Multiparameter flow cytometry of bacteria: implications for diagnostics and therapeutics

BACKGROUND: Flow cytometric studies of antibiotic susceptibilities of bacteria have typically measured a single fluorescence parameter, such as membrane potential (indicating viability), or permeability to nucleic acid stains such as propidium (indicating nonviability). Cytometry of bacteria stained simultaneously with a membrane potential dye and a permeability indicator reveals unanticipated complexity. METHODS: Aliquots of cultures of three bacterial species were stained with the potential-sensitive dye hexamethylindocarbocyanine [DiIC1(3)] and the permeability indicator TO-PRO-3, in the presence and absence of a proton ionophore which collapses the potential gradient. They were analyzed using a dual-laser flow cytometer. RESULTS: Cultures grown under suboptimal conditions appear to contain cells that take up TO-PRO-3 while maintaining membrane potential, although some events showing both high DiIC1(3) fluorescence and high TO-PRO-3 fluorescence may represent clumps. CONCLUSIONS: Variations in metabolic patterns between species and within organisms under suboptimal culture conditions or following antibiotic exposure may make it difficult to develop flow cytometric clinical assays for antibiotic susceptibility. However, transient permeabilization of otherwise resistant organisms by sublethal doses of antibiotics may make it possible to treat infections by such organisms with suitably derivatized, otherwise toxic molecules; multiparameter cytometry should be useful in pursuing this approach to therapy.     

Characterization and reconstitution of a cell free system for NAD(+)-dependent deoxyhypusine formation on the 18 kDa eIF-4D precursor

Deoxyhypusine formation on the 18 kDa eIF-4D precursor is due to a covalent linkage between a lysine residue of the protein and the aminobutyl moiety derived from spermidine. The deoxyhypusine is then hydroxylated to form hypusine. This post-translational modification represents one of the most specific spermidine-dependent biochemical events in eukaryotic cells. Deoxyhypusine formation can be performed in vitro at pH 9.5 and is greatly stimulated by NAD+. Using the labeling of the 18 kDa protein by [3H]spermidine as an assay for deoxyhypusine formation, we found that (i) significant deoxyhypusine formation can be demonstrated in vitro at pH 7.2 only if NAD+ is present, (ii) deoxyhypusine formation was sensitive to buffer composition; buffers made of basic amino acids and Tris were inhibitory, (iii) sulfhydryl reagents and metal ions such as Cu2+ and Fe3+ were potent inhibitors of deoxyhypusine formation and (iv) the 18 kDa protein substrate was heat-stable. The in vitro activity of deoxyhypusine formation, which depends on the presence of both enzyme and protein substrate, can be separated from the product, eIF-4D, by a one-step Cibacron blue dye affinity column. Taking advantage of this finding, we have developed a simple procedure, based on the use of Cibacron blue dye, for partially purifying both the deoxyhypusine-forming enzyme and the 18 kDa protein substrate. When the partially purified enzyme and protein substrate were mixed in the presence of 1 mM NAD+ and [3H]spermidine, the 18 kDa protein was radiolabeled, no labeling could be detected if any one component was absent. Using partially purified enzyme, we have also determined the half-life of the protein substrate in alpha-difluoromethyl ornithine (DFMO)-treated NB-15 cells and found it to be longer than 10 h.     

Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.     

Partition of membrane probes in a gel/fluid two-component lipid system: a fluorescence resonance energy transfer study

A non-ideal lipid binary mixture (dilauroylphosphatidylcholine/distearoylphosphatidylcholine), which exhibits gel/fluid phase coexistence for wide temperature and composition ranges, was studied using photophysical techniques, namely fluorescence anisotropy, lifetime and resonance energy transfer (FRET) measurements. The FRET donor, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanol amine, and a short-tailed FRET acceptor, 1,1'-didodecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC12(3)), were shown to prefer the fluid phase by both intrinsic anisotropy, lifetime and FRET measurements, in agreement with published reports. The other studied FRET acceptor, long-tailed probe 1,1'-dioctadecil-3,3,3',3'-tetramethylindocarbocyanine (DiIC18(3)), is usually reported in the literature as partitioning mainly to the gel. While intrinsic lifetime studies indeed indicated preferential partition of DiIC18(3) into a rigidified environment, FRET analysis pointed to an increased donor-acceptor proximity as a consequence of phase separation. These apparently conflicting results were rationalized on the basis of segregation of DiIC18(3) to the gel/fluid interphase. In order to fluid-located donors sense these interphase-located acceptors, fluid domains should be small (not exceed approximately 10-15 nm). It is concluded that membrane probes which apparently prefer the gel phase may indeed show a non-random distribution in this medium, and tend to locate in an environment which simultaneously leads to less strict packing constraints and to favorable hydrophobic matching interactions.     

