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PubMed1986

Effects of cell density and extracellular matrix on the lateral diffusion of major histocompatibility antigens in cultured fibroblasts.

We have studied the effect of cell density on the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membranes of fibroblasts using fluorescence recovery after photobleaching. The percent recovery of fluorescence was decreased in fibroblasts grown in confluent cultures. While recovery of fluorescence was measurable in greater than 90% of the cells from sparse cultures, measurable recovery was detected in only 60-80% of the cells from dense cultures; no mobile antigens were detectable in 20-40% of cells examined. The diffusion coefficient on human skin fibroblast cells that did show recovery was the same for cells grown in sparse or dense conditions. In WI-38, VA-2, and c1 1d cultures the diffusion coefficients of mobile antigens were smaller in cells from dense cultures. Changes in lateral diffusion occurred with increased cell-cell contact and with age of cell culture but were not observed in growth-arrested cells or in sparse cells cultured in medium conditioned by confluent cells. Decreased mobile fractions of MHC antigens were observed when cells were plated on extracellular matrix materials derived from confluent cultures. Treatment of the extracellular matrix materials with a combination of proteolytic enzymes or by enzymes that degrade proteoglycans abolished this effect. Matrices produced by cells from other cell lines were less effective in inducing changes in mobile fractions and purified matrix components alone did not induce changes in lateral diffusion. Finally, there were no differences in the proportion of MHC antigens that were resistant to Triton X-100 extraction in sparse and dense cells. These results suggest that cell-cell interactions mediated through extracellular matrix materials can influence the lateral diffusion of at least part of the population of MHC antigens.

Animals↗

PubMed1988

Tumor necrosis factor and immune interferon act in concert to slow the lateral diffusion of proteins and lipids in human endothelial cell membranes.

Vascular endothelial surface-related activities may depend on the lateral mobility of specific cell surface macromolecules. Previous studies have shown that cytokines induce changes in the morphology and surface antigen composition of vascular endothelial cells in vitro and at sites of immune and inflammatory reactions in vivo. The effects of cytokines on membrane dynamic properties have not been examined. In the present study, we have used fluorescence photobleaching recovery (FPR) to quantify the effects of the cytokines tumor necrosis factor (TNF) and immune interferon (IFN-gamma) on the lateral mobilities of class I major histocompatibility complex protein, of an abundant 96,000 Mr mesenchymal cell surface glycoprotein (gp96), and of a phospholipid probe in cultured human endothelial cell (HEC) membranes. Class I protein and gp96 were directly labeled with fluorescein-conjugated monoclonal antibodies; plasma membrane lipid mobility was examined with the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE). In untreated, confluent HEC monolayers, diffusion coefficients were 30 x 10(-10) cm2 s-1 for class I protein, 14 x 10(-10) cm2 s-1 for gp96, and 80 x 10(-10) cm2 s-1 for Fl-PE. Fractional mobilities were greater than 80% for each probe. Cultures treated at visual confluence for 3-4 d with either 100 U/ml TNF or 200 U/ml IFN-gamma did not exhibit significant changes in protein or lipid mobilities despite significant changes in cell morphology and membrane antigen composition. In HEC cultures treated concomitantly with TNF and IFN-gamma, however, diffusion coefficients decreased by 71-79% for class I protein, 29-55% for gp96, and 23-38% for Fl-PE. Fractional mobilities were unchanged. By immunoperoxidase transmission electron microscopy, plasma membranes of untreated and cytokine-treated HEC were flat and stained uniformly for class I antigen. "Line" FPR measurements on doubly treated HEC demonstrated isotropic diffusion of class I protein, gp96, and Fl-PE. Finally, although TNF and IFN-gamma retarded the growth of HEC cultures and disrupted the organization of cell monolayers, the slow diffusion rates of gp96 and Fl-PE in confluent doubly treated monolayers were not reproduced in sparse or subconfluent untreated monolayers. We conclude that the slowing of protein and lipid diffusion induced by the combination of TNF and IFN-gamma is not due to plasma membrane corrugations, to anisotropic diffusion barriers, or to decreased numbers of cell-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Membrane↗

PubMed1995

Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether.

