Scaling Relationship and Optimization of Double-Pulse Electroporation

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.     

Scaling Relationship and Optimization of Double-Pulse Electroporation

The efficacy of electroporation is known to vary significantly across a wide variety of biological research and clinical applications, but as of this writing, a generalized approach to simultaneously improve efficiency and maintain viability has not been available in the literature. To address that discrepancy, we here outline an approach that is based on the mapping of the scaling relationships among electroporation-mediated molecular delivery, cellular viability, and electric pulse parameters. The delivery of Fluorescein-Dextran into 3T3 mouse fibroblast cells was used as a model system. The pulse was rationally split into two sequential phases: a first precursor for permeabilization, followed by a second one for molecular delivery. Extensive data in the parameter space of the second pulse strength and duration were collected and analyzed with flow cytometry. The fluorescence intensity correlated linearly with the second pulse duration, confirming the dominant role of electrophoresis in delivery. The delivery efficiency exhibited a characteristic sigmoidal dependence on the field strength. An examination of short-term cell death using 7-Aminoactinomycin D demonstrated a convincing linear correlation with respect to the electrical energy. Based on these scaling relationships, an optimal field strength becomes identifiable. A model study was also performed, and the results were compared with the experimental data to elucidate underlying mechanisms. The comparison reveals the existence of a critical transmembrane potential above which delivery with the second pulse becomes effective. Together, these efforts establish a general route to enhance the functionality of electroporation.     

Concentrations of circulating hormones during the interval between pulses of a PGF2α metabolite in mares and heifers

The temporal relationship of several hormones to a metabolite of prostaglandin F2α (PGFM) was studied in mares and heifers from the beginning of the first PGFM pulse during luteolysis to the end of the second pulse. Mares (n=7) were selected with a 9-h interval between the peaks of the two pulses. In mares, estradiol-17β (estradiol) increased (P<0.05) within each PGFM pulse and plateaued for a mean of 6h between the pulses, resulting in a stepwise estradiol increase. Progesterone decreased linearly (P<0.0001) throughout the intra-pulse and inter-pulse intervals of PGFM. In heifers (n=6), inter-pulse intervals were variable, and therefore Hours 1-4 of the first pulse (Hour 0=PGFM peak) and Hours -4 to -1 of the second pulse were used to represent the mean 8-h interval between peaks of the two pulses. Estradiol increased (P<0.05) during the ascending portion of each PGFM pulse and then decreased (P<0.05) beginning at Hour -1 of the first PGFM pulse and Hour 0 of the second pulse. The 1-h delay during the second pulse was accompanied by an apparent increase in PRL. A transient decrease in estradiol occurred in individuals between PGFM pulses at a mean of 5h after the first PGFM peak, concomitant with a transient LH increase (P<0.05). Results indicated that estradiol plateaued in mares and fluctuated in heifers during the interval between PGFM pulses. Heifers also showed temporal relationships between estradiol and LH and apparently between estradiol and PRL.     

Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores.

It has been shown recently that electrically induced DNA transfer into cells is a fast vectorial process with the same direction as DNA electrophoresis in an external electric field (Klenchin, V. A., S. I. Sukharev, S. M. Serov, L. V. Chernomordik, and Y. A. Chizmadzhev. 1991. Biophys. J. 60:804-811). Here we describe the effect of DNA interaction with membrane electropores and provide additional evidences for the key role of DNA electrophoresis in cell electrotransfection. The assay of electrically induced uptake of fluorescent dextrans (FDs) by cells shows that the presence of DNA in the medium during electroporation leads to a sharp increase in membrane permeability to FDs of M(r) < 20,000. The permeability increases with DNA concentration and the effect is seen even if FD is added to the cell suspension a few minutes after pulse application. The longer the DNA fragment, the greater the increase in permeability. The use of a two-pulse technique allows us to separate two effects provided by a pulsed electric field: membrane electroporation and DNA electrophoresis. The first pulse (6 kV/cm, 10 microseconds) creates pores efficiently, whereas transfection efficiency (TE) is low. The second pulse of much lower amplitude, but substantially longer (0.2 kV/cm, 10 ms), does not cause poration and transfection by itself but enhances TE by about one order of magnitude. In two-pulse experiments, TE rises monotonously with the increase of the second pulse duration. By varying the delay duration between the two pulses, we estimate the lifetime of electropores (which are DNA-permeable in conditions of low electric field) as tens of seconds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanistic analysis of electroporation-induced cellular uptake of macromolecules