Neuronal influence on B and H human blood-group antigen expression in rat cochlear cultures

Summary The presence of B and H human blood-group antigens was analyzed by immunocytochemistry in rat cochleas developing either in vivo or in vitro. Developing animals, on embryonic day (E) 18 and postnatal day (P) 3, were used for in vivo studies. For in vitro studies, cochleas were removed at E18 and placed for 3 or 8 days in organotypic culture either directly or after partial spiral ganglion removal. Results from epithelial regions from cochleas developing in vivo were similar to those observed in corresponding areas of direct organotypic cultures where the innervation from spiral ganglion neurons was present. Antibodies to human blood group antigens, anti B and anti AB, selectively labeled hair cells. The intensity of labeling was weak at E18, but increased at P3 in vivo or after 3–8 days in organotypic culture. Anti H antibodies showed weak labeling of the apical surface of hair cells and other epithelial cells at E18; this labeling also increased at P3 or after 3–8 days in culture. In contrast, the non-innervated regions from organotypic cultures, where ganglia were partially removed, exhibited an epithelial disorganization and no hair cell labeling with any of the antibodies studied. The present findings suggest that human blood-group antigen expression on developing cochlear hair cells of rats may be related to afferent nerve fiber influence.     

Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling

Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling.The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDASE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling.     

The response of cultured trigeminal and spinal cord motoneurons to nerve growth factor

Dissociated neurons from the trigeminal (V) region of the metencephalic basal plate or the ventral spinal cord from chick embryos of Day 4 (V basal plate) or Day 5 (spinal cord) were cultured on a laminin substratum either in the presence of nerve growth factor (NGF) or in control medium. Assessment was made of neuronal survival, the amount of neurite elaborated, and the percentage of neurons initiating neurites. The presence of motoneurons was verified by retrograde labeling with the fluorescent dye diI. NGF was found to significantly increase the quantity of neuritic processes produced by the spinal cord dissociates at both 24 and 48 hr in vitro. The percentage of neurons initiating neuritic processes was significantly increased by NGF in the trigeminal population at 48 hr in vitro. Neuronal survival was not enhanced by NGF in either group. Both trigeminal and spinal cord neurons were also found to specifically bind 125I-NGF in culture. These results provide direct evidence for an influence of NGF on process formation of early embryonic motoneurons in culture.     

Organization of the sea urchin egg endoplasmic reticulum and its reorganization at fertilization

The ER of eggs of the sea urchin Lytechinus pictus was stained by microinjecting a saturated solution of the fluorescent dicarbocyanine DiIC18(3) (DiI) in soybean oil; the dye spread from the oil drop into ER membranes throughout the egg but not into other organelles. Confocal microscopy revealed large cisternae extending throughout the interior of the egg and a tubular membrane network at the cortex. Since diffusion of DiI is confined to continuous bilayers, the spread of the dye supports the concept that the ER is a cell-wide, interconnected compartment. In time lapse observations, the internal cisternae were seen to be in continuous motion, while the cortical ER was stationary. After fertilization, the internal ER appeared to become more finely divided, beginning as a wave apparently coincident with the calcium wave and becoming most marked by 2-3 min. By 5-8 min the ER returned to an organization similar to that of the unfertilized egg. The cortical network also changed at fertilization; it became disrupted and eventually recovered. DiI labeling allowed continuous observations of the ER during pronuclear migration and mitosis. DiI-stained membranes accumulated in the region of the microtubule array surrounding the sperm nucleus and centriole (the sperm aster) as it migrated to the center of the egg; this accumulation persisted near the centrosomes and zygote nucleus throughout pronuclear fusion and the first two mitotic cycles. We have used a new method to observe the spatial and temporal organization of the ER in a living cell, and we have demonstrated a striking reorganization of the ER at fertilization.     

