Fungi anaesthesia

Abstract

Electrical activity of fungus Pleurotus ostreatus is characterised by slow (h) irregular waves of baseline potential drift and fast (min) action potential likes spikes of the electrical potential. An exposure of the myceliated substrate to a chloroform vapour lead to several fold decrease of the baseline potential waves and increase of their duration. The chloroform vapour also causes either complete cessation of spiking activity or substantial reduction of the spiking frequency. Removal of the chloroform vapour from the growth containers leads to a gradual restoration of the mycelium electrical activity.

Full text

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Fungi anaesthesia
Andrew Adamatzky1* & Antoni Gandia2
Electrical activity of fungus Pleurotus ostreatus is characterised by slow (h) irregular waves of baseline
potential drift and fast (min) action potential likes spikes of the electrical potential. An exposure of the
myceliated substrate to a chloroform vapour lead to several fold decrease of the baseline potential
waves and increase of their duration. The chloroform vapour also causes either complete cessation of
spiking activity or substantial reduction of the spiking frequency. Removal of the chloroform vapour
from the growth containers leads to a gradual restoration of the mycelium electrical activity.
Most living cells are sensitive to anaesthetics1–3. First experiments on anaesthesia of plants have be done by
Claude Bernard in late 1800s4. Later experiments on amoeba5 shown that weak concentration of narcotics causes
the amoebae to spread out and propagate in a spread condition while narcotic concentrations led to cessation
of movements. During last century the experimental evidences mounted up including anaesthesia of yeasts1,
various aquatic invertebrates6, plants3,7, protists8, bronchial ciliated cells9. A general consensus now is that any
living substrate can be anaesthetised3. e question remains, however, how exactly species without a nervous
system would respond to exposure to anaesthetics.
In present paper we focus on fungi anaesthesia. Why fungi? Fungi are the largest, most widely distributed, and
oldest group of living organisms10. Smallest fungi are microscopic single cells. e largest (15 hectares) mycelium
belongs to Armillaria gallica (synonymous with A. bulbosa, A. inata, and A. lutea)11 and the largest fruit body
belongs to Phellinus ellipsoideus (formerly Fomitiporia ellipsoidea) which weighs half-a-ton12.
Fungi exhibit a high degree of protocognitive abilities. For example, they are capable for ecient exploration
of conned spaces13–17. Moreover, optimisation of the mycelial network18 is similar to that of the slime mould
Physarum polycephalum19 and transport networks20. erefore, we can speculate that the fungi can solve the
same range of computational problems as P. polycephalum21, including shortest path22–26, Voronoi diagram27,
Delaunay triangulation, proximity graphs and spanning tree, concave hull and, possibly, convex hull, and, with
some experimental eorts, travelling salesman problem28. e fungi’s protocognitive abilities and computational
potential make them fruitful substrates for anaesthesia because they might show us how non-neuron awareness
is changing under eects of narcotics.
We use extracellular electrical potential of mycelium as indicator of the fungi activity. Action potential-like
spikes of electrical potential have been discovered using intra-cellular recording of mycelium of Neurospora
crassa29 and further conrmed in intra-cellular recordings of action potential in hyphae of Pleurotus ostreatus
and A. gallica30 and in extra-cellular recordings of fruit bodies of and substrates colonised by mycelium of P.
ostreatus31. While the exact nature of the travelling spikes remains uncertain we can speculate, by drawing analo-
gies with oscillations of electrical potential of slime mouldPhysarum polycephalum32–35, that the spikes in fungi
are triggered by calcium waves, reversing of cytoplasmic ow, translocation of nutrients and metabolites. Stud-
ies of electrical activity of higher plants can brings us even more clues. us, the plants use the electrical spikes
for a long-distance communication aimed to coordinate the activity of their bodies36–38. e spikes of electrical
potential in plants relate to a motor activity39–42, responses to changes in temperature43, osmotic environment44,
and mechanical stimulation45,46.
e paper is structured as follows. We present the experimental setup in “Methods” section. Analysis of the
electrical activity of the intact non-anesthetised and anaesthetised fungi is given in “Results” section. “Discus-
sion” section present some critique and directions for further research.
Methods
A commercial strain of the fungus Pleurotus ostreatus (collection code 21-18, Mogu S.r.l., Italy), previously
selected for its superior tness growing on the targeted substrate, was cultured on sterilised hemp shives con-
tained in plastic (PP5) lter patch microboxes (SacO2, Belgium) that were kept in darkness at ambient room
temperature c.22
◦
C. Aer one week of incubation, a hemp brick well colonised by the fungus was manually
crumbled and spread on rectangular fragments, c.
12
×
12 cm2
, of moisturised non-woven hemp pads. When
these fragments were colonised, as visualised by white and healthy mycelial growth on surface, they were used
for experiments.