Our previous results indicated that the plasma membrane of cultured normal rat kidney fibroblastic cell is compartmentalized for diffusion of receptor molecules, and that long-range diffusion is the result of successive intercompartmental jumps (Sako, Y. and Kusumi, A. 1994. J. Cell Biol. 125:1251-1264). In the present study, we characterized the properties of intercompartmental boundaries by tagging transferrin receptor (TR) with either 210-nm-phi latex or 40-nm-phi colloidal gold particles, and by dragging the particle-TR complexes laterally along the plasma membrane using laser tweezers. Approximately 90% of the TR-particle complexes showed confined-type diffusion with a microscopic diffusion coefficient (Dmicro) of approximately 10(-9) cm2/s and could be dragged past the intercompartmental boundaries in their path by laser tweezers at a trapping force of 0.25 pN for gold-tagged TR and 0.8 pN for latex-tagged TR. At lower dragging forces between 0.05 and 0.1 pN, particle-TR complexes tended to escape from the laser trap at the boundaries, and such escape occurred in both the forward and backward directions of dragging. The average distance dragged was half of the confined distance of TR, which further indicates that particle-TR complexes escape at the compartment boundaries. Since variation in the particle size (40 and 210 nm, the particles are on the extracellular surface of the plasma membrane) hardly affects the diffusion rate and behavior of the particle-TR complexes at the compartment boundaries, and since treatment with cytochalasin D or vinblastin affects the movements of TR (Sako and Kusumi as cited above), argument has been advanced that the boundaries are present in the cytoplasmic domain. Rebound of the particle-TR complexes when they escape from the laser tweezers at the compartment boundaries suggests that the boundaries are elastic structures. These results are consistent with the proposal that the compartment boundaries consist of membrane skeleton or a membrane-associated part of the cytoskeleton (membrane skeleton fence model). Approximately 10% of TR exhibited slower diffusion (Dmicro approximately 10(-10)-10(-11) cm2/s) and binding to elastic structures.

Animals↗

PubMed1982

Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints.

Measurements of lateral molecular diffusion on blebs formed on the surfaces of isolated muscle cells and myoblasts are reported. These blebbed membranes retain integral proteins but apparently separate from the detectable cytoskeleton. On blebs, acetylcholine receptors, concanavalin A receptors, and stearoyldextran extrinsic model receptor molecules are free to diffuse with a diffusion coefficient (D) approximately 3 x 10(-9) cm2/s, which is close to the value predicted for hydrodynamic drag in the lipid membrane. In contrast, diffusion of these typical receptors in intact cell membranes is constrained to D approximately less than 10(-10) cm2/s with substantial fractions virtually nondiffusible (D less than 10(-12) cm2/s). Lipid analog diffusion is also slightly enhanced in blebs as expected of evanescent lipid protein interaction. This strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane.

Animals↗

PubMed1982

Surface functions during mitosis. III. Quantitative analysis of ligand-receptor movement into the cleavage furrow: diffusion vs. flow.

The surface distribution of concanavalin A (Con A) bound to cell membrane receptors varies dramatically as a function of mitotic phase. The lectin is distributed diffusely on cells labeled and observed between mid-prophase and early anaphase, whereas cells observed in late anaphase or telophase demonstrate a marked accumulation of Con A-receptor complexes over the developing cleavage furrow (Berlin, Oliver, and Walter. 1978. Cell. 15:327-341). In this report, we first use a system based on video intensification fluorescence microscopy to describe the simultaneous changes in cell shape and in lectin-receptor complex topography during progression of single cells through the mitotic cycle. The video analysis establishes that fluorescein succinyl Con A (F-S Con A)-receptor complex redistribution begins coincident with the first appearance of the cleavage furrow and is essentially complete within 2-3 min. This remarkable redistribution of surface fluorescence occurs during only a modest change in cell shape from a sphere to a belted cylinder. It reflects the translocation of complexes and not the accumulation of excess labeled membrane in the cleavage furrow: first, bound fluorescent cholera toxin which faithfully outlines the plasma membrane is not accumulated in the cleavage furrow, and, second, electron microscopy of peroxidase-Con A labeled cells undergoing cleavage shows that there is a high linear density of lectin within the furrow while Con A is virtually eliminated from the poles. The rate of surface movement of F-S Con A was quantitated by photon counting during a repetitive series of laser-excited fluorescence scans across dividing cells. Results were analyzed in terms of two alternative models of movement: a flow model in which complexes moved unidirectionally at constant velocity, and a diffusion model in which complexes could diffuse freely but were trapped at the cleavage furrow. According to these models, the observed rates of accumulation were attainable at either an effective flow velocity of approximately 1 micron/min, or an effective diffusion coefficient of approximately 10(-9) cm2/s. However, in separate experiments the lectin-receptor diffusion rate measured directly by the method of fluorescence recovery after photobleaching (FRAP) on metaphase cells was only approximately 10(-10) cm2/s. Most importantly, photobleaching experiments during the actual period of F-S Con A accumulation showed that lectin-receptor movement during cleavage occurs unidirectionally. These results rule out diffusion and make a process of oriented flow of ligand-receptor complexes the most likely mechanism for ligand-receptor accumulation in the cleavage furrow.

