Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons

Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25 kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons.     

A low molecular weight factor is a significant mediator of non-opsonic neutrophil activation by Helicobacter pylori

Helicobacter pylori is believed to trigger neutrophil activation through several factors, including the H. pylori neutrophil-activating protein (HpNAP). The aim of this study was to characterise the factors within H. pylori cell-free extracts that stimulate neutrophil activation. Neutrophil activation was found to be dose-dependent and exhibited considerable variation between different clinical isolates. Activity was attributable to more than one protein factor. A low mol. wt fraction of <3 kDa was found to contribute a large proportion of the neutrophil-stimulating activity within H. pylori cell-free extract. Additional activity was provided by a high mol. wt fraction, possibly representing HpNAP. An inhibition ELISA and neutralisation experiments failed to identify or exclude formyl methionyl leucyl phenylalanine as the active factor within the low mol. wt fraction. The importance of the putative, low mol. wt neutrophil-activating factor may have been overlooked by those studies that have used concentrated H. pylori extracts.     

Protective effect of ultra low molecular weight heparin on glutamate-induced apoptosis in cortical cells

PURPOSE: To investigate the effect of ultra low molecular weight heparin (ULMWH) on glutamate induced apoptosis in rat cortical cells and to explore the possible mechanisms. MATERIALS AND METHODS: Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was first analyzed with Hoechst 33258 and then confirmed by DNA fragmentation. The concentration of free intracellular calcium ([Ca(2+)](i)) was determined with fura-2/AM fluorometry. The expression of Bcl-2 family protein and caspase-3 were evaluated with Western blot. RESULTS: Typical apoptotic morphological change in rat cortical cells treated with 100 micromol/L glutamate for 24h was detected by Hoechst 33258 staining, which was then confirmed by the DNA ladder of agarose gel electrophoresis. The apoptotic rate of the glutamate treated cells was up to 33.21%, and 24 h of treatment with glutamate increased [Ca(2+)](i), down-regulated Bcl-2 expression, up-regulated Bax expression, and increased caspase-3 activation in rat cortical cells. Our research demonstrated that ULMWH pretreatment can prevent the glutamate-induced apoptosis, attenuate the increase of [Ca(2+)](i) not only in medium containing Ca(2+) but also in Ca(2+)-free medium, up-regulate the expression of Bcl-2, down-regulate the expression of Bax, and decrease caspase-3 activation. CONCLUSION: ULMWH has neuroprotective capacity to antagonize glutamate-induced apoptosis in cortical cells, through decrease of Ca(2+) release and modulation of apoptotic processes.     

Tyrosine phosphorylation of a low molecular weight protein induced by RANTES in T-lymphocytes

The beta-chemokine RANTES, a T-lymphocyte activator, chemoattractant, and inducer of homotypic aggregation, is considered to exert extensive effects on T lymphocytes through either G protein-coupled or protein tyrosine kinase (PTK) signaling pathway. In the present study, we analyzed RANTES-induced signal transduction through PTK as an early event in T-lymphocyte activation. Tyrosine phosphorylation is detected by immunoblots in the human T-cell line H9 after incubation with human recombinant RANTES. The tyrosine phosphorylation of a protein with a molecular mass of about 25 kD is measurable as early as 30 s and maximal at 1-5 min; and is a dose-dependent effect. The phosphorylation response can be abrogated by the tyrosine-kinase inhibitor herbimycin A (HA) but is insensitive to heterotrimeric Galphai protein inhibitor pertussis toxin (Ptx). This phenomenon is also observed in a visible homotypic aggregation response after incubation serum-starved H9 cells with RANTES. The phosphorylation response can not be down-regulated by preincubation with either anti-CC chemokine receptor 5 (CCR5) antibody or HIV-1Bal supernatants. Our results suggest that tyrosine phosphorylation of a protein with molecular mass of about 25 kD via Src-family PTK(s) is an early event in T-lymphocyte activation associated with the homotypic aggregation in response to RANTES.     

Electrophysiological characterization of embryonic hippocampal neurons cultured in a 3D collagen hydrogel

Rat embryonic hippocampal neurons were cultured in (1) 3D collagen hydrogels as ‘entrapped’ evenly distributed cells, (2) at the interface of two collagen layers (sandwich model), and (3) on the surface of collagen coated coverslips (2D model). In the ‘entrapment’ model the neuronal processes grew out of the plane of the cell body and extended into the collagen matrix, in contrast to the sandwich model where the cells and their processes rarely left the plane in which they were seeded. Hippocampal neurons ‘entrapped’ in the 3D collagen gel grew the same number, but shorter, processes and exhibited improved survival compared to neurons cultured in the 2D model. There was no difference in the electrophysiological properties of the neurons cultured in the 3D compared to the 2D model except in the resting membrane potential and in the duration of the after-hyperpolarization. Spontaneous postsynaptic currents were recorded in 14- and 21-day-old 3D cultures evidencing functional synapse formation. Our results indicate that the physiological characteristics of 3D neuronal cultures are similar to traditional 2D cultures. However, functional 3D networks of hippocampal neurons will be necessary for multi-level circuit formation, which could be essential for understanding the basis of physiological learning and memory.     