OPEN
1Unconventional Computing Laboratory, UWE, Bristol, UK. 2Institute for Plant Molecular and Cell Biology,
CSIC-UPV, Valencia, Spain. *email: andrew.adamatzky@uwe.ac.uk
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Electrical activity of the colonised hemp pads was recorded using pairs of iridium-coated stainless steel
sub-dermal needle electrodes (Spes Medica S.r.l., Italy), with twisted cables and ADC-24 (Pico Technology,
UK) high-resolution data logger with a 24-bit A/D converter. To keep electrodes stable we have been placing
a polyurethane pad under the fabric. e electrodes were arranged in a line (Fig.1a,b). e pairs of electrodes
were pierced through the fabric and into the polyurethane pad.
e fungal substrates pierced with electrodes was placed into 20cm by 10cm by 10cm plastic boxes with
tight lids.
We recorded electrical activity at one sample per second. During the recording, the logger has been doing
as many measurements as possible (typically up to 600 per second) and saving the average value. We set the
acquisition voltage range to 156mV with an oset accuracy of 9
µ
V at 1Hz to maintain a gain error of 0.1%.
Each electrode pair was considered independent with the noise-free resolution of 17 bits and conversion time
of 60ms. Each pair of electrodes, called channels, reported a dierence of the electrical potential between the
electrodes. Distance between electrodes was 1-2cm. In each trial, we recorded eight electrode pairs, channels,
simultaneously.
To study the eect of chloroform we soaked a piece of lter paper c. 4cm by 4cm in chloroform (Sigma
Aldrich, analytical standard) and placed the piece of paper inside the plastic container with the recorded fungal
substrate.
e humidity of the fungal colonies was 70–80% (MerlinLaser Protimeter, UK). e experiments were con-
ducted in a room with ambient temperature 21
◦
C and in the darkness of protective growing tents (Fig.1c).
We have conducted ten experiments, in each experiments we recorded electrical activity of the fungi via eight
channels, i.e. 80 recordings in total.
Figure1. Experimental setup. (a, b)Exemplar locations of electrodes. (a)Top view. (b)Side view. (c)Setup in
the grow tent.
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Results
Myceliated hemp pad exhibit patterns of electrical activity similar to that of spiking neural tissue. Examples of
action potential like spikes, solitary and in trains, are shown in Fig.2.
Application of the chloroform to the container with fungi substantially aected the electrical activity of the
fungi. An example of an extreme, i.e. where almost all electrical activity of mycelium seized, response is shown
in Fig.3. In this example, the introduction of the chloroform leads to the suppression of the spiking activity and
reduction of deviation in values of the electrical potential dierences recorded on the channels.
e intact, non-anesthetised, mycelium composite (control ) shows median amplitude of the irregular move-
ments of the baseline potential is 0.45mV (average 0.64mV,
σ=0.64
), median duration 29,850s (average
67,507s,
σ
=
29,850
). Aer exposure to chloroform the baseline potential movements show median amplitude
reduced to 0.16mV (average 0.18mV,
σ=0.12
) and median duration increased to 38,507s (average 38,114s,
σ
=
38,507
). For the eight channels (pairs of dierential electrodes) recorded exposure to chloroform led to
nearly three times decrease in amplitude of the dris of baseline potential and nearly 1.3 increase in duration
of the dris. Before exposure to chloroform the mycelium composite produced fast (i.e. less than 10–20min)
spikes. Median amplitude of the spikes was 0.48mV (average 0.52mV,
σ=0.2
). Median duration of spikes was
62s (average 63s,
σ=18
), median distance between the spike 214s (average 189s,
σ=90
). Aer exposure to
Figure2. Example of spiking activity recorded from a control sample; an intact, non-anesthetised, myceliated
hemp pad. Spikes are shown by arrows.
Figure3. An example showing how the electrical activity of fungi changes when chloroform is introduced. e
moment of the chloroform introduction is shown by arrow.
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chloroform the mycelium composite did not show any spiking activity above level of background noise, which
was for this particular recording c. 0.05mV.
In some cases the spiking activity is diminished gradually with decreased frequency and lowered amplitude,
as exemplied in Fig.4. Typically, the intact (control) spiking frequency is a spike per 70min while aer inhala-
tion of chloroform a spike per 254min in the rst 40-50hours and decreased to nearly zero aer. e median
amplitude of intact mycelium spikes is 0.51mV, average 0.74mV (
σ
=0.59). Anaesthetised mycelium shows,
spikes with median amplitude 0.11mV, average 0.2mV (
σ=0.2
). Spikes are not distributed uniformly but
gathered in trains of spikes. In the intact mycelium there is a media of 3 spikes in the train, average number of
spikes is 4.2 (
σ=4.4
). Median duration of a spike train is 84min, average 112min (
σ=32
). Media interval
between trains is 53min, average 55s (
σ=29
). Anaesthetised mycelium emits trains with median number of
2 spikes, average 2.5 spikes, average 2.5 spikes (
σ=0.84
). A median duration of such trains is 29min, average
51min (
σ=22
). e trains appear much more rarely than the trains in the intact mycelium: median interval
between trains is 227min.