Animals↗

PubMed1961

Studies on transformations of Hemophilus influenzae. II. The molecular weight of transforming DNA by sedimentation and diffusion measurements.

The sedimentation and diffusion coefficients have been determined for Hemophilus influenzae transforming activity and DNA using P(32)-labeled DNA. The methods employed the Spinco fixed boundary separation cell for measurements of the sedimentation coefficient and the Northrop-Anson diffusion cell to determine the diffusion coefficient. There was a very close correlation between the amount of DNA and transforming activity sedimented or diffused. The sedimentation coefficient (s(20 degrees )), for both biological activity and DNA was 27 and the diffusion coefficient (D(20 degrees )) 1 x 10(-8) cm(2)/sec. The molecular weight calculated from these coefficients gave a value of 16 million. There was no difference in the sedimentation coefficients for the two unlinked markers, streptomycin and erythromycin resistance, and the diffusion coefficients for single markers or the linked markers, streptomycin and cathomycin, were the same.

DNA↗

PubMed1962

Diffusion barrieres in the squid nerve fiber. The axolemma and the Schwann layer.

The squid nerve barriers are formed by (a) the axolemma (membrane of the axon proper), a membrane 80 A thick perforated by cylindrical pores 4.0 to 4.5 A radius, and (b) the Schwann layer, constituted of numerous cells forming a layer one cell thick, crossed by 60 A wide slit channels. If a molecule present in the axoplasm has to reach the extraneural space, it has to pass (a) the pores, and (b) the channels, in series, and the diffusion rate will depend on the effective diffusion areas per unit path length, A(pd)/Deltax for the axolemma, and A(cd)/Deltax for the Schwann layer. By addition, A(nd)/Deltax, the transneural effective area for diffusion per unit path length is obtained. The diffusion rates of C(14)-ethylene glycol (2.2 A radius), and C(14)-glycerol (2.8 A radius) were measured. The diffusion rate of H(3)-labeled water (1.5 A radius) has been previously determined. The results expressed in terms of A(nd)/Deltax (mean values +/- SD, referred to 1 cm(2) of nerve surface) are 5.3 +/- 1.4 cm for water, 2.5 +/- 0.4 cm for ethylene glycol, and 0.29 +/- 0.03 cm for glycerol. Theoretical values for A(nd)/Deltax of 2.5 and 0.83 cm for ethylene glycol and glycerol have been calculated. The agreement between the theoretical values for A(nd)/Deltax and the experimental ones supports the diffusion barrier model described above.

Animals↗

PubMed1974

Red cell membrane permeability deduced from bulk diffusion coefficients.

The permeability coefficients of dog red cell membrane to tritiated water and to a series of[(14)C]amides have been deduced from bulk diffusion measurements through a "tissue" composed of packed red cells. Red cells were packed by centrifugation inside polyethylene tubing. The red cell column was pulsed at one end with radiolabeled solute and diffusion was allowed to proceed for several hours. The distribution of radioactivity along the red cell column was measured by sequential slicing and counting, and the diffusion coefficient was determined by a simple plotting technique, assuming a one-dimensional diffusional model. In order to derive the red cell membrane permeability coefficient from the bulk diffusion coefficient, the red cells were assumed to be packed in a regular manner approximating closely spaced parallelopipeds. The local steady-state diffusional flux was idealized as a one-dimensional intracellular pathway in parallel with a one-dimensional extracellular pathway with solute exchange occurring within the series pathway and between the pathways. The diffusion coefficients in the intracellular and extracellular pathways were estimated from bulk diffusion measurements through concentrated hemoglobin solutions and plasma, respectively; while the volume of the extracellular pathway was determined using radiolabeled sucrose. The membrane permeability coefficients were in satisfactory agreement with the data of Sha'afi, R. I., C. M. Gary-Bobo, and A. K. Solomon (1971. J. Gen. Physiol. 58:238) obtained by a rapid-reaction technique. The method is simple and particularly well suited for rapidly permeating solutes.

