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When Is the Brain Dead? Living-Like Electrophysiological Responses and Photon Emissions from Applications of Neurotransmitters in Fixed Post-Mortem Human Brains

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The structure of the post-mortem human brain can be preserved by immersing the organ within a fixative solution. Once the brain is perfused, cellular and histological features are maintained over extended periods of time. However, functions of the human brain are not assumed to be preserved beyond death and subsequent chemical fixation. Here we present a series of experiments which, together, refute this assumption. Instead, we suggest that chemical preservation of brain structure results in some retained functional capacity. Patterns similar to the living condition were elicited by chemical and electrical probes within coronal and sagittal sections of human temporal lobe structures that had been maintained in ethanol-formalin-acetic acid. This was inferred by a reliable modulation of frequency-dependent microvolt fluctuations. These weak microvolt fluctuations were enhanced by receptor-specific agonists and their precursors (i.e., nicotine, 5-HTP, and L-glutamic acid) as well as attenuated by receptor-antagonists (i.e., ketamine). Surface injections of 10 nM nicotine enhanced theta power within the right parahippocampal gyrus without any effect upon the ipsilateral hippocampus. Glutamate-induced high-frequency power densities within the left parahippocampal gyrus were correlated with increased photon counts over the surface of the tissue. Heschl’s gyrus, a transverse convexity on which the primary auditory cortex is tonotopically represented, retained frequency-discrimination capacities in response to sweeps of weak (2μV) square-wave electrical pulses between 20 Hz and 20 kHz. Together, these results suggest that portions of the post-mortem human brain may retain latent capacities to respond with potential life-like and virtual properties.
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RESEARCH ARTICLE
When Is the Brain Dead? Living-Like
Electrophysiological Responses and Photon
Emissions from Applications of
Neurotransmitters in Fixed Post-Mortem
Human Brains
Nicolas Rouleau
1,2
, Nirosha J. Murugan
1,2
, Lucas W. E. Tessaro
2,3
, Justin N. Costa
2,4
,
Michael A. Persinger
1,2,3,4
*
1Biomolecular Sciences Program, Laurentian University, Sudbury, Ontario, Canada, 2Behavioural
Neuroscience Program, Laurentian University, Sudbury, Ontario, Canada, 3Human Studies Program,
Laurentian University, Sudbury, Ontario, Canada, 4Department of Biology, Laurentian University, Sudbury,
Ontario, Canada
*mpersinger@laurentian.ca
Abstract
The structure of the post-mortem human brain can be preserved by immersing the organ
within a fixative solution. Once the brain is perfused, cellular and histological features are
maintained over extended periods of time. However, functions of the human brain are not
assumed to be preserved beyond death and subsequent chemical fixation. Here we present
a series of experiments which, together, refute this assumption. Instead, we suggest that
chemical preservation of brain structure results in some retained functional capacity. Pat-
terns similar to the living condition were elicited by chemical and electrical probes within cor-
onal and sagittal sections of human temporal lobe structures that had been maintained in
ethanol-formalin-acetic acid. This was inferred by a reliable modulation of frequency-depen-
dent microvolt fluctuations. These weak microvolt fluctuations were enhanced by receptor-
specific agonists and their precursors (i.e., nicotine, 5-HTP, and L-glutamic acid) as well as
attenuated by receptor-antagonists (i.e., ketamine). Surface injections of 10 nM nicotine
enhanced theta power within the right parahippocampal gyrus without any effect upon the
ipsilateral hippocampus. Glutamate-induced high-frequency power densities within the left
parahippocampal gyrus were correlated with increased photon counts over the surface of
the tissue. Heschl’s gyrus, a transverse convexity on which the primary auditory cortex is
tonotopically represented, retained frequency-discrimination capacities in response to
sweeps of weak (2μV) square-wave electrical pulses between 20 Hz and 20 kHz. Together,
these results suggest that portions of the post-mortem human brain may retain latent capac-
ities to respond with potential life-like and virtual properties.