应用激光共聚焦扫描技术对海马脑片神经元内钙的观察

用微量注射法将荧光剂Ｆｌｕｏ－３注入大鼠海马,对神经元进行在体荧光标记,可清晰地标记多个神经细胞。联合应用激光共聚焦扫描显微镜,观察大鼠海马脑片ＣＡ１锥体细胞在青霉素,谷氨酸模拟致痫及缺糖缺氧时胞内钙的变化。结果显示：无镁时,谷氨酸和青霉素可致海马ＣＡ１锥体细胞胞内钙的缓慢增加;离体缺糖缺氧时ＣＡ１锥体细胞胞内钙亦增多。     

Compositional analysis of growing axons from rat sympathetic neurons

We describe culture systems for neurons of an adrenergic autonomic ganglion which: (a) permit cultivation of neurons without supporting cells, (b) permit separate harvest of somal and axonal material, and (c) permit direct access to the neuronal surface. The antimetabolites used to suppress supporting cell growth did not have demonstrable effects on neuronal polypeptide synthesis. Rapid neurite outgrowth, which characterized these cultures, was prevented by colchicine or cycloheximide and resumed promptly after their withdrawal. Axons separated from cell bodies showed no incorporation of label from leucine or fucose, but did exhibit incorporation of glucosamine. The major polypeptides present in this neuron, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, are described. No major differences in polypeptide content were observed when soma and axons were compared. Likewise, there were no differences detected in polypeptides synthesized by neurons in suspension or neurons actively extending processes. Analysis of the polypeptides within the neurites after labeling with amino acids indicated transport at a number of different rates; certain of these polypeptides corresponded in size and transport characteristics to polypeptides observed in the rabbit optic nerve after labeling of retinal ganglion cells. Tubulin and actin have been definitively identified in this cell type (18); we found proteins similar in size and proportionate amounts to be among the rapidly transported soluble polypeptides. The prominent polypeptides observed after several methods of surface labeling are described.     

Retrograde Loading of Nerves,Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method^1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons^3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.     

Fluorescent carbocyanine dyes allow living neurons of identified origin to be studied in long-term cultures

A prerequisite for many studies of neurons in culture is a means of determining their original identity. We needed such a technique to study the interactions in vitro between a class of spinal cord neurons, sympathetic preganglionic neurons, and their normal target, neurons from the sympathetic chain. Here, we describe how we use two highly fluorescent carbocyanine dyes, which differ in color but are otherwise similar, to identify neurons in culture. The long carbon chain carbocyanine dyes we use are lipid-soluble and so become incorporated into the plasma membrane. Neurons can be labeled either retrogradely or during dissociation. Some of the labeled membrane gradually becomes internalized and retains its fluorescence, allowing identification of cells for several weeks in culture. These dyes do not affect the survival, development, or basic physiological properties of neurons and do not spread detectably from labeled to unlabeled neurons. It seems likely that cells become retrogradely labeled mainly by lateral diffusion of dye in the plane of the membrane. If so, carbocyanine dyes may be most useful for retrograde labeling over relatively short distances. An additional feature of carbocyanine labeling is that neuronal processes are brightly fluorescent for the first few days in culture, presumably because dye rapidly diffuses into newly inserted membrane. We have used carbocyanine dyes to identify sympathetic preganglionic neurons in culture. Our results indicate that preganglionic neurons can survive in the absence of their target cells and that several aspects of their differentiation in the absence of target appear normal.     

Tissue distribution, autoradiography, and metabolism of 4-(2'-methoxyphenyl)-1-[2' -[N-2"-pyridinyl)-p-[(18)F]fluorobenzamido]ethyl]piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist for positron emission tomography: An In vivo study in rats

The in vivo behavior of 4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-p-[(18)F]fluorobenzamido ]ethyl]-piperazine (p-[(18)F]MPPF), a new serotonin 5-HT(1A) antagonist, was studied in awake, freely moving rats. Biodistribution studies showed that the carbon-fluorine bond was stable in vivo, that this compound was able to cross the blood-brain barrier, and that a general diffusion equilibrium could account for the availability of the tracer. The great quantity of highly polar metabolites found in plasma did not contribute to the small amounts of metabolites found in hippocampus, frontal cortex, and cerebellum. Exvivo p-[(18)F]MPPF and in vitro 8-hydroxy-2-(di-n-[(3)H]propylamino)tetralin autoradiography were compared both qualitatively and quantitatively. Qualitative evaluation proved that the same brain regions were labeled and that the p-[(18)F]MPPF labeling is (a) in total agreement with the known distribution of 5-HT(1A) receptors in rats and (b) characterized by very low nonspecific binding. Quantitative comparison demonstrated that the in vivo labeling pattern obtained with p-[(18)F]MPPF cannot be explained by differences in regional blood flow, capillary density, or permeability. The 5-HT(1A) specificity of p-[(18)F]MPPF and binding reversibility were confirmed in vivo with displacement experiments. Thus, this compound can be used to evaluate parameters characterizing 5-HT(1A) binding sites in the brain.     

Involvement of activated caspase-3-like proteases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons

Excessive activation of glutamate receptors mediates neuronal death in a number of neurodegenerative diseases. The intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Following mild insults via NMDA receptor activation, central neurons undergo apoptosis, but with more fulminant insults, necrosis intervenes. Caspases are important in several forms of apoptosis in vivo and in vitro. Previously, we have demonstrated that caspases are important in excitotoxicity-mediated apoptosis of cerebrocortical neurons. To determine the possible activation of caspase-3 in NMDA-induced neuronal apoptosis, we used an affinity-labeling technique: Biotinylated N-acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD.CHO) preferentially labels conformationally active caspase-3-like proteases, allowing us to visualize affinity-labeled caspases with streptavidin-fluorescein isothiocyanate under confocal microscopy. NMDA-induced apoptosis of cerebrocortical neurons was associated with a time-dependent increase in conformationally active caspase-3-like proteases. The activation of caspases was apparent within 20 min of NMDA stimulation and was localized primarily in the cytosol. However, following incubation of neurons for 18-24 h, conformationally active caspase-3-like proteases were also detectable in nuclei. Double labeling with propidium iodide to detect chromatin condensation indicated that affinity-labeled caspase-3-like proteases were specifically expressed in apoptotic cells. To further confirm this, we used an antibody specific for the conformationally active fragment of caspase-3 and found largely concordant results. Moreover, preincubation with DEVD.CHO prevented NMDA-induced apoptosis. Our results suggest that caspase-3-like proteases play a major role in excitotoxin-induced neuronal apoptosis.     

Apoptosis is induced by radiofrequency fields through the caspase-independent mitochondrial pathway in cortical neurons

Joubert, V., Bourthoumieu, S., Leveque, P. and Yardin, C. Apoptosis is Induced by Radiofrequency Fields through the Caspase-Independent Mitochondrial Pathway in Cortical Neurons. Radiat. Res. 169, 38-45 (2008). In the present study, we investigated whether continuous-wave (CW) radiofrequency (RF) fields induce neuron apoptosis in vitro. Rat primary neuronal cultures were exposed to a CW 900 MHz RF field with a specific absorption rate (SAR) of 2 W/kg for 24 h. During exposure, an increase of 2 degrees C was measured in the medium; control experiments with neurons exposed to 39 degrees C were then performed. Apoptosis was assessed by condensation of nuclei with 4',6-diamino-2-phenylindole (DAPI) staining observed with an epifluorescence microscope and fragmentation of DNA with TdT-mediated dUTP nick-end labeling (TUNEL) analyzed by flow cytometry. A statistically significant difference in the rate of apoptosis was found in the RF-field-exposed neurons compared to the sham-, 37 degrees C- and 39 degrees C-exposed neurons either 0 or 24 h after exposure using both methods. To assess whether the observed apoptosis was caspase-dependent or -independent, assays measuring caspase 3 activity and apoptosis-inducing factor (AIF) labeling were performed. No increase in the caspase 3 activity was found, whereas the percentage of AIF-positive nuclei in RF-field-exposed neurons was increased by three- to sevenfold compared to other conditions. Our results show that, under the experimental conditions used, exposure of primary rat neurons to CW RF fields may induce a caspase-independent pathway to apoptosis that involves AIF.     