Amides↗

PubMed1975

Oxygen diffusion in biological and artificial membranes determined by the fluorochrome pyrene.

Quenching of pyrene fluorescence by oxygen was used to determine oxygen diffusion coefficients in phospholipid dispersions and erythrocyte plasma membranes. The fluorescence intensity and lifetime of pyrene in both artificial and natural membranes decreases about 80% in the presence of 1 atm O2, while the fluorescence excitation and emission spectra and the absorption spectrum are unaltered. Assuming the oxygen partition coefficient between membrane and aqueous phase to be 4.4, the diffusion coefficients for oxygen at 37 degrees C are 1.51 X 10(-5) cm2/s in dimyristoyl lecithin vesicles, 9.32 X 10(-6) cm2/s in dipalmitoyl lecithin vesicles, and 7.27 X 10(-6) cm2/s in erythrocyte plasma membranes. The heats of activation for oxygen diffusion are low (less than 3 kcal/degree-mol). A dramatic increase in the diffusion constant occurs at the phase transition of dimyristoyl and dipalmitoyl lecithin, which may result from an increase in either the oxygen diffusion coefficient, partition coefficient, or both. The significance of the change in oxygen diffusion below and above the phase transition for biological membranes is discussed.

Cell Membrane↗

PubMed1976

Proton transport by phosphate diffusion--a mechanism of facilitated CO2 transfer.

We have measured CO2 fluxes across phosphate solutions at different carbonic anhydrase concentrations, bicarbonate concentration gradients, phosphate concentrations, and mobilities. Temperature was 22-25 degrees C, the pH of the phosphate solutions was 7.0-7.3. We found that under physiological conditions of pH and pCO2 a facilitated diffusion of CO2 occurs in addition to free diffusion when (a) sufficient carbonic anhydrase is present, and (b) a concentration gradient of HCO3- is established along with a pCO2 gradient, and (c) the phosphate buffer has a mobility comparable to that of bicarbonate. When the phosphate was immobilized by attaching 0.25-mm-long cellulose particles, no facilitation of CO2 diffusion was detectable. A mechanism of facilitated CO2 diffusion in phosphate solutions analogous to that in albumin solutions was proposed on the basis of these findings: bicarbonate diffusion together with a facilitated proton transport by phosphate diffusion. A mathematical model of this mechanism was formulated. The CO2 fluxed predicted by the model agree quantitatively with the experimentally determined fluxes. It is concluded that a highly effective proton transport mechanism acts in solutions of mobile phosphate buffers. By this mechanism; CO2 transfer may be increased up to fivefold and proton transfer may be increased to 10,000-fold.

Carbon Dioxide↗

PubMed1990

Diffusion limitation in the block by symmetric tetraalkylammonium ions of anthrax toxin channels in planar phospholipid bilayer membranes.

Current flow through the channel formed in planar phospholipid bilayer membranes by the PA65 fragment of anthrax toxin is blocked, in a voltage-dependent manner, by tetraalkylammonium ions (at micromolar concentrations), which bind to a blocking site within the channel lumen. We have presented evidence that diffusion plays a significant role in the kinetics of blocking by tetrabutylammonium ion (Bu4N+) from the cis (toxin-containing) side of the membrane (Blaustein, R. O., E. J. A. Lea, and A. Finkelstein. 1990. J. Gen. Physiol. 96:921-942); in this paper we examine the implications and consequences of diffusion control for binding kinetics. As expected for a diffusion-affected reaction, both the entry rate constant (kcis1) of Bu4N+ from the cis solution to the blocking site and the exit rate constant (kcis-1) of Bu4N+ from the blocking site to the cis solution are reduced if the viscosity of that medium is increased by the addition of dextran. In conformity with both thermodynamics and kinetic arguments, however, the voltage-dependent equilibrium binding constant, Keq (= kcis-1/kcis1), is not altered by the dextran-induced viscosity increase of the cis solution. The entry rate constants (kcis1) for tetrapentylammonium (Pe4N+), tetrahexylammonium (Hx4N+), and tetraheptylammonium (Hp4N+) are also diffusion controlled, and all of them, including that for Bu4N+, attain a voltage-independent plateau value at large positive cis voltages consistent with diffusion limitation. Although the plateau value of kcis1 for Hx4N+ is only a factor of 3 less than that for Bu4N+, the plateau value for Hp4N+ is a factor of 35 less. This precipitous fall in value indicates, from diffusion-limitation theory, that the diameter of the channel entrance facing the cis solution is not much larger than the diameter of Hp4N+, i.e., approximately 12 A.