PLOS ONE | DOI:10.1371/journal.pone.0167231 December 1, 2016 1 / 26
a11111
OPEN ACCESS
Citation: Rouleau N, Murugan NJ, Tessaro LWE,
Costa JN, Persinger MA (2016) When Is the Brain
Dead? Living-Like Electrophysiological Responses
and Photon Emissions from Applications of
Neurotransmitters in Fixed Post-Mortem Human
Brains. PLoS ONE 11(12): e0167231. doi:10.1371/
journal.pone.0167231
Editor: Sam Doesburg, Hospital for Sick Children,
CANADA
Received: June 12, 2016
Accepted: November 10, 2016
Published: December 1, 2016
Copyright: ©2016 Persinger et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
presented within the paper and its Supporting
Information files.
Funding: The authors received no specific funding
for this work.
Competing Interests: The authors have declared
that no competing interests exist.
Introduction
The fundamental principle that integrates anatomy and physiology can be effectively summa-
rized as “structure dictates function”. This means the functional capacities of biological sub-
strata are determined by the chemical composition, geometry, and spatial orientation of
structural subcomponents [1,2]. As the heterogeneity of structure increases within a given
organ, so does the functional heterogeneity. Nowhere is this more evident than in the human
brain. It can be described as a collection of partially-isolated networks which function in
concert to produce consciousness, cognition, and behaviour. It also responds to its multi-
variate, diversely energetic environment by producing non-isotropic reflections within its
micrometer and nanometer spaces. The specific spatial aggregates of these dendritic alterations
result in processes that have been collectively described as memory: the representation of
experience.
When structures of the brain undergo changes sufficient to terminally disrupt these func-
tional processes and the individual is ultimately observed to lose the capacity to respond to sti-
muli [3], the brain is said to be clinically dead. This state has been assumed to be largely
irreversible. It should be noted that the specific criteria which must be achieved in order to
ascribe death to an individual are not universal and exhibit a significant degree of non-consen-
sus [4]. The precise point beyond which the brain is no longer “living”, a threshold which
remains unidentified, is perhaps less definite than has been historically assumed. Without life
support systems, either endogenously in the form a cardiovascular network or exogenously in
the form of mechanical aids, the brain degenerates progressively until full decomposition and
dissolution. Complete loss of structure is strongly correlated with the complete loss of func-
tion. When the brain is dead and the tissue has lost its structural integrity, the individual is
assumed to no longer be represented within what remains of the organ.
If, however, the brain is immersed within certain chemical solutions before degeneration
and decomposition, the intricate and multiform structures of the human brain can be pre-
served [57] for decades or perhaps centuries. The gyri and sulci which define the convex and
concave landscapes of the brain’s outer surface as well as the cytoarchitectural features of the
cerebral cortex remain structurally distinct. The deep nuclei and surrounding tract systems
remain fixed in space, unchanging in time. Though structurally intact, the functions of the
brain are, however, still considered to be absent. It has been assumed that the chemical micro-
environment (e.g., pH, nutrient content, ionic gradients, charge disparities, etc.) of both cells
and tissues within the preserved brain must be altered to such a degree to prevent degradation
that these spaces no longer represent those which underlie the cellular processes which give
rise to normal human cognition and behaviour.
The principle of anatomy and physiology which describes the relationship between struc-
ture and function would hold that in the presence of structural integrity so too must there be a
functional integrity. If the structure-function relationship is a physical determinant, functional
capacities should scale with structural loss and vice versa. Therefore the maintenance of struc-
ture subsequent to clinical death by chemical fixation could potentially regain some basic func-
tion of the tissue to the extent to which structure and function are intimately related. Here we
present lines of evidence that indicate brains preserved and maintained over 20 years in etha-
nol-formalin-acetic acid (EFA) [8], a chemical fixative, retain basic functions as inferred by
microvolt fluctuations and paired photon emissions within the tissue. They are both reliably
induced and systematically controlled by the display of electrical and chemical probes which
include the basic inhibitory and excitatory neurotransmitters or their precursors. Each of these
profiles exhibit dosage-dependence and magnitude dependences that are very similar to those
displayed by the living human brain.
When Is the Human Brain Dead?
PLOS ONE | DOI:10.1371/journal.pone.0167231 December 1, 2016 2 / 26
Materials & Methods
Tissue Samples
Human brain tissue samples fixed in EFA (72% ethanol, 18% dH
2
O, 5% acetic acid, 5% formal-
dehyde) were subjected to a series of experimental procedures. The aim was to elicit stimulus-
response patterns characteristic of structure-function relationships observed in the living
human central nervous system. Three (n = 3) caudal coronal sections and four (n = 4) hemi-
spheric sections severed within the midline sagittal plane along the medial longitudinal fissure
at the level of the corpus callosum were used throughout the course of the study. Coronal sec-
tions were selected based upon exclusionary criteria including the presence of the basilar
artery, Ammon’s horn, and the parahippocampal gyrus. Of the four sagittal sections, two were
left hemispheres and two were right hemispheres. All samples were originally obtained from
separate full human brain specimens and were therefore independently sourced. All specimens
had been obtained from anonymous donors or from accredited companies (North Carolina
Biological Supplies) over 20 years ago. The brains had been stored in secure areas and handled
appropriately and respectfully.
Measurement Device: Quantitative Electroencephalography
A Mitsar quantitative electroencephalography (QEEG) amplifier was equipped with needle
electrodes which were inserted directly into the brain tissue (Fig 1). Weak microvolt
Fig 1. Coronal sections of human brain tissue fixed in EFA. Each section was equipped with a needle electrode
inserted into the grey matter of the left parahippocampal gyrus (Pr) referenced (Ref) to the basilar artery (A). The
hippocampal body (HB) and parahippocampal gyrus (PHG) served as the regions of interest (B). Cytoarchitecture
of the hippocampal body fixed in EFA can be visualized under x40 (C) and x200 (D) magnification in stained
(Toluidine Blue-O) sections.
doi:10.1371/journal.pone.0167231.g001
When Is the Human Brain Dead?
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fluctuations (μV) were measured within WinEEG software throughout the course of all experi-
ments outlined here using an HP ENVY laptop computer running Windows 8. Each experi-
ment involved specific needle electrode placement and referencing procedures which were
contingent upon the type of sample. In all cases, notch filters were applied to exclude voltage
fluctuations whose frequency spectra were sourced between 50 and 70 Hz as well as 110 and
130 Hz in order to reduce environmental noise. Low and high cut filters of 1.6Hz and 50Hz
were applied. Electrode impedance was regulated to <5k. Data were extracted as power den-
sities (PDs) in 30 second segments with 2 second epochs. Each extraction consisted of power
measures within delta (1.5Hz– 4Hz), theta (4Hz– 7.5Hz), alpha (7.5Hz– 14Hz), beta1 (14Hz–
20Hz), beta2 (20Hz– 30Hz), and gamma (30Hz– 40Hz) band ranges.
In the case of coronal sections, needle electrodes were inserted into the basilar artery which
served as an electrical reference (Fig 1). A single electrode was then inserted into the region of
interest. The left and right parahippocampal gyri and hippocampal bodies served as the areas
of interest throughout the course of the study. Only one structure was ever probed for a given
trial due to limitations of the referencing procedure. This means that it was not possible to
infer in real-time whether or not hemispheric analogues or adjacent structures were simulta-
neously responding to the same stimulus. Once the needle electrode was inserted into the
region of interest and was referenced to an average of electrodes inserted into the basilar
artery, microvolt fluctuations could be recorded. Sagittal sections of fixed human brain tissue
were probed similarly. The basilar artery always served as the electrical reference point. In the
case of the sagittal sections, the primary loci of interest were the transverse temporal gyri. Nee-
dle electrodes were inserted directly into the transverse temporal gyri within both the postero-
medial and antero-lateral subdivisions. The precise stimulation procedure carried out over the
course of electrophysiological measurement is outlined elsewhere.
Measurement Device: Photomultiplier Tube
Photon measures were obtained within a darkened chamber concurrently with QEEG for trials
involving applications of glutamate to the brain. Raw photon counts were recorded using a sin-
gle photomultiplier tube (PMT) that was suspended 10cm above the brain specimen. The PMT
was a DM0090C model from Sens-Tech Sensor Technologies, with a spectral response range
between 300–850 nm (visible light). Sens-Tech Counter timer software recorded digital output
from the photomultiplier tube at a 50 Hz sampling rate for 3000 readings (20 msec data points
for 60 seconds) on a Lenovo ThinkPad laptop that was positioned outside of the enclosed cham-
ber via USB output cables. To remove the contributions from dark counts (i.e., those associated
with the intrinsic photoelectric circuitry), counts measured when the brain tissue was present
were subtracted from baseline conditions when no tissue was present. It was determined that
the use of equipment measuring electric potential differences (QEEG) and photon counts over
time would serve as a measure of internal validity, confirming the presence of systematic
response patterns which could be observed by both measurement devices independently.
Procedures
Chemical Application. Within the context of the living brain, chemical signals in the
form of neurotransmitters are transduced at the level of the receptor into miniature inhibitory
and excitatory post-synaptic potentials or IPSPs and EPSPs [9]. They undergo summation
within the post-synaptic cell resulting in further propagation of electrochemical signals. The
proximal cause of ionic inflow to the cell is receptor-modulated by agonistic and antagonistic
ligands interacting at the level of the plasma membrane. Here, we have designed a series of
experiments which involved the application of neurotransmitters, their precursors, or known
When Is the Human Brain Dead?
PLOS ONE | DOI:10.1371/journal.pone.0167231 December 1, 2016 4 / 26
modulators of receptors within the central nervous system to coronal sections of human brain
tissue fixed in EFA. L-glutamic acid (glutamate), 5-Hydroxy-L-tryptophan (5-HTP), ()-nico-
tine, and ketamine were obtained from Sigma-Aldrich (USA) and serially diluted into various
concentrations ranging between 1 M and 1 nM.
Experiments involving the application of chemical compounds to the tissue were associated
with a specific injection protocol. Each injection was preceded by washing the surface of the
coronal slice with 10% ethanol-formalin-acetic acid (EFA) which was followed by a 30 second
baseline condition during which electrophysiological recordings were obtained. A 1 mL ali-
quot of the solution was injected on to the surface of coronal slices at the parahippocampal-
hippocampal interface. The point of injection was therefore crudely distributed over both
regions of interest. Therefore, upon injection of the compound, any changes in microvolt fluc-
tuations observed within either the parahippocampal gyrus or hippocampal body was not nec-
essarily due to stimulation of the probed area alone. Instead, adjacent regions, whose efferent
and afferent connections likely contributed to local activity, should be considered as potential
sources of any differences in addition to the probed area.
Electrical Stimulation. The human primary auditory cortex is localized within the medial
two-thirds of the transverse temporal gyrus or Heschl’s gyrus (HG) whereas the antero-lateral
component is designated as an adjacent, non-primary region [10]. Morphometric analyses
have revealed the reliable presence of tonotopic subfields along HG which run perpendicular
to the classical postero-medial-to-antero-lateral cytoarchitectonic organizational divisions
[11]. These “tonotopic maps” are frequency-representing gradients within the tissue which
process primary auditory information received by way of the medial geniculate nucleus of the
thalamus. An experimental verification of preserved frequency-discrimination within the pos-
tero-medial component of Heschl’s gyrus in chemically fixed brain specimens could support a
structural-functional model in post-mortem tissue.
Square and sine wave-forms were generated using Audacity’s (2.0.5) tone-generating tool
on an HP ENVY laptop running Windows 8. Each signal consisted of 30 seconds of a square or
sine wave with an associated frequency of 20 Hz, 100 Hz, 500 Hz, 1 kHz, 2 kHz, 5 kHz, 10 kHz,
12 kHz, 15 kHz, or 20 kHz. We selected the 20 Hz– 20 kHz range to reflect the operating range
of the human auditory pathways. Though the relationship between pressure waves and their
transduced electrical equivalents is not 1:1, the large band range would accommodate our prac-
tical, methodological needs to demonstrate frequency-dependent discrimination. Amplitude of
the signal within Audacity was set to 0.8 (a.u.). The signal output was regulated to 10% of maxi-
mum audio card output. A coaxial cable coupled to an electronic breadboard jumper cable by
an alligator clip served as a stimulating probe which was inserted into the tissue. The voltage
equivalent at the level of the needle probe positioned within ~2 mm adjacent to the stimulating
probe, 2 μV, was measured directly by the electrophysiological recording device [12].
The measurement procedure involved inserting both the data collecting needle probe from
the electrophysiological recording device and the stimulating probe into either the postero-
medial or antero-lateral division of the right or left HG. The needle probes were separated by
~ 2 mm where the stimulating probe was always the lateral-most probe. Each trial consisted of
a 30 second baseline followed by 270 seconds of stimulation. The 270 second stimulation
period was further divided into 9 periods, each with an associated frequency. Frequencies were
counterbalanced to eliminate order effects.
Methods of Analysis
Power densities (PDs) were extracted from WinEEG 2.93.59 (07.2013) and imported to SPSS
v19 for subsequent analysis. The spectral analysis technique and resulting power values were
When Is the Human Brain Dead?
PLOS ONE | DOI:10.1371/journal.pone.0167231 December 1, 2016 5 / 26
selected so as to isolate frequency-dependent signatures from the overall signal. Alternative
signal processing techniques of raw data extractions were employed when analyzing the tis-
sue’s response to electrical stimulation so as to infer information processing disparities as a
function of the probed region. Fractal geometry, when applied to statistical analyses, refers to a
method of generating a ratio which represents an index of complexity–how the detail in the
pattern or signal under analysis changes with respect to the scale or level of discourse at which
it is being measured and examined. The Higuchi Fractal Dimension (HFD) algorithm is one
such method of determining statistical complexity and was employed in the present study
involving electrical stimulation of HG as has been employed in other studies which have exam-
ined electroencephalographic complexity [13]. Additional data processing was conducted
prior to the HFD analysis. It was assumed that the effect of the stimulus on the tissue as
inferred by the recorded QEEG signal would be best illustrated if a difference was taken
between each of the segments corresponding to the periods of frequency-specific stimulation
and the baseline electrophysiological recordings from the fixed brain. Thus each of the seg-
ments underwent the following transform prior to HFD analyses:
ND ¼ ðRSxy RSBLÞ
Where ND is the new data file generated, RS
xy
is the raw signal segment for frequency xy,
and RS
BL
is the raw signal baseline (no frequency), for each individual trial.
Results
Surface Injections of Nicotine
Analyses of variance (ANOVAs) revealed statistically significant three-way interactions
between structure, hemisphere, and concentration for differences of theta (4Hz– 7.5Hz) and
alpha (7.5Hz– 14Hz) PDs (S1 File). Alpha effects were weak, and were eliminated when
accounting for multiple comparisons. The three-way theta power interaction was conspicuous
[F(10,130) = 4.06, p <.001, η
2
= .22]. Right parahippocampal theta power differed as a func-
tion of concentration [F(10,32) = 8.02, p <.001, η
2
= .78] (Fig 2). The effect was dose-depen-
dent with a general linear increase (r = .60). The effect was not present for the immediately
adjacent hippocampal body (p>.05) or contralateral structures (p>.05) which was the major
source of the interaction. The control (water) condition was associated with differences in
power as a function of structure; however, there were no within-structure differences in theta
power as a function of hemisphere (p>.05).
The primary sources of variance associated with increases in theta power within the right
parahippocampal gyrus following surface injections of nicotine were between sham condition
(M = 59.43, SEM = 3.49) and a number of concentrations, namely: 10 nM (M = 120.20, SEM =
6.63), 1 uM (M = 102.77, SEM = 12.03), 10 uM (M = 79.70, SEM = 1.90), 1 mM (M = 83.23,
SEM = 6.48), 10 mM (M = 110.33, SEM = 9.21), 100 mM (M = 109.93, SEM = 6.19), and 1 M
(M = 132.47, SEM = 7.00). Effect sizes ranged between 75% and 96%. Theta power was also
observed to linearly increase as a function of concentration, r = .53, p <.001; rho = .50, p <
.005 (Fig 3). This relationship was strengthened when removing trials involving the peak 10
nM concentration (r = .71, p <.001). In fact, power fluctuations were essentially non-linear
below concentrations of 1 μM after which the relationship strengthened markedly. After Bon-
feronni correction for multiple comparison (α= .005), three concentration ranges could be
discerned, each defined by a “peak” wherein the average was visually increased as seen in Fig 2.
Three concentrations, 10 nM, 1 uM, and 1M, were selected across the range of the set for fur-
ther experimentation where the time-course of theta fluctuations would be monitored post-
injection.
When Is the Human Brain Dead?
PLOS ONE | DOI:10.1371/journal.pone.0167231 December 1, 2016 6 / 26
A grand mean of the “peak” concentration theta power responses to surface injections of
nicotine (M = 95.06, SEM = 7.47) were increased relative to baseline conditions (M = 78.41,
SEM = 3.34) [t(34) = 2.04, p = .05, r
2
= .11]. An ANOVA revealed that 10 nM injections over
the surface of the parahippocampi induced time-dependent fluctuations in theta power, F
(5,35) = 2.60, p <.05, η
2
= .30. These disparities were likely driving the differences observed
Fig 3. Concentration Dependence: Nicotine. Theta (4Hz– 7.5Hz) PDs within the right parahippocampal gyrus as
a function of the concentration of nicotine injected over the surface of the tissue.
doi:10.1371/journal.pone.0167231.g003
Fig 2. Nicotine Response. Theta (4Hz– 7.5Hz) PDs as a function of nicotine concentration within the right
parahippocampal gyrus (PHG) and hippocampus (HB). Significant differences from sham (Water) after correction
(α= .005) are indicated.
doi:10.1371/journal.pone.0167231.g002
When Is the Human Brain Dead?
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with the grand-means. Other concentrations did not demonstrate robust time-dependent
effects (p>.05). Homogeneous subsets identified two groups wherein the major sources of var-
iances were between theta power 20 minutes post-injection (which was indistinguishable from
baseline theta power) and the 1 minute immediately following injection (r
2
= .27, p <.05).
These differences in theta power within the PHG are visualized in Fig 4A. A clear initial
increase was noted, followed by a decrease to baseline levels over time.
Further analysis of hemispheric effects indicated that theta power increased significantly
within right hemispheric structures (parahippocampal gyri and hippocampal bodies) 5 min-
utes post-injection of 10 nM nicotine (M = 91.75, SEM = 9.08) relative to baseline conditions
(M = 63.43, SEM = 7.04), t(10) = 2.46, p <.05, r
2
= .38 (Fig 4B). The effect was specific to the
theta band and did not generalize to left hemispheric structures [t(10) = .38, p>.05]. Left hemi-
spheric structures did not demonstrate time-dependent changes relative to baseline. As is
apparent in Fig 4B, the high degree of variability associated with left hemispheric theta power
1 minute post-injection may have masked an early-phase homologous effect.
PDs collected serially over several weeks of experimentation were plotted over time in order
to discern any long-term effects associated with repeated and protracted exposures to surface
injections of various concentrations of nicotine. Time, in this case, was represented by trial
order. Investigating linear relationships between band-specific power and trial order (implicitly
time) within the tissue revealed a negative correlation for the theta-band PDs during the sham
condition (r = -.62, p <.05). The relationship is plotted in Fig 5. Selecting for the first and last
three sham trials, a significant decrease in theta power was observed, t(4) = 5.40, p = .006, r
2
=
.88. Three conspicuous trials can be seen in Fig 5 which can be interpreted as “peaks” within the
negative trend which are represented at points 5, 8 and 9. Each trial was determined to have
originated from separate coronal slices (n = 3). Trials 8 and 9 were preceded by injections of 10
mM nicotine. Considering that conditions associated with each trial were randomized and
order-effects were unlikely due to chance alone, this observation could be relevant.