In all ten but one experiment the container remained closed for over 3–4days. By that time all kinds of
electrical activity in mycelium bound substrate extinguished and the mycelium never recovered to a functional
state. In experiment illustrated in Fig.5 we removed a source of chloroform aer 16h and kept the container
open and well ventilated for an hour to remove any traces of chloroform from the air. e intact mycelium shows
median frequency of spiking as one spike per 27min, average 24min. Median amplitude of the spikes is 3.4mV,
average 3.25mV (
σ=1.45
). e anaesthetised mycelium demonstrates electrical spiking activity reduced in
amplitude: median amplitude of spikes is 0.24mV, average 0.32mV (
σ=0.2
), and low frequency of spiking:
median distance between spikes is 38min, average 40min. Electrical activity of the mycelium restores to above
noise level c.60h aer the source of the chloroform is removed from the enclosure (insert in Fig.5). Frequency
of spikes is one spike per 82min (median), average 88min. e amplitudes of recovering spikes are 0.96mV
in median (average 0.93mV,
σ=0.08
) which are three times less than of the spikes in the mycelium before the
narcosis but nearly ve times higher than of the spike of the anaesthetised mycelium.
Discussion
We demonstrated that the electrical activity of the fungus Pleurotus ostreatus is a reliable indicator of the fungi
anaesthesia. When exposed to a chloroform vapour the mycelium reduces frequency and amplitude of its spik-
ing and, in most cases, cease to produce any electrical activity exceeding the noise level (Table1a). When the
chloroform vapour is eliminated from the mycelium enclosure the mycelium electrical activity restores to a level
similar to that before anaesthesia (Table1b).
e fungal responses to chloroform are similar to that recorded by us with slime mould Physarum polycepha-
lum (unpublished results). A small concentration of anaesthetic leads to reduced frequency and amplitude of
electrical potential oscillation spikes of the slime mould, and some irregularity of the electrical potential spikes
(Fig.6a). Large amounts of anaesthetic causes the electrical activity to cease completely and never recover
(Fig.6b). Similar electrical responses to anaesthetic again highlights the fact that slime mould exhibits same-
degree anities with fungi as with protozoa, and slime mould has been classied as fungi previously47–49.
Potential, mV
1.0
0.5
0
0.5
1.0
1.5
Time, sec
011052 1053 1054 105
Figure4. Example of reduced frequency of spiking under eect of chloroform vapour. Moment when a source
of chloroform vapour was added into the container is shown by arrow.
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Results presented in the paper contribute towards lling up the gaps in the taxonomy-related studies of
anaesthesia, the living creatures from Protists to fungi to plants to insects to mammals are susceptible to anaes-
thetics. Eects of chloroform on fungi might also implicitly indicate presence of potassium channels, which are
inhibited by anaesthetics50–52.
With regards to directions of future research, as far as we are aware, the present paper is the rst in the eld,
and therefore it rather initiates the research than brings any closure or conclusions. We know that anaesthetics
block electrical activity of fungi (as well as slime moulds) however we do not know exact biophysical mechanisms
of these actions. e study of biophysics and molecular biology of fungi anaesthesia could be a scope for future
research. Another direction of studies could be the analysis of the decision making abilities of fungi under the
inuence of anaesthetics. An experiment could be constructed when fungal hyphae are searching for an optimal
path in a labyrinth when subjected to increasing doses of chloroform vapour. ere may be an opportunity to
make a mapping from concentrations of anaesthetic to geometry of the mycelium search path.
Figure5. Example of electrical activity of mycelium colonised hemp pad before, during and aer stimulation
with chloroform vapour. Arrow labelled ‘ON’ shows moment when a source of chloroform vapour was added
into the enclosure, ‘OFF’ when the source of chloroform was removed. e spiking activity of the mycelium
recovering from anaesthesia is zoomed in.
Table 1. Anaesthesia induced changes in electrical activity of fungi. (a)Containers with chloroform remain
sealed. (b)Lid from the container is removed aer 16h.
a b
Intact Anaesthetised Intact Anaesthetised Recovered
Baseline potential 0.64mV 0.18mV Spike amplitude 3.25mV 0.32 mV 0.93mV
Duration of oscillations
29 ·103
28 ·103
Spike frequency 24min 40min 88min
Spike amplitude 0.4mV 0
Spike duration 62s n/a
Spike frequency 214s n/a
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Received: 19 June 2021; Accepted: 13 December 2021
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Acknowledgements
is project has received funding from the European Union’s Horizon 2020 research and innovation pro-
gramme FET OPEN “Challenging current thinking” under grant agreement No 858132. e authors would like
to acknowledge the collaboration of Mogu S.r.l. providing the living materials used in the experiments. At the
time of experiments AG was aliated with Mogu S.r.l., Inarzo, Italy. All the experiments have been conducted
in the Unconventional Computing Lab, UWE, Bristol.
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Author contributions
A.A. and A.G. conducted and analysed experiments. A.A. and A.G. wrote the paper.
Competing interests
e authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to A.A.
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