Antigens, Bacterial↗

PubMed1997

Measurement of the ferric diffusion coefficient in agarose and gelatine gels by utilization of the evolution of a radiation induced edge as reflected in relaxation rate images.

A method has been developed to determine the diffusion coefficients of ferric ions in ferrous sulphate doped gels. A radiation induced edge was created in the gel, and two spin-echo sequences were used to acquire a pair of images of the gel at different points of time. For each of these image pairs, a longitudinal relaxation rate image was derived. From profiles through these images, the standard deviations of the Gaussian functions that characterize diffusion were determined. These data provided the basis for the determination of the ferric diffusion coefficients by two different methods. Simulations indicate that the use of single spin-echo images in this procedure may in some cases lead to a significant underestimation of the diffusion coefficient. The technique was applied to different agarose and gelatine gels that were prepared, irradiated and imaged simultaneously. The results indicate that the diffusion coefficient is lower in a gelatine gel than in an agarose gel. Addition of xylenol orange to a gelatine gel lowers the diffusion coefficient from 1.45 to 0.81 mm2 h-1, at the cost of significantly lower Rl sensitivity. The addition of benzoic acid to the latter gel did not increase the Rl sensitivity.

Benzoates↗

PubMed1998

Comparison of finite-difference transport and diffusion calculations for photon migration in homogeneous and heterogeneous tissues.

We analyse the limits of the diffusion approximation to the time-independent equation of radiative transfer for homogeneous and heterogeneous biological media. Analytical calculations and finite-difference simulations based on diffusion theory are compared with discrete-ordinate, finite-difference transport calculations. The influence of the ratio of absorption and transport scattering coefficient (mu(a)/mu'(s)) on the accuracy of the diffusion approximation are quantified and different definitions for the diffusion coefficient, D, are discussed. We also address effects caused by void-like heterogeneities in which absorption and scattering are very small compared with the surrounding medium. Based on results for simple homogeneous and heterogeneous systems, we analyse diffusion and transport calculation of light propagation in the human brain. For these simulations we convert density maps obtained from magnetic resonance imaging (MRI) to optical-parameter maps (mu(a) and mu'(s)) of the brain. We show that diffusion theory fails to describe accurately light propagation in highly absorbing regions, such as haematoma, and void-like spaces, such as the ventricles and the subarachnoid space.

Absorption↗

PubMed2002

Diffusion tensor MR imaging of principal directions: a tensor tomography approach.

A novel approach to reconstructing the principal directions of a diffusion tensor field directly from magnetic resonance imaging (MRI) data using a tensor tomography data acquisition approach was developed. If tensor eigenvalues are assumed to be known, the reconstruction of principal directions requires fewer measurements than the reconstruction of the full tensor field. The tensor tomography data acquisition method (rotating diffusion gradients) leads to a unique reconstruction of principal directions, whereas the conventional MRI acquisition technique (stationary diffusion gradients) leads to an ambiguous reconstruction of principal directions when the same number of measurements are used. A computer-generated phantom was used to simulate the diffusion tensor field in the mid-ventricular region of the myocardium. The principal directions of the diffusion tensor field were assumed to align with the fibre structure of the myocardium. An iterative algorithm was used to reconstruct the principal directions. Computer simulations verify that the proposed method provides accurate reconstruction of the principal directions of a diffusion tensor field.

Algorithms↗

PubMed2005

Coupled radiative transfer equation and diffusion approximation model for photon migration in turbid medium with low-scattering and non-scattering regions.

In this paper, a coupled radiative transfer equation and diffusion approximation model is extended for light propagation in turbid medium with low-scattering and non-scattering regions. The light propagation is modelled with the radiative transfer equation in sub-domains in which the assumptions of the diffusion approximation are not valid. The diffusion approximation is used elsewhere in the domain. The two equations are coupled through their boundary conditions and they are solved simultaneously using the finite element method. The streamline diffusion modification is used to avoid the ray-effect problem in the finite element solution of the radiative transfer equation. The proposed method is tested with simulations. The results of the coupled model are compared with the finite element solutions of the radiative transfer equation and the diffusion approximation and with results of Monte Carlo simulation. The results show that the coupled model can be used to describe photon migration in turbid medium with low-scattering and non-scattering regions more accurately than the conventional diffusion model.