Pulsed electric field has been widely used as a nonviral gene delivery platform. The delivery efficiency can be improved through quantitative analysis of pore dynamics and intracellular transport of plasmid DNA. To this end, we investigated mechanisms of cellular uptake of macromolecules during electroporation. In the study, fluorescein isothiocyanate-labeled dextran (FD) with molecular weight of 4,000 (FD-4) or 2,000,000 (FD-2000) was added into suspensions of a murine mammary carcinoma cell (4T1) either before or at different time points (ie, 1, 2, or 10 sec) after the application of different pulsed electric fields (in high-voltage mode: 1.2-2.0 kV in amplitude, 99 microsec in duration, and 1-5 pulses; in low-voltage mode: 100-300 V in amplitude, 5-20 msec in duration, and 1-5 pulses). The intracellular concentrations of FD were quantified using a confocal microscopy technique. To understand transport mechanisms, a mathematical model was developed for numerical simulation of cellular uptake. We observed that the maximum intracellular concentration of FD-2000 was less than 3% of that in the pulsing medium. The intracellular concentrations increased linearly with pulse number and amplitude. In addition, the intracellular concentration of FD-2000 was approximately 40% lower than that of FD-4 under identical pulsing conditions. The numerical simulations predicted that the pores larger than FD-4 lasted <10 msec after the application of pulsed fields if the simulated concentrations were on the same order of magnitude as the experimental data. In addition, the simulation results indicated that diffusion was negligible for cellular uptake of FD molecules. Taken together, the data suggested that large pores induced in the membrane by pulsed electric fields disappeared rapidly after pulse application and convection was likely to be the dominant mode of transport for cellular uptake of uncharged macromolecules.     

A general odorant background affects the coding of pheromone stimulus intermittency in specialist olfactory receptor neurones

In nature the aerial trace of pheromone used by male moths to find a female appears as a train of discontinuous pulses separated by gaps among a complex odorant background constituted of plant volatiles. We investigated the effect of such background odor on behavior and coding of temporal parameters of pheromone pulse trains in the pheromone olfactory receptor neurons of Spodoptera littoralis. Effects of linalool background were tested by measuring walking behavior towards a source of pheromone. While velocity and orientation index did drop when linalool was turned on, both parameters recovered back to pre-background values after 40 s with linalool still present. Photo-ionization detector was used to characterize pulse delivery by our stimulator. The photo-ionization detector signal reached 71% of maximum amplitude at 50 ms pulses and followed the stimulus period at repetition rates up to 10 pulses/s. However, at high pulse rates the concentration of the odorant did not return to base level during inter-pulse intervals. Linalool decreased the intensity and shortened the response of receptor neurons to pulses. High contrast (>10 dB) in firing rate between pulses and inter-pulse intervals was observed for 1 and 4 pulses/s, both with and without background. Significantly more neurons followed the 4 pulses/s pattern when delivered over linalool; at the same time the information content was preserved almost to the control values. Rapid recovery of behavior shows that change of perceived intensity is more important than absolute stimulus intensity. While decreasing the response intensity, background odor preserved the temporal parameters of the specific signal.     

Short-term synaptic plasticity in the dentate gyrus of monkeys

The hippocampus plays an important role in learning and memory. Synaptic plasticity in the hippocampus, short-term and long-term, is postulated to be a neural substrate of memory trace. Paired-pulse stimulation is a standard technique for evaluating a form of short-term synaptic plasticity in rodents. However, evidence is lacking for paired-pulse responses in the primate hippocampus. In the present study, we recorded paired-pulse responses in the dentate gyrus of monkeys while stimulating to the medial part of the perforant path at several inter-pulse intervals (IPIs) using low and high stimulus intensities. When the stimulus intensity was low, the first pulse produced early strong depression (at IPIs of 10-30 ms) and late slight depression (at IPIs of 100-1000 ms) of field excitatory postsynaptic potentials (fEPSPs) generated by the second pulse, interposing no depression IPIs (50-70 ms). When the stimulus intensity was high, fEPSPs generated by the second pulse were depressed by the first pulse at all IPIs except for the longest one (2000 ms). Population spikes (PSs) generated by the second pulse were completely blocked or strongly depressed at shorter IPIs (10-100 or 200 ms, respectively), while no depression or slight facilitation occurred at longer IPIs (500-2000 ms). Administration of diazepam slightly increased fEPSPs, while it decreased PSs produced by the first pulse. It also enhanced the facilitation of PSs produced by the second stimulation at longer IPIs. The present results, in comparison with previous studies using rodents, indicate that paired-pulse responses of fEPSPs in the monkey are basically similar to those of rodents, although paired-pulse responses of PSs in the monkey are more delayed than those in rodents and have a different sensitivity to diazepam.     

Light-Induced resetting of the circadian pacemaker: quantitative analysis of transient versus steady-state phase shifts.

The suprachiasmatic nuclei of the hypothalamus contain the major circadian pacemaker in mammals, driving circadian rhythms in behavioral and physiological functions. This circadian pacemaker's responsiveness to light allows synchronization to the light-dark cycle. Phase shifting by light often involves several transient cycles in which the behavioral activity rhythm gradually shifts to its steady-state position. In this article, the authors investigate in Syrian hamsters whether a phase-advancing light pulse results in immediate shifts of the PRC at the next circadian cycle. In a first series of experiments, the authors aimed a light pulse at CT 19 to induce a phase advance. It appeared that the steady-state phase advances were highly correlated with activity onset in the first and second transient cycle. This enabled them to make a reliable estimate of the steady-state phase shift induced by a phase-advancing light pulse on the basis of activity onset in the first transient cycle. In the next series of experiments, they presented a light pulse at CT 19, which was followed by a second light pulse aimed at the delay zone of the PRC on the next circadian cycle. The immediate and steady-state phase delays induced by the second light pulse were compared with data from a third experiment in which animals received a phase-delaying light pulse only. The authors observed that the waveform of the phase-delay part of the PRC (CT 12-16) obtained in Experiment 2 was virtually identical to the phase-delay part of the PRC for a single light pulse (obtained in Experiment 3). This finding allowed for a quantitative assessment of the data. The analysis indicates that the delay part of the PRC-between CT 12 and CT 16-is rapidly reset following a light pulse at CT 19. These findings complement earlier findings in the hamster showing that after a light pulse at CT 19, the phase-advancing part of the PRC is immediately shifted. Together, the data indicate that the basis for phase advancing involves rapid resetting of both advance and delay components of the PRC.     

Thermal equilibration between the M and N intermediates in the photocycle of bacteriorhodopsin.

The stages in the photocycle of bacteriorhodopsin (BR) involving the M and N intermediates are investigated using a double pulse excitation method. A first (cycling) pulse at 532 nm is followed, with an appropriate time delay, by a second pulse (337, 406, 446, or 470 nm) which induces the M-->BR back-photoreaction. After depletion by the second pulse a repopulation of M in the millisecond range is observed which is interpreted in terms of a thermal N-->M relaxation. It is thus concluded that a (thermal) M<-->N equilibrium accounts for the biphasic decay of M in the BR photocycle. Other models for this stage of the light-driven proton-pump are therefore unnecessary.     

Thresholds for Phosphatidylserine Externalization in Chinese Hamster Ovarian Cells following Exposure to Nanosecond Pulsed Electrical Fields (nsPEF)

High-amplitude, MV/m, nanosecond pulsed electric fields (nsPEF) have been hypothesized to cause nanoporation of the plasma membrane. Phosphatidylserine (PS) externalization has been observed on the outer leaflet of the membrane shortly after nsPEF exposure, suggesting local structural changes in the membrane. In this study, we utilized fluorescently-tagged Annexin V to observe the externalization of PS on the plasma membrane of isolated Chinese Hamster Ovary (CHO) cells following exposure to nsPEF. A series of experiments were performed to determine the dosimetric trends of PS expression caused by nsPEF as a function of pulse duration, τ, delivered field strength, E_D, and pulse number, n. To accurately estimate dose thresholds for cellular response, data were reduced to a set of binary responses and ED50s were estimated using Probit analysis. Probit analysis results revealed that PS externalization followed the non-linear trend of (τ*E_D ²)⁻¹ for high amplitudes, but failed to predict low amplitude responses. A second set of experiments was performed to determine the nsPEF parameters necessary to cause observable calcium uptake, using cells preloaded with calcium green (CaGr), and membrane permeability, using FM1-43 dye. Calcium influx and FM1-43 uptake were found to always be observed at lower nsPEF exposure parameters compared to PS externalization. These findings suggest that multiple, higher amplitude and longer pulse exposures may generate pores of larger diameter enabling lateral diffusion of PS; whereas, smaller pores induced by fewer, lower amplitude and short pulse width exposures may only allow extracellular calcium and FM1-43 uptake.     

Quantitative study of molecular transport due to electroporation: uptake of bovine serum albumin by erythrocyte ghosts.

Electroporation is believed to involve the creation of aqueous pathways in lipid bilayer membranes by transient elevation of the transmembrane voltage to approximately 1 V. Here, results are presented for a quantitative study of the number of bovine serum albumin (BSA) molecules transported into erythrocyte ghosts caused by electroportion. 1) Uptake of BSA was found to plateau at high field strength. However, this was not necessarily an absolute maximum in transport. Instead, it represented the maximum effect of increasing field strength for a particular pulse protocol. 2) Maximum uptake under any conditions used in this study corresponded to approximately one-fourth of apparent equilibrium with the external solution. 3) Multiple and longer pulses each increased uptake of BSA, where the total time integral of field strength correlated with uptake, independent of inter-pulse spacing. 4) Pre-pulse adsorption of BSA to ghost membranes appears to have increased transport. 5) Most transport of BSA probably occurred by electrically driven transport during pulses; post-pulse uptake occurred, but to a much lesser extent. Finally, approaches to increasing transport are discussed.     

A long-lived fusogenic state is induced in erythrocyte ghosts by electric pulses

Treatment of erythrocyte ghosts in random positions in a suspension with membrane fusion-inducing direct current electric field pulses causes the membranes to become fusogenic. Significant fusion yields are observed if the membranes are dielectrophoretically aligned into membrane-membrane contact with a weak alternating electric field as much as 5 min after the application of the pulses. This demonstrates that a long-lived membrane structural alteration is involved in this fusion mechanism. Other experiments indicate that the areas on the membrane which become fusogenic after treatment with the pulses may be very highly localized. The locations of these fusogenic areas coincide with where the trans-membrane electric field strength was greatest during the pulse. The fusogenic membrane alteration, or components thereof, in these areas laterally diffuses very slowly or not at all, or, to be fusogenic, must be present at concentrations in the membrane above a certain threshold. The loss of soluble 0.9-3-nm-diameter fluorescent probes from resealed cytoplasmic compartments of randomly positioned erythrocyte ghosts occurs through electric field pulse-induced pores only during a pulse but not between pulses or after a train of pulses if the probe diameter is 1.2 nm or greater. For a given pulse treatment of membranes in random positions in suspensions, an increase in ionic strength of the medium results in (a) a decrease in loss during the pulse, (b) no difference in loss between pulses, and (c) an increase in fusion yield when membrane-membrane contact is established. The latter two results (b and c) are incompatible with a fusion mechanism that proposes a simple relationship between electric field-induced pores and fusion.     

Species Recognition During Substrate-Borne Communication in <Emphasis Type="Italic">Nezara viridula</Emphasis> (L.) (Pentatomidae: Heteroptera)

The information code in the temporal and spectral characteristics of the substrate-borne communication signals produced by insects has been primarily studied in insects in the suborder Auchenorrhyncha. In the present study we investigated which of the female calling song (FCS) parameters in Nezara viridula (L.) (Heteroptera, Pentatomidae) are essential for recognition by conspecific males. In playback experiments we measured male vibrational responsiveness to FCS signals varying in the durations of pulse trains and inter-pulse train intervals, repetition times, duty cycles, and dominant frequencies, and determined the preference range for each specific parameter. Males were able to distinguish songs of different temporal and frequency parameters and responded best to values characteristic of the song of conspecific females. Signal recognition is achieved on the basis of two temporal filters tuned to the durations of the pulse train and inter-pulse train interval. Males responded best to the dominant frequency characteristic of conspecific songs, which are tuned to the resonant properties of the herbaceous plants used for intraspecific signal transmission during communication.     

A delay in membrane fusion: lag times observed by fluorescence microscopy of individual fusion events induced by an electric field pulse

Low light level video microscopy of the fusion of DiI- (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) labeled rabbit erythrocyte ghosts with unlabeled rabbit erythrocyte ghosts, held in stable apposition by dielectrophoresis in sodium phosphate buffers, showed reproducible time intervals (delays) between the application of a single fusogenic electric pulse and the earliest detection of fluorescence in the unlabeled adjacent membranes. The delay increased over the range 0.3-4 s with a decrease in (i) the electric field strength of the fusion-inducing pulse from 1000 to 250 V/mm, (ii) the decay half-time of the fusogenic pulse in the range 1.8-0.073 ms, and (iii) the dielectrophoretic force which brings the membranes into close apposition. A change in the buffer viscosity from 1.8 to 10 mP.s caused the delay to increase from 0.36 to 3.7 s (in glycerol solutions) or to 5.2 s (in sucrose solutions). The delay decreased 2-3 times with an increase in temperature from 21 to 37 degrees C. It did not differ significantly for "white" ghosts [0.013 mM hemoglobin (Hb)] or "red" ghosts (0.15 mM Hb) or buffer strength over the range 5-60 mM (sodium phosphate, pH 8.5). The calculated activation energy, 17 kcal/mol, does not depend on the field strength. The yield of fused cells was high when the delay was short. The delay in electrofusion resembles the delays in pH-dependent fusion of vesicular stomatitis viruses with erythrocyte ghosts [Clague, M. J., Schoch, C., Zech, L., & Blumenthal, R. (1990) Biochemistry 29, 1303-1308] and of fibroblasts expressing influenza hemagglutinin and red blood cells [Morris, S. J., Sarkar, D.P., White, J. M., & Blumenthal, R. (1989) J. Biol. Chem. 264, 3972-3978].(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of feedback during singing and flight in Drosophila melanogaster

ABSTRACT. During Drosophila courtship 'pulse song', muscle potentials occur at two points during the cycle of neuromuscular events which result in a sound pulse being produced. The dorsal longitudinal, second and third dorsal ventral and axillary muscles show potentials 18 ms before each sound pulse while the first dorsal ventral, basalar and sternobasalar muscles fire 3 ms after the onset of each pulse. The timing of these events remains unaltered in animals with the antennae removed, indicating that acoustic feedback is not an important factor. Courting vestigial flies, in the absence of detectable wing base movements, produce indirect muscle potentials at the appropriate song inter-pulse intervals. Thus proprioceptive feedback is also unimportant in determining the intervals between pulses. During putative 'sine song', 'pulse song' and flight in vestigial flies, however, the timing of basalar muscle potentials is abnormal. Also, if the wing is driven externally at a frequency different from that of normal flight, basalar and, to a lesser extent, first dorsal ventral muscles, are phase locked to the driving frequency. These two results suggest that the timing of those muscles which fire at the beginning of the sound pulses is set by proprioceptive feedback. A model of song production is proposed which takes into account the data from this and from previously published papers.     

2-ns Electrostimulation of Ca2+ Influx into Chromaffin Cells: Rapid Modulation by Field Reversal