低分子肝素治疗重度子痫前期患者外周血vWF、AT的变化

目的探讨低分子肝素能否改变重度子痫前期患者血浆中vWF含量,AT的活性水平,为低分子肝素用于重度子痫前期的治疗提供新的理论基础。方法选择2010年3月1日～2010年11月30日在天津医科大学宝坻临床学院、天津医科大学总医院分娩的重度子痫前期病人39例为研究组。根据入院顺序随机分成2组,传统治疗组20人,给予传统常规的治疗。低分子肝素治疗组19人,在传统常规治疗的基础上给予低分子肝素钙0.4m1,每天一次,皮下注射,两组分别于入院时及治疗3～5天后抽取孕妇静脉血,检测血浆vWF：Ag和AT：A及常规凝血试验。结果两组重度子痫前期患者治疗前血浆中vWF：Ag与同期对照组比较明显升高,而AT：A与同期对照组比较明显减少,低分子肝素治疗后血浆中vWF：Ag与治疗前比较明显降低,而AT：A与治疗前比较明显增高,传统方法治疗后血浆vWF：Ag和AT：A与治疗前比较差异无统计学意义。结论低分子肝素结合传统疗法治疗重度子痫前期患者,可改变患者血浆中vWF含量,AT的活性水平。     

Plasma lipolytic activity and substrate oxidation after intravenous administration of heparin and a low molecular weight heparin fragment

This study examines the effects of heparin and a low molecular weight heparin (LMWH) fragment on plasma lipolytic activity and substrate oxidation. Indirect calorimetry was performed continuously in healthy male subjects receiving a constant infusion of fat emulsion (0.2 g min-1) and glucose (0.8 g min-1) during a period of 4 h. After 2 h an infusion of heparin (n = 6) or LMWH (n = 6) (100 antifactor Xa units kg-1) or saline (n = 6) was given over 1 h. Plasma concentration of the fat emulsion decreased by 76 +/- 5% with heparin and by 12 +/- 7% with LMWH (P less than 0.01). In the case of LMWH the initial fall was followed by a consistent rise in fat emulsion concentration for the entire remaining study period. Compared to the control experiments, plasma FFA increased five times with heparin and three times with LMWH (P less than 0.05). The average respiratory quotient (RQ) and energy expenditure (EE) increased constantly during the study period and did not differ significantly between the groups. In all groups the average increase in glucose oxidation was 40-50%, while fat oxidation decreased to a comparable extent. Infusions of heparin and LMWH had no effect on RQ or EE. A microcalorimetric study on isolated rat adipocytes in buffer solutions containing glucose, fat emulsion, heparin or LMWH was also made. The heat output from the adipocytes was not influenced by the presence of heparin or LMWH. In conclusion, infusion of heparin resulted in a pronounced increase in FFA availability, whereas LMWH exerted a less marked lipolytic effect. However, the heparin-induced elevations in plasma FFA were not accompanied by measurable rises in lipid oxidation rate.     

Non-competitive inhibition of GABA currents by phenothiazines in cultured chick spinal cord and rat hippocampal neurons

The gamma-aminobutyric acid (GABA) inhibiting properties of several classes of antipsychotic medications were studied using gigaseal whole-cell voltage-clamp techniques in cultured chick spinal cord and rat hippocampal neurons. At doses above 1 microM trifluoperazine, chlorpromazine and thioridazine blocked GABA currents in a non-competitive fashion decreasing the maximal transmitter response without altering the half-maximal effective concentration. In contrast, haloperidol was ineffective against GABA at concentrations up to 100 microM. Among the agents studied trifluoperazine was the most potent GABA inhibitor with half maximal effect at 12 microM. Trifluoperazine (100 microM) also inhibited glycine-gated chloride currents in spinal cord neurons to an extent comparable to GABA (85 +/- 6% inhibition) but reduced glutamate currents by less than 35% in either spinal cord or hippocampal neurons.     

