Cortical Neurons in Distinguishing Taste Qualities by Individual and Correlative Activities

To examine the possibility that different taste qualities arerepresented by the correlative activity of cortical gustatoryneurons, we made simultaneous recordings of neuron pairs duringapplication of four basic tastes into the oral cavity of anesthetizedrats and the following observations were made: (i) in 30 of67 pairs of taste responsive neurons, peaks (troughs in a fewcases) were produced in the cross-correlograms (CCs) duringstimulation with some tastants; (ii) the correlative dischargesoccupied 6–8% of the total spikes discharged by individualneurons during taste stimulation and occurred, in a considerablenumber of cases, even during stimulation with tastants to whichone or both of the component neurons of a pair were apparentlynon-responding (often sucrose and quinine); (iii) the numberof tastants to which a neuron pair responded with a significantcorrelative activity was often greater than the number of tastantsto which the component neurons of the same pair responded withsignificant changes in discharge rate; (iv) there was no significantdifference between the correlative (formation of peaks or troughsin the CC) and individual (change in discharge rate of individualneurons) ways of coding in the sensitivity to distinguish betweentwo taste qualities ranked to be adjacent on the basis of thenumber of spikes composing the response; and (v) the peaks ortroughs appearing in two CCs during stimulations with two kindsof tastants were compared with regard to overlapping of theirdelay ranges and widths. The spikes in the non-overlapping portionof each peak (suppressed spike number in the case of troughs)are supposed to be able to contribute to two-taste discrimination:the correlated discharges occurring with a delay time that correspondsto the overlapping portion can in no way be judged differently,but the spikes falling in the non-overlapping portion may contributeto the differentiation. The ratio of the non-overlapping portionto the entire peak (or trough) was 0.35 on average. It is concludedthat temporal coding of taste qualities seems to operate effectivelyin the gustatory cortex. Chem. Senses 22: 363–373, 1997.     

Intracellular Calcium Spikes in Rat Suprachiasmatic Nucleus Neurons Induced by BAPTA-Based Calcium Dyes

Background

Circadian rhythms in spontaneous action potential (AP) firing frequencies and in cytosolic free calcium concentrations have been reported for mammalian circadian pacemaker neurons located within the hypothalamic suprachiasmatic nucleus (SCN). Also reported is the existence of “Ca²⁺ spikes” (i.e., [Ca²⁺]_c transients having a bandwidth of 10∼100 seconds) in SCN neurons, but it is unclear if these SCN Ca²⁺ spikes are related to the slow circadian rhythms.

Methodology/Principal Findings

We addressed this issue based on a Ca²⁺ indicator dye (fluo-4) and a protein Ca²⁺ sensor (yellow cameleon). Using fluo-4 AM dye, we found spontaneous Ca²⁺ spikes in 18% of rat SCN cells in acute brain slices, but the Ca²⁺ spiking frequencies showed no day/night variation. We repeated the same experiments with rat (and mouse) SCN slice cultures that expressed yellow cameleon genes for a number of different circadian phases and, surprisingly, spontaneous Ca²⁺ spike was barely observed (<3%). When fluo-4 AM or BAPTA-AM was loaded in addition to the cameleon-expressing SCN cultures, however, the number of cells exhibiting Ca²⁺ spikes was increased to 13∼14%.

Conclusions/Significance

Despite our extensive set of experiments, no evidence of a circadian rhythm was found in the spontaneous Ca²⁺ spiking activity of SCN. Furthermore, our study strongly suggests that the spontaneous Ca²⁺ spiking activity is caused by the Ca²⁺ chelating effect of the BAPTA-based fluo-4 dye. Therefore, this induced activity seems irrelevant to the intrinsic circadian rhythm of [Ca²⁺]_c in SCN neurons. The problems with BAPTA based dyes are widely known and our study provides a clear case for concern, in particular, for SCN Ca²⁺ spikes. On the other hand, our study neither invalidates the use of these dyes as a whole, nor undermines the potential role of SCN Ca²⁺ spikes in the function of SCN.     

Intracellular Calcium Homeostasis Changes Induced in Rat Spinal Cord Neurons by Extracellular Acidification

Changes in intracellular Ca²⁺ induced by extracellular acidification to pH = 6 were studied in isolated rat spinal dorsal horn neurons using indo-1 fluorescent technique. In all neurons such treatment induced a decrease of basal [Ca²⁺]_i level by 20.8%, preceded in some of them by temporary increase. The changes were completely reversible. The depolarization-induced [Ca²⁺]_i transients became strongly and also reversibly depressed. If tested after termination of acidification, they demonstrated substantial prolongation of their decay phase, reaching 310% at 120 sec after the application of depolarization. To analyze the mechanisms of such changes, mitochondrial protonophore CCCP has been applied between the end of acidification and the depolarizing pulse. This completely eliminated the described slowing of the transients' decay. To the contrary, application of caffeine to induce Ca²⁺ release from the endoplasmic reticulum did not show significant changes in the corresponding [Ca²⁺]_i transients. A conclusion is made that in mammalian neurons extracellular acidification, apart from inhibiting voltage-operated Ca²⁺ channels, also substantially alters the Ca²⁺ exchange function of mitochondria responsible for rapid accumulation of ions and their delayed release back into the cytosol.     

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H₂DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H₂DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H₂O₂. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions.