ABSTRACT
The giant (2–3 × 10−2 m long) internodal cells of the aquatic plant, Chara, exhibit a rapid (>100 × 10−6 m s−1) cyclic cytoplasmic streaming which stops in response to mechanical stimuli. Since the streaming – and the stopping of streaming upon stimulation – is easily visible with a stereomicroscope, these single cells are ideal tools to investigate mechanosensing in plant cells, as well as the potential for these cells to be anesthetized. We found that dropping a steel ball (0.88 × 10−3 kg, 6 × 10−3 m in diameter) through a 4.6 cm long tube (delivering ca. 4 × 10−4 J) reliably induced mechanically-stimulated cessation of cytoplasmic streaming. To determine whether mechanically-induced cessation of cytoplasmic streaming in Chara was sensitive to anesthesia, we treated Chara internodal cells to volatilized chloroform in a 9.8 × 10−3 m3 chamber for 2 minutes. We found that low doses (15,000–25,000 ppm) of chloroform did not always anesthetize cells, whereas large doses (46,000 and higher) proved lethal. However, 31,000 ppm chloroform completely, and reversibly, anesthetized these cells in that they did not stop cytoplasmic streaming upon mechanostimulation, but after 24 hours the cells recovered their sensitivity to mechanostimulation. We believe this single-cell model will prove useful for elucidating the still obscure mode of action of volatile anesthetics.
Introduction
Plants, like all sentient organisms, sense and respond to environmental and biological stimuli including light, touch, gravity, electricity, temperature, chemical gradients, and volatile hormones.Citation1,Citation2 Plants are awareCitation1 if not conscious (in a restrictive sense, consciousness requires language).Citation2 Similarly, plants, like all sentient organisms, may be rendered insensitive to stimuli (i.e., they may be anesthetized)Citation3–6 Indeed, Claude BernardCitation7 considered the ability to be anesthetized to be a causa sine qua non for life.
Although the term anesthesia was first proposed by Oliver Wendell Holmes in a letter to W. T. G. Morton on November 21, 1846,Citation8 the effects later to be known as anesthesia were first described by Michael Faraday in 1818.Citation9 The first surgeries performed under anesthesia, a painless tooth extraction, and a tumor removed from the neck were carried out in Boston in September and October of 1846, by W.T.G. Morton and John Collins Warren, respectively.Citation10,Citation11 Shortly thereafter, surgical anesthesia became widespread and, in 1878, Claude Bernard demonstrated that plants could be anesthetized as well.Citation7,Citation12
At present, although it is clear that anesthesia results in loss of consciousness in animals and the inhibition of responsiveness to stimuli in both animals and plants, the mechanism of this inhibition is still obscure. In general, two models have emerged for the mechanism of action of general anesthetics: a physical effect on the plasma membrane; and specific binding to protein targets, specifically neurotransmitter-gated, and voltage-gated ion channels.Citation11
A number of physiological and developmental responses in plants were already demonstrated to be subject to anesthesia in the 19th century.Citation7,Citation12 Recently, the effects of anesthetics on the leaf movement of Mimosa pudica and Drosera capensis, trap closure of Dionaea muscipula, the rotational movement of pea tendrils, and dormancy breaking of Lepidium sativum seeds have been described.Citation6 While a diverse set of plant responses have been shown to be subject to anesthesia, all previous reports describe the effects of anesthetics on complex systems. We believe that a relatively simple, single-cell model may be useful for the elucidation of anesthetics’ mode of action since the site of stimulus, anesthesia, and response upon recovery from anesthesia, must all be in the same cell.
The giant (ca. 3–6 × 10−2 m long and 0.5 × 10−3 m in diameter), single, internodal cells of Chara exhibit a rapid, ca. 100 × 10−6 m s−1,Citation13 actin-myosin basedCitation14 rotational cytoplasmic streaming. Chara internodal cells respond to mechanical stimulation by immediately halting cytoplasmic streaming.Citation15–21 The stopping of cytoplasmic streaming is referred to as mechanically-induced, excitation-cessation coupling (E-C coupling).Citation22 Here, we describe a system for reliably stimulating Chara internodal cells, such that they repeatably stop cytoplasmic streaming, in order to assess whether this well-known physiological response in a single cell is subject to anesthesia. Our goal is to develop a model system which will enable the elucidation of the mechanisms involved in the action of anesthetics.
Methods
Chemicals
Chloroform was purchased from Sigma-Aldrich (St Louis, MO, USA). Concentrations of chloroform in parts per million (ppm) were calculated from the ideal gas law assuming 100% volatilization of chloroform. NaCI; KCl and CaCl2 were purchased from Research Organics (Cleveland, OH, USA).
Culture conditions
Chara corallina Klein ex Willd, em. R.D.W. (=Chara australis R. Brown) was grown in an aquarium containing soil/water medium under continuous fluorescent light at 21–22°C in a temperature-controlled room. The soil contained a low fraction (ca. 3%) of organic matter. Under these conditions, CaCO3 does not deposit on the cell wall and there is not any visible banding pattern, therefore cytoplasmic streaming is easily visible. See Wayne and StavesCitation23 for details of the culture conditions.
Experimental set-up
The subapical (most expanded) internodal cells were used for experiments. Typically, these cells were 3–5 × 10−2 m long and had a diameter of approximately 0.5 × 10−3 m. The internodal cells were removed from the culture by cutting through the internodal cells above and below the selected cell, leaving the nodes intact. To avoid damaging the intact internodal cells, they were manipulated by grasping the fragments remaining from the cut internodal cells with forceps.
Chara internodal cells were placed in a plastic Petri dish containing artificial pond water (APW: 0.1 mol m−3 NaCI; KCl and CaCl2). The cells were allowed to recover for 15–30 minutes after being cut from the cultures before being subjected to any tests.
Mechanostimulation
The internodal cells were removed from the recovery dish and placed in a Plexiglas chamber 10 × 10−2 m long, 2.5 × 10−2 m wide and 0.3 × 10−2 m high containing no medium. The chamber was moved to a stereo microscope (Leica-MS5) where the cells were observed to ensure that cytoplasmic streaming was present and visible. Once streaming was confirmed, the chamber was placed under a rigid plastic tube (inner diameter, 7 × 10−3m) of various lengths and a steel ball (6 × 10−3 m in diameter, 0.88 × 10−3kg) was allowed to fall the length of the tube onto the Chara internodal cell (). The cell chamber was then immediately (ca. 10 s) moved back to the stereomicroscope and the cell was observed to determine whether streaming had stopped (successful mechanostimulation) or continued (unsuccessful mechanostimulation).
Figure 1. Our experimental set up for mechanical stimulation of a single Chara internodal cell. Magnetic balls (6 × 10−3 m in diameter, 0.88 × 10−3 kg) were dropped through tubes (inner diameter = 7 × 10−3 m) of varying lengths.
Anesthesia
To test the efficacy of an anesthetic treatment, internodal cells were subjected to mechanostimulation as described above with the steel ball falling from a height we had determined caused the cessation of cytoplasmic streaming in 100% of test cases without killing the cells. After the initial mechanostimulation (which we verified caused streaming cessation as described above) cells were allowed to recover for several minutes in a Petri dish containing APW. Upon recovery, defined as the resumption of cytoplasmic streaming at normal rates, the cells were placed inside a 9.8 × 10−3 m3 glass vacuum chamber on a disk of filter paper dampened with APW which was placed on top of the top half of a glass Petri dish. The appropriate amount of chloroform was sprayed with a glass syringe onto the surface of a Kimwipe placed in the bottom half of a glass Petri dish on the opposite side of the vacuum chamber (). A small rechargeable fan (Treva) was turned on to speed chloroform volatilization and distribution of the vaporized chloroform in the chamber, and the lid to the vacuum chamber was quickly replaced. After 2 minutes, the cell was removed from the chamber, cytoplasmic streaming was confirmed, within 15 seconds, as described above, and the cell was mechanostimulated as described above.
Figure 2. The set-up for anesthetizing cells. Chara internodal cells were placed inside a 9.8 × 10−3 m3 glass vacuum chamber on a disk of filter paper dampened with APW which was placed on the top of the top half of a glass Petri dish. The appropriate amount of chloroform was sprayed with a glass syringe onto the surface of a Kimwipe placed in the bottom half of a glass Petri dish on the opposite side of the vacuum chamber. A small rechargeable fan (Treva) was turned on to speed the volatilization and distribution of the vaporized chloroform in the chamber.
Recovery from anesthesia
To determine whether anesthetized cells were able to recover sensitivity to mechanical stimulation, anesthetized cells were placed in a Petri dish containing APW for 24 hours. After this time, cells were again mechanostimulated, as described above. If mechanically-induced E-C coupling was observed, the cell was considered to have recovered from the effects of anesthesia.
Statistics
Mean values ± the standard error of the mean are reported for all experiments. The standard error was calculated using the Wald method for determining confidence intervals from unweighted data from Bernoulli trials.Citation24
Results
Cytoplasmic streaming
Under our growth and experimental conditions, we found that, consistent with previous reportsCitation13 Chara internodal cells exhibit rapid (ca. 110–120 × 10−6 m s−1) cytoplasmic streaming (data not shown). The motive force for the streaming is generated from myosin, attached to organelles, moving along actin cables embedded in the gel endoplasm.Citation25 The vacuole, making up the greatest volume of the internodal cells, also exhibits streaming. This is because large, streaming organelles (such as nuclei) have a diameter larger than the thickness of the cytoplasm, thus the tonoplast bulges into the vacuole as the organelles move along the actin cables and the vacuolar contents stream as if they were moved by a peristaltic pump ().
Figure 3. A representation of the rapid (ca.100 × 10−6 m s−1) cytoplasmic streaming in Chara internodal cells. The motive force is generated by myosin attached to organelles which move along actin cables embedded in the gel endoplasm just interior to the stationary chloroplasts.Citation25 Note that single and clumps of nuclei push the tonoplast into the vacuole and their movement acts like a peristaltic pump, causing the contents of the vacuole to stream as well.
Mechanical stimulation
We find that as we increased the length of the tube through which the 6 mm magnetic ball fell there was a dose-dependent response resulting in mechanically-stimulated excitation-cessation coupling, EC-coupling (i.e. the mechanical stimulation caused an excitation which resulted in the cessation of cytoplasmic streaming). A fraction of the cells tested exhibited E-C coupling with a tube length of 3.7 × 10−2 m (3.2 × 10−4 J), while the steel ball falling through a 4.6 × 10−2 m tube (delivering 4 × 10−4 J) reliably induced E-C coupling in 100% of the cells tested (). When the energy associated with the impacting ball exceeded 4.4 × 10−4 J (5.1 × 10−2 m) the impacts were lethal to some (40%) of the cells while by 14.5 × 10−4 J (14.5 × 10−2 m) 100% of the cells were killed by the falling ball.
Figure 4. The effect of tube length on mechanically-induced cessation of cytoplasmic streaming and survival of Chara internodal cells. Steel balls (0.88 × 10−3 kg, 6 × 10−3 m diameter) were dropped through tubes of various lengths and impacted the internodal cells as illustrated in . As the distance the ball dropped increased, the fraction of cells which stopped streaming increased until 100% of the cells stopped streaming at a tube length of 4.6 × 10−2 m. Longer tube lengths resulted in death to some (until 11.9 × 10−2 m) or all (16.8 × 10−2 m and longer) cells. N = 7–8.
Chloroform-induced anesthesia
To determine whether E-C coupling in Chara was subject to anesthesia, we treated Chara internodal cells with volatilized chloroform in a 9.8 × 10−3 m3 vacuum chamber. We find that as we increase the concentration of chloroform, there is a dose-dependent effect on the establishment of anesthesia in Chara internodal cells. After a 2-minute treatment at 23,250 ppm chloroform, the majority (60%) of the cells tested exhibited anesthesia (i.e. cytoplasmic streaming did not stop after being struck with a ball delivering 4 × 10−4 J on impact with the cell). At 31,000 ppm, 100% of the tested cells were anesthetized. After a 2-minute treatment with this concentration of chloroform, 100% of the cells were alive after 24 hours and exhibited E-C coupling when mechanostimulated by a ball delivering 4 × 10−4 J on impact with the cell (i.e. they recovered after anesthesia). After treatment with higher concentrations of chloroform (46,500 ppm) 100% of the cells were anesthetized and living after treatment but none of the cells recovered sensitivity to stimulation or were alive after 24 hours ().