Cellular effects of nanosecond-pulsed electric field exposures can be attenuated by an electric field reversal, a phenomenon called bipolar pulse cancellation. Our investigations of this phenomenon in neuroendocrine adrenal chromaffin cells show that a single 2-ns, 16 MV/m unipolar pulse elicited a rapid, transient rise in intracellular Ca²⁺ levels due to Ca²⁺ influx through voltage-gated calcium channels. The response was eliminated by a 2-ns bipolar pulse with positive and negative phases of equal duration and amplitude and fully restored (unipolar-equivalent response) when the delay between each phase of the bipolar pulse was 30 ns. Longer interphase intervals evoked Ca²⁺ responses that were greater in magnitude than those evoked by a unipolar pulse (stimulation). Cancellation was also observed when the amplitude of the second (negative) phase of the bipolar pulse was half that of the first (positive) phase but progressively lost as the amplitude of the second phase was incrementally increased above that of the first phase. When the amplitude of the second phase was twice that of the first phase, there was stimulation. By comparing the experimental results for each manipulation of the bipolar pulse waveform with analytical calculations of capacitive membrane charging/discharging, also known as accelerated membrane discharge mechanism, we show that the transition from cancellation to unipolar-equivalent stimulation broadly agrees with this model. Taken as a whole, our results demonstrate that electrostimulation of adrenal chromaffin cells with ultrashort pulses can be modulated with interphase intervals of tens of nanoseconds, a prediction of the accelerated membrane discharge mechanism not previously observed in other bipolar pulse cancellation studies. Such modulation of Ca²⁺ responses in a neural-type cell is promising for the potential use of nanosecond bipolar pulse technologies for remote electrostimulation applications for neuromodulation.     

A systematic study of field inversion gel electrophoresis.

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model.     

Modeling electroporation in a single cell

Electroporation uses electric pulses to promote delivery of DNA and drugs into cells. This study presents a model of electroporation in a spherical cell exposed to an electric field. The model determines transmembrane potential, number of pores, and distribution of pore radii as functions of time and position on the cell surface. For a 1-ms, 40 kV/m pulse, electroporation consists of three stages: charging of the cell membrane (0-0.51 micros), creation of pores (0.51-1.43 micros), and evolution of pore radii (1.43 micros to 1 ms). This pulse creates approximately 341,000 pores, of which 97.8% are small ( approximately 1 nm radius) and 2.2% are large. The average radius of large pores is 22.8 +/- 18.7 nm, although some pores grow to 419 nm. The highest pore density occurs on the depolarized and hyperpolarized poles but the largest pores are on the border of the electroporated regions of the cell. Despite their much smaller number, large pores comprise 95.3% of the total pore area and contribute 66% to the increased cell conductance. For stronger pulses, pore area and cell conductance increase, but these increases are due to the creation of small pores; the number and size of large pores do not increase.     

Rapid optimization of electroporation conditions for plant cells,protoplasts, and pollen

The optimization of electroporation conditions for maximal uptake of DNA during direct gene transfer experiments is critical to achieve high levels of gene expression in transformed plant cells. Two stains, trypan blue and fluorescein diacetate, have been applied to optimize electroporation conditions for three plant cell types, using different square wave and exponential wave electroporation devices. The different cell types included protoplasts from tobacco, a stable mixotrophic suspension cell culture from soybean with intact cell walls, and germinating pollen from alfalfa and tobacco. Successful electroporation of each of these cell types was obtained, even in the presence of an intact cell wall when conditions were optimized for the electroporation pulse. The optimal field strength for each of these cells differs, protoplasts having the lowest optimal pulse field strength, followed by suspension cells and finally germinating pollen requiring the strongest electroporation pulse. A rapid procedure is described for optimizing electroporation parameters using different types of cells from different plant sources.     

Effects of odor flux and pulse rate on chemosensory tracking in turbulent odor plumes by the blue crab, Callinectes sapidus

The ability of animals to track through chemical plumes is often related to properties of evanescent odor bursts and to small-scale mixing process that determine burst properties. However, odor plumes contain variation over a range of scales, and little is known about how variation in the properties of the odor signal on the scale of one to several seconds affects foraging performance. We examined how flux and pulse rate interact to modulate the search behavior of blue crabs, Callinectes sapidus, locating odor sources in controlled flume flows. Experimental treatments consisted of continuous plumes and plumes with discrete odor pulses at intervals of 2.5 s and 4 s at two fluxes. Crabs experienced diminished search success and reduced search efficiency as flux decreased and the inter-pulse interval lengthened. There often were significant interactions between flux and pulse length, and neither property completely determined search behavior. Thus, over the time span of several seconds, the blue crab chemosensory system is not a simple flux detector. The sensitivity of blue crabs to inter-pulse intervals in the range of several seconds indicates that larger-scale mixing processes, which create odor variation on comparable scales, may exert a significant impact on foraging success in nature.