Animals↗

PubMed1991

The diffusion of beta-lactam antibiotics through mixed gels of cystic fibrosis-derived mucin and Pseudomonas aeruginosa alginate.

The rates of diffusion through purified extracellular alginate from Pseudomonas aeruginosa were measured for twelve beta-lactam antibiotics. The diffusion rate was reduced as the antibiotic molecular weight increased, but the range of diffusion rates exhibited by a common anti-pseudomonal penicillins was relatively small. The diffusion of ticarcillin through 1.0% w/v mixtures of alginate and purified mucus glycoprotein (mucin) from sputa of cystic fibrosis patients showed that, at equivalent concentrations, alginate represented the greater barrier to penetration. However if the mucin concentration was increased to 4.0% w/v, a more realistic physiological concentration, the diffusion of ticarcillin was retarded to a greater extent than in 1% w/v alginate, and the effect was compounded by other sputum components such as DNA. The results suggest that the antibiotic diffusion barrier represented by mucin may be significant in vitro, particularly for nebulized antibiotics.

Alginates↗

PubMed1996

The 5' and 3' splice sites come together via a three dimensional diffusion mechanism.

We present evidence that the splice sites in mammalian pre-mRNAs are brought together via a three dimensional diffusion mechanism. We tested two mechanisms for splice site pairing: a lateral diffusion ('scanning') model and the currently favored three dimensional diffusion ('jumping') model. Two lines of evidence that distinguish between these two models are presented. The first utilized bipartite splicing substrates tethered by double-stranded RNA stems predicted to provide either a moderate or severe block to splice site pairing via a scanning mechanism. Splice site pairing via a jumping mechanism was expected to be unaffected or affected minimally. The second approach utilized a flexible poly(ethylene glycol) moiety within the intron. This insertion was predicted to reduce scanning efficiency but not the efficiency of a three dimensional diffusion mechanism. The best explanation for the data with the bipartite RNAs is that splice site pairing occurs through three dimensional diffusion. Kinetic analysis of the poly(ethylene glycol) containing substrate showed that neither the lag phase nor the initial rates of mRNA production and spliceosome assembly were affected by this insertion. Therefore, both experimental approaches supported the three dimensional diffusion model of splice site pairing.

Base Sequence↗

PubMed2007

Determination of the EC50 amnesic concentration of etomidate and its diffusion profile in brain tissue: implications for in vitro studies.

BACKGROUND: Etomidate is a widely used general anesthetic that has become a useful tool to investigate mechanisms of anesthetic action in vivo and in brain slices. However, the free aqueous concentration of etomidate that corresponds to amnesia in vivo and the diffusion profile of etomidate in brain slices are not known. METHODS: The authors assessed the effect of intraperitoneally injected etomidate on contextual fear conditioning in mice. Etomidate concentrations in brain tissue were obtained by high-performance liquid chromatography. Uptake studies in 400-microm-thick brain slices were used to calculate the diffusion and partition coefficients of etomidate. A diffusion model was used to calculate the expected concentration profile within a brain slice as a function of time and depth. The predicted rate of drug equilibration was compared with the onset of electrophysiologic effects on inhibitory circuit function in recordings from hippocampal brain slices. RESULTS: Etomidate impaired contextual fear conditioning with an ED50 dose of 11.0+/-0.1 mg after intraperitoneal injection, which corresponded to an EC50 brain concentration of 208+/-9 ng/g. The brain:artificial cerebrospinal fluid partition coefficient was 3.35, yielding an EC50,amnesia aqueous concentration of 0.25 microm. The diffusion coefficient was approximately 0.2x10 cm/s. The development of etomidate action in hippocampal brain slices was compatible with the concentration profile predicted by this diffusion coefficient. CONCLUSIONS: The free aqueous concentration of etomidate corresponding to amnesia, as defined by impaired contextual fear conditioning in mice, is 0.25 microM. Diffusion of etomidate into brain slices requires approximately an hour to reach 80% equilibration at a typical recording depth of 100 microm. This information will be useful in designing and interpreting in vitro studies using etomidate.

Amnesia↗