Identification of Specific Sensory Neuron Populations for Study of Expressed Ion Channels

Sensory neurons transmit signals from various parts of the body to the central nervous system. The soma for these neurons are located in the dorsal root ganglia that line the spinal column. Understanding the receptors and channels expressed by these sensory afferent neurons could lead to novel therapies for disease. The initial step is to identify the specific subset of sensory neurons of interest. Here we describe a method to identify afferent neurons innervating the muscles by retrograde labeling using a fluorescent dye DiI (1,1''-dioctadecyl-3,3,3'',3''-tetramethylindocarbocyanine perchlorate). Understanding the contribution of ion channels to excitation of muscle afferents could help to better control excessive excitability induced by certain disease states such as peripheral vascular disease or heart failure. We used two approaches to identify the voltage dependent ion channels expressed by these neurons, patch clamp electrophysiology and immunocytochemistry. While electrophysiology plus pharmacological blockers can identify functional ion channel types, we used immunocytochemistry to identify channels for which specific blockers were unavailable and to better understand the ion channel distribution pattern in the cell population. These techniques can be applied to other areas of the nervous system to study specific neuronal groups.     

Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails

To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1'- didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in their alkyl tail length or unsaturation; these differences are expected to affect their distribution in membrane domains of varying fluidity or curvature. All three DiIs initially enter sorting endosomes containing endocytosed transferrin. DiIC16(3), with two long 16-carbon saturated tails is then delivered to late endosomes, whereas FAST DiI, with two cis double bonds in each tail, and DiIC12(3), with saturated but shorter (12-carbon) tails, are mainly found in the endocytic recycling compartment. We also find that DiOC16(3) (3,3'- dihexadecyloxacarbocyanine perchlorate) and FAST DiO (3, 3'-dilinoleyloxacarbocyanine perchlorate) behave similarly to their DiI counterparts. Furthermore, whereas a phosphatidylcholine analogue with a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore attached at the end of a 5-carbon acyl chain is delivered efficiently to the endocytic recycling compartment, a significant fraction of another derivative with BODIPY attached to a 12-carbon acyl chain entered late endosomes. Our results thus suggest that endocytic organelles can sort membrane components efficiently based on their preference for association with domains of varying characteristics.     

Effects of cholesterol depletion and increased lipid unsaturation on the properties of endocytic membranes

Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC(12) and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC(16) and DiIC(18)) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F. R. (1999) J. Cell Biol. 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties. There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as DiIC(16), were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change in overall membrane properties, and not cholesterol levels per se, by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes.     

Persistence of an amine uptake system in cultured rat sympathetic neurons which use acetylcholine as their transmitter

Cultures of dissociated rat superior cervical ganglion neurons (SCGN) were treated with the sympatholytic agent, guanethidine. When treated within the first couple of weeks in vitro, the neurons were rapidly destroyed. The cells grew less susceptible to the toxic effects of guanethidine with age in vitro. Moreover, the apparent affinity, Km, of the transport molecule for norepinephrine (NE) and guanethidine remained essentially unchanged between 2 and 7 wk in culture, as did the maximum velocity of transport (Vmax). This is at a time when previous studies have shown these neurons to be using acetylcholine (ACh) as their neurotransmitter. Cultures which were grown without supporting cells and from which cholinergic synaptic interactions were recorded physiologically were processed for autoradiography after incubation with [3H]NE. All cell bodies and processes seen had silver grains accumulated over them. These experiments show that sympathetic neurons in vitro maintain their amine uptake system relatively unchanged, even though they use ACh as their transmitter. The implications of these findings are discussed.