Extracellular matrix-induced synthesis of a low molecular weight collagen by fetal calf ligament fibroblasts

Fetal calf ligamentum nuchae fibroblasts, cultured from animals of different gestational age, synthesize a unique, low molecular weight collagen termed FCL-1 (Sage, H., Mecham, R., Johnson, C., and Bornstein, P., 1983, J. Cell Biol. 97:1933-1938). Previous studies on the elastogenic differentiation of these cells in vitro demonstrated that the extracellular matrix (ECM) protein elastin was specifically induced in undifferentiated fibroblasts when they were grown on ligament ECM isolated from animals at later stages of development (Mecham, R.P., Madaras, J.G., and Senior, R.M., 1984. J. Cell Biol. 98:1804-1812). To investigate the expression of FCL-1 as a function of developmental age, we grew fetal calf ligament fibroblasts from an 85 d (first trimester) animal (FCL 85d) on three different substrata: ligament from a 120 d (second trimester) animal, ligament from a 270 d (term) animal, and unmodified plastic tissue culture dishes. FCL 270d fibroblasts were grown on plastic substrata and served as a differentiated cellular control. Analysis of metabolically radiolabeled proteins from both the culture media and the cell layers showed that the synthesis of FCL-1 was selectively increased in those cells cultured on ligament ECM. For FCL 85d fibroblasts grown on 120 d and 270 d ligaments, FCL-1 comprised 17% and 22%, respectively, of the culture medium proteins that precipitated at concentrations of ammonium sulfate from 20-50%. FCL 85d and 270d fibroblasts grown on plastic substrata yielded values of 2.5% and 1.0%, respectively. This effect appeared to be specific for this collagen and did not reflect a general increase in the synthesis of connective tissue proteins of the ECM (e.g., types I and III procollagen). As percent of total newly-synthesized cellular protein, the output of FCL-1 was 10-fold higher by FCL 85d cells grown on 270d ligament ECM (5.8%) as compared to that of the same cellular population grown on a plastic surface (0.56%). The presence of the ligament ECM also altered the levels and distribution of secreted proteins between the culture medium and the cell layer. These studies provide evidence for differential expression of the novel collagen FCL-1 by FCL fibroblasts during development and suggest that such expression is affected, at least in part, by interaction of the cell with a ligament ECM.     

Presenilin 2 is secreted in mouse primary neurons: a release enhanced by apoptosis

Presenilin 1 (PS1) and presenilin 2 (PS2) are homologous transmembrane proteins genetically associated with Alzheimer disease. As previously reported by our group for PS1, here we demonstrated that, in mouse primary neurons and microglial cells, PS2 C-terminal fragment (CTF) is released by shedding into the extracellular compartment as a soluble form and that this release is 4.07-fold increased during apoptosis. When compared with PS1, PS2 seems to be more susceptible to apoptosis since its secretion is increased 2.8-fold more than PS1. During apoptosis either proteins were colocalized especially within shedded vesicles. The present study suggest an active role for the presenilins CTF on putative target cells.     

Lipid substitution on low molecular weight (0.6-2.0 kDa) polyethylenimine leads to a higher zeta potential of plasmid DNA and enhances transgene expression

Cationic polymers are desirable gene carriers because of their better safety profiles than viral delivery systems. Low molecular weight (MW) polymers are particularly attractive, since they display little cytotoxicity, but they are also ineffective for gene delivery. To create effective carriers from low MW polymers palmitic acid (PA) was substituted on 0.6-2.0 kDa polyethylenimines (PEIs) and their efficiency for plasmid DNA (pDNA) delivery was evaluated. The extent of lipid substitution was dependent on the lipid/PEI feed ratio and the polymer MW. While the hydrodynamic size of the polymer/pDNA complexes (polyplexes) increased or decreased depending on the extent of lipid substitution, the ζ potential of the assembled complexes was consistently higher as a result of lipid substitution. Lipid substitution generally increased the in vitro toxicity of the PEIs, but it was significantly lower than that of the 25 kDa branched PEI. The in vitro transfection efficiency of the lipid-substituted polymers was higher than that of native PEIs, which were not at all effective. The delivery efficiency was proportional to the extent of lipid substitution as well as the polymer MW. This correlated with the increased uptake of lipid-substituted polyplexes, based on confocal microscopic investigations with FITC-labeled pDNA. The addition of chloroquine further increased the transfection efficiency of lipid-substituted PEIs, indicating that endosomal release was a limiting factor affecting the efficiency of these carriers. This study indicates that lipid substitution on low MW PEIs makes their assembly more effective, resulting in better delivery of pDNA into mammalian cells.     