Table 1. The effect of chloroform treatment on the induction of anesthesia of Chara internodal cells as indicated by the continuation of cytoplasmic streaming after a mechanical stimulus. Cells were treated with chloroform as shown in for 2 minutes and then stimulated by a steel ball falling 4.6 × 10−2 m before striking the cell. At 31,000 ppm, 100% of the cells were anesthetized and recovered after 24 hours. At higher concentrations, the cells were anesthetized but were dead within 24 hours. N = 10.
Discussion
We wished to determine whether we could establish a single-cell model for investigating the mechanism of anesthetics. In order to accomplish this, we first had to calibrate their response to a stimulus (mechanical stimulation) in order to, second, determine whether anesthesia reduced or eliminated responsiveness.
Mechanical stimulation
It is well established that it is an action potential that is responsible for mechanically-induced EC coupling in characean intenodal cells. Upon mechanically-induced distortion of the plasma membrane, Chara internodal cells generally generate an action potentialCitation26,Citation27 causing an influx of Ca2+ from the external medium (providing the external Ca2+ concentration is sufficiently highCitation21,Citation27–31) and a consequent increase of the Ca2+ concentration of the cytoplasm. The increased Ca2+ concentration activates a protein kinase which phosphorylates myosin and inhibits its interaction with myosin, immediately halting cytoplasmic streaming and causing the cytoplasm to gel.Citation31,Citation32 This has the obvious adaptive benefit of potentially allowing wound healing and avoiding the loss of the streaming, fluid cytoplasm after wounding.Citation33
We found that dropping a 6 mm magnetic ball through a 4.6 cm tube (delivering 4 × 10−4 J in kinetic energy) reliably induced mechanically-stimulated excitation-cessation coupling (EC-coupling) in non-anesthetized characean cells. ShimmenCitation26 described an apparatus which dropped a 1.3 × 10−3 kg glass tube from various heights onto a Chara internodal cell. He reported that dropping the tube from heights of 1–4 × 10−2 m produced receptor potentials of increasing magnitude, while when the tube was dropped from 5 × 10−2 m (corresponding to a kinetic energy (KE = mass * g * height) of 6.37 × 10−4 J) an action potential was generated. Thus, our results that mechanically-induced EC-coupling needs a minimum kinetic energy stimulation of 4 × 10−4 J are consistent with the results of ShimmenCitation26 and the assumption that a mechanically-induced action potential is essential for the response. These energetic requirements for mechanostimulation are larger than the 1.1 × 10−8 J we reported previously,Citation21 but our previous report used a different stimulation method and, while the results were reproducible, the present method makes fewer assumptions when calculating the energy associated with the falling ball. Staves and WayneCitation21 made the argument that there were at least two different classes of mechanoreceptors in the plasma membrane of Chara based in part on the amount of energy required for gravity sensing and mechanosensing. For gravity sensing, the energy available to a characean internodal cell as a consequence of its orientation in a gravitational field is approximately 10−15 J.Citation34 If 1.1 × 10−8 J were required for sensing mechanostimulation, it is unlikely that the same mechanoreceptor would be active over the range of energies required for both gravity sensing and for sensing mechanical stimulation. Since our data suggest that there is an even larger difference between the energies required for gravity sensing and sensing mechanical stimulation, the suggestion that the mechanoreceptors involved in the two processes are differentCitation21 is strengthened.
Anesthesia
We find that 1 × 10−6 m3 (1 ml) of chloroform, volatilized in a 9.8 × 10−3 m3 vacuum chamber (31,000 ppm), completely, and reversibly, induced anesthesia (i.e. inhibited the mechanically-induced cessation of cytoplasmic streaming) in Chara internodal cells. To our knowledge, this is the first report of anesthesia in single plant cells. Since, under our culture conditions, cytoplasmic streaming is easily visible with a stereomicroscope, and calibrated mechanical stimulations of the large, non-corticating, internodal cells of Chara induce an immediate stopping of streaming, we believe that these single cells are ideal tools to investigate the mechanisms of chloroform-based and related anesthetics. Since it has been demonstrated that the generation of an action potential is essential for E-C couplingCitation21,Citation26 we assume, and must confirm, that chloroform anesthesia prevents the generation of an action potential.