In humans the oxygen uptake slow component is reduced by prior exercise of high as well as low intensity

The aim of the study was to examine to what extent prior high- or low-intensity cycling, yielding the same amount of external work, influenced the oxygen uptake (V˙O₂) slow component of subsequent high-intensity cycling. The 12 subjects cycled in two protocols consisting of an initial 3 min period of unloaded cycling followed by two periods of constant-load exercise separated by 3 min of rest and 3 min of unloaded cycling. In protocol 1 both periods of exercise consisted of 6 min cycling at a work rate corresponding to 90% peak oxygen uptake (V˙O_2peak). Protocol 2 differed from protocol 1 in that the first period of exercise consisted of a mean of 12.1 (SD 0.8) min cycling at a work rate corresponding to 50% V˙O_2peak. The difference between the 3rd min V˙O₂ and the end V˙O₂ (ΔV˙O₂₍₆₋₃₎) was used as an index of the V˙O₂ slow component. Prior high-intensity exercise significantly reduced ΔV˙O₂₍₆₋₃₎. The ΔV˙O₂₍₆₋₃₎ was also reduced by prior low-intensity exercise despite an unchanged plasma lactate concentration at the start of the second period of exercise. The reduction was more pronounced after prior high- than after prior low-intensity exercise (59% and 28%, respectively). The results of this study show that prior exercise of high as well as low intensity reduces the V˙O₂ slow component and indicate that a metabolic acidosis is not a necessary condition to elicit a reduction in ΔV˙O₂₍₆₋₃₎. Accepted: 8 July 2000     

Galactosylated low molecular weight chitosan as a carrier delivering oligonucleotides to Kupffer cells instead of hepatocytes in vivo

The in vivo cellular localization of oligodeoxynucleotides (ODNs) delivered by galactosylated low molecular weight chitosan (gal-LMWC) was investigated. The gal-LMWCs preference for Kupffer cells was confirmed by in vivo and in vitro experiments. Furthermore, asialoglycoprotein receptor (ASGPr) was studied as a possible surface lectin which may involved in the endocytosis of the gal-LMWC/ODN complexes. Results showed that the gal-LMWC/ODN complex accumulated in liver when injected intravenously (i.v.). Further studies revealed that 50.6% of the complex was taken up by Kupffer cells in liver, 33.2% was taken up by endothelial cells, and only 16.2% of the complex was taken up by parenchymal cells. In vitro results also confirmed the affinity of gal-LMWC to murine Kupffer cells. Inhibition of the transfection by lactose and N-acetyl galactosamine (GalNAc) suggested that the particles might enter macrophages via ASGPr and the inhibition by LMWC implied that there might be other lectins involved in the endocytosis. In summary, our studies revealed that gal-LMWC/ODN complex is inclined to enter into Kupffer cells rather than into liver parenchymal cells in vivo. Galactosylation may not be a proper means for targeting chitosan/DNA nanoparticles to hepatocytes but it does have the potential to be a Kupffer cells targeting strategy especially for delivering drugs for antiinflammation.     

Enhancement of human parainfluenza virus-induced cell fusion by pradimicin, a low molecular weight mannose-binding antibiotic

Oligosaccharides, especially mannose residues, expressed on the cell surface, are thought to be important for virus-induced membrane fusion. We examined the effect of mannose-binding compounds, pradimicin derivative BMY-28 864 (PRM) and concanavalin A (Con A), on cell fusion of human parainfluenza type 2 virus (hPIV2)-infected HeLa cells. Syncytium formation of hPIV2-infected HeLa cells was suppressed in the presence of Con A. On the other hand, PRM enhanced cell fusion of hPIV2-infected HeLa cells. These effects were blocked by addition of mannose-rich mannan. However, PRM shows little effect on virus growth and the expression of viral glycoproteins on the cell surface in hPIV2-infected HeLa cells. Fluorescein-isothiocyanate-labeled pradimicin and Con A bound to both uninfected and hPIV2-infected mononuclear cells, indicating that these compounds have an affinity to several cellular component(s). In contrast to Con A, PRM had little affinity to the viral glycoproteins. It is inferred from these results that the enhancement of hPIV2-induced cell fusion is probably due to the interaction between PRM and cellular component(s). Received: 25 May 1997     

Differential effects of the protein kinase inhibitor K-252a on the in vitro survival of chick embryonic neurons

The effects of the protein kinase inhibitor K-252a on the in vitro survival of different populations of chick embryonic neurons were tested. Following dissociation from the corresponding ganglia by trituration, the neurons were cultured on laminin-coated dishes in both the presence and absence of their respective neurotrophic factor. K-252a alone promoted long-term neuronal survival of dorsal root ganglion (DRG) and ciliary ganglion (CG) neurons in a dose-dependent fashion. No such effect was seen with sympathetic ganglion (SG) neurons. In addition, K-252a did not interfere with nerve growth factor (NGF)- or ciliary neuronotrophic factor (CNTF)-mediated survival of DRG or CG neurons, respectively, but completely blocked the NGF- and CNTF-induced survival of SG neurons. High potassium-induced survival of SG neurons was not affected by K-252a. These results point to differences between various neuronal populations in the signal transduction mechanism for neurotrophic factors.     