It is interesting to note that the time of exposure and concentration of chloroform required to anesthetize Chara internodal cells is similar to that required for human anesthesia (a few minutes 22,500 ppm).Citation35 Indeed, it appears that organisms from bacteria to yeast, and people to plants are all subject to anesthesia within a relative narrow (one order of magnitude) range of concentrations of volatile anesthetics.Citation36 This has led to the suggestion that anesthetic responsiveness may date back to the Last Common AncestorCitation37 and thus may be as fundamental to life as glycolysis and the genetic apparatus. If so, Claude BernardCitation7 was prescient when, writing of volatile anesthetics, he claimed “what is alive must sense and can be anesthetized, the rest is dead.”
In previous work, in which chloroform was applied directly to characean (Chara or Nitella) cells, Kiihne,Citation38 Ewart,Citation16 Osterhout and Harris,Citation39 and NicholsCitation40 reported that cytoplasmic streaming stopped, and cells were often killed in response to the treatment. This is likely the result of the chloroform permeabilizing the plasma membrane. With short-term and localized treatment of chloroform, Ca2+ from the external medium could move into the cell down its electrochemical gradient and induce a cessation of cytoplasmic streaming.Citation31,Citation32 Longer term stopping of cytoplasmic streaming probably reflected more cellular damage and dead cells.
Through his work with animals and increasing exposure time to volatile anesthetics, Claude BernardCitation12 recognized three stages of anesthesia. In the first stage, the animal became unconscious and no longer felt pain or perceived cold, but all vital functions continued (the condition we consider is achieved under surgical general anesthesia). In the second stage, respiratory movements stopped and in the third stage, heart beating and ciliary movement ceased.Citation41 The treatments we describe in this paper result in an anesthetized state equivalent to Bernard’s first stage. The results of the treatments of Kiihne,Citation38 Ewart,Citation16 Osterhout and Harris,Citation39 and NicholsCitation40 appear to be equivalent to Bernard’s third stage, or death.
Regarding the controversy over the mechanism of volatile anesthetics,Citation11 our finding that Chara internodal cells could be anesthetized without stopping cytoplasmic streaming indicates that chloroform’s mode of action is not simply causing the plasma membrane to become permeable to ions. If this were the case, streaming would immediately stop upon the influx of Ca2+ from the external medium.Citation31,Citation32 The general consensus currently supports the receptor theory for the mechanism of anesthesia, wherein the anesthetic binds to a receptor on the plasma membrane.Citation11,Citation42,Citation43 However, the wide variety of volatile anestheticsCitation11,Citation42 which affect a wide variety of responses in essentially all organismsCitation4,Citation11,Citation44 and the finding that general and local anesthetics all cause depression of the freezing point of transitions in biomembranes (an effect reversed by applying hydrostatic pressure)Citation45 argues against the anesthetic receptor theory.Citation6 Membrane proteins (muscarinic and nicotinic receptors, NMDA receptors, 5-HT receptors, GABA receptors, and potassium channels)Citation46 have, indeed, been shown to be targets of anesthetics. However, it is possible that anesthetics may act indirectly on these proteins by changing the properties of the membrane, perhaps the thickness of the lipid bilayer.Citation6,Citation47 Our results are consistent with, but do not confirm, this model.
In summary, we developed a carefully calibrated mechanical stimulation and showed that the giant internodal cells of characean algae can be anesthetized by concentrations of chloroform previously shown to induce anesthesia in animal cells. We have provided evidence that chloroform-induced anesthesia in Chara does not simply arise from a loss of integrity of the plasma membrane. Considering that the question of the mechanism of anesthesia is not settled, we propose that these large internodal cells are ideal subjects for further studies on the mechanism of action of anesthetics.
Acknowledgments
The authors would like to acknowledge and thank Sheila A. Blackman for her careful review and lively discussion of the manuscript and Ian Staves for his insight in determining confidence intervals from Bernoulli trials.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
References
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