Characterization of alternative pathway inhibition by a serum derived, low molecular weight complement inhibitor.

Normal human serum contains an inhibitor of complement which is distinguished by its small size of 500 daltons, the low molecular weight inhibitor (LMWI). When LMWI was present during incubation of zymosan or cobra venom factor with serum, formation of complement reactive complexes was blocked as measured by failure of these mixtures to lyse susceptible erythrocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH). Addition of LMWI to pre-formed complexes had no effect on their subsequent haemolytic activity. When dialysis was used to remove LMWI from reaction mixtures, it was shown that LMWI had not irreversibly altered any of the complement components. Purified components were used to demonstrate that LMWI prevented factor D activation of cobra venom factor-factor B complexes. LMWI also strongly inhibited binding of 125I-factor B to human erythrocytes bearing C3b and had little or nor effect on binding of 125I-factor H to the C3b bearing cells. Factor B binding to C3b was equally inhibited on normal and PNH erythrocytes. Thus, a dialysable fraction from normal human serum prevents activation of human complement by blocking formation, but not the activity of the C3/C5 convertase. These low molecular weight inhibitors result in inhibition of factor B binding to C3b and inhibition of factor D activation of C3bB complexes.     

GABA-induced calcium signaling in cultured enteric neurons is reinforced by activation of cholinergic pathways

GABA is an important inhibitory transmitter in the CNS. In the enteric nervous system, however, both excitatory and inhibitory actions have been reported. Here, we investigated the effects of GABA on the intracellular Ca2+ concentration of guinea-pig myenteric neurons (at 35 degrees C) using Fura-2-AM. Neurons were identified by 75 mM K+ depolarization (5 s), which evoked a transient intracellular Ca2+ concentration increase. GABA (10 s) induced a dose dependent (5 nM-1 microM) transient intracellular Ca2+ concentration rise in the majority of neurons (500 nM GABA: 251+/-17 nM, n=232/289). Interestingly, the response to 5 microM GABA (n=18) lasted several minutes and did not fully recover. GABA response amplitudes were significantly (P<0.001) reduced by GABAA and GABAB receptor antagonists (10 microM) bicuculline and phaclofen. The GABAA agonist isoguvacine (10 microM) and GABAB agonist baclofen (10 microM) induced similar responses as 50 nM GABA, while the GABAC agonist cis-4-aminocrotonic acid (CACA) (10 microM) only elicited small responses in a minority of neurons. Removal of extracellular Ca2+ abolished all responses while depletion of intracellular Ca2+ stores by thapsigargin (5 microM) did not alter the responses to 500 nM GABA (n=13), but reduction of Ca2+ influx through voltage-dependent Ca2+ channels did. The nicotinic antagonist hexamethonium (100 microM) also reduced GABA responses by almost 70% suggesting that GABA stimulates cholinergic pathways, while the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and the 5-HT3 receptor blocker ondansetron only had minor effects. Conclusion: GABA elicits transient intracellular Ca2+ concentration responses in the majority of myenteric neurons through activation of GABAA and GABAB receptors and much of the response can be attributed to facilitation of ACh release. Thus GABA may act mainly as a modulator that sets the state of excitability of the enteric nerve network. A concentration of 5 microM GABA, although frequently used in pharmacological experiments, seems to cause a detrimental response reminiscent of the neurotoxic effects glutamate has in the CNS.     

Alphaxalone activates a Cl- conductance independent of GABAA receptors in cultured embryonic human dorsal root ganglion neurons.

Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5alpha-pregnane-3alpha-ol-11,20-dione) effects on GABAA receptors and on Cl- currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl- currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 microM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 +/- 0.15). The alphaxalone-induced whole cell current was carried largely by Cl- ions. Its reversal potential was close to the theoretical Cl- equilibrium potential, changing with a shift in the external Cl- concentration as predicted by the Nernst equation for Cl- ions. And because the alphaxalone-current was not suppressed by the competitive GABAA receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 microM), it did not appear to result from activation of GABAA receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl- concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 microM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl- channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 microM alphaxalone revealed a single conductance state with a chord conductance of approximately 29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl- channels whose electrophysiological and pharmacological properties are distinct from those of Cl- channels associated with GABAA receptors.