Surface Injections of 5-Hydroxy-L-trypotophan
Applications of various concentrations of 5-HTP to the coronal sections revealed a number of
features (S2 File). Significant increases in theta (4Hz– 7.5Hz) PDs within the right hippocam-
pal body were noted for the 100 nM (M = 82.67, SEM = 10.81) and 100 μM (M = 81.53,
SEM = 10.21) concentrations relative to the water control (M = 39.47, SEM = 5.32) where
p<.05 and effect sizes were 76% and 77% respectively (Fig 6). Other frequencies were
Fig 4. Time Dependence: Nicotine. Theta (4Hz– 7.5Hz) PDs as a function of time (min) from injection (time = 0 or
between -1 and 1) of 10 nM nicotine for hippocampal (HB) and parahippocampal (PHG) loci (A) as well as between left
(Left) and right (Right) hemispheres (B).
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unaffected and the contralateral hippocampal body did not demonstrate similar response pat-
terns (p>.05).
The right parahippocampal gyrus displayed increased gamma (30Hz– 40Hz) activity upon
injection of 10 nM 5-HTP (M = 1.58, SEM = .06) relative to water control [(M = 1.23, SEM =
.03), t(4) = 4.98, p = .008, r
2
= .86 (Fig 7)]. The contralateral parahippocampal gyrus did not
express similar differences as a function of any concentrations of 5-HTP (p>.05). Other
Fig 5. Repeated Exposure: Nicotine. Theta (4Hz– 7.5Hz) PDs upon injection of water (control) as a function of
time.
doi:10.1371/journal.pone.0167231.g005
Fig 6. 5HTP Response: Right Hippocampus. Theta (4Hz– 7.5Hz) PDs within the right hippocampal gyrus as a
function of the molar concentration of 5-HTP applied to the surface of coronal sections of human brain tissue.
Significant differences are indicated (p>.05).
doi:10.1371/journal.pone.0167231.g006
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frequency-specific microvolt fluctuations remained unaffected by surface injections of 5-HTP
applied to the right parahippocampal gyrus (p>.05). Dose-dependent linear relationships or
changes in microvolt potentials over time could not be identified for coronal sections exposed
to 5-HTP. This feature was observed for all of the other applied chemical compounds.
Surface Injections of Glutamate
An ANOVA revealed a three-way interaction between structure, hemisphere, and concentra-
tion for global (1.5 Hz– 40 Hz) PDs [F(8, 107) = 3.02, p <.01, η
2
= .20] (S3 File). Selecting for
the left parahippocampal gyrus, global power (1.5Hz– 40Hz) increased upon injection of con-
centrations of 100 nM (M = 108.76, SEM = 6.07) of glutamate relative to water (sham) control
[(M = 70.23, SEM = 2.49) [t(4) = -5.88, p <.005, r
2
= .90 (Fig 8)]. This effect was largely due to
increases in delta (1.5Hz– 4Hz) activity between the same conditions [t(4) = -4.99, p <.01,
r
2
= .86]. Conspicuous high-frequency modulations by nanomolar-range glutamate can be
observed in Figs 9and 10. The left parahippocampal gyrus demonstrated increased theta band
PDs upon surface injections of 10 μM concentrations (M = 89.37, SEM = 1.29) of glutamate
relative to the sham condition [(M = 69.20, SEM = 4.10), t(4) = -4.70, p <.01, r
2
= .85]. Finally,
increases of 0.3 to 0.5 μVHz
-1
were observed for probed left parahippocampi within the
gamma band upon surface injections of 10 mM and 1 mM glutamate respectively with associ-
ated effect sizes of 68% and 70%. The anatomically adjacent left hippocampal body did not dis-
play significantly different spectral power upon surface injections of any concentration of
glutamate relative to the sham condition (p>.05).
A conspicuous linear relationship between gamma activity within the left parahippocampal
gyrus and the concentration of the injected material was also observed [r = .45, p <.05; rho =
.47, p <.05 (Fig 9)]. Linear relationships between concentration of the injected material and
Fig 7. 5HTP Response: Right Parahippocampal Gyrus. Gamma (30Hz– 40Hz) PDs within the right
parahippocampal gyrus as a function of the molar concentration of 5-HTP applied to the surface of coronalsections
of human brain tissue. Significant differences are indicated (p>.05).
doi:10.1371/journal.pone.0167231.g007
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spectral power within any band could not be identified within alternative structures (p>.05).
Selecting for concentrations between 10
8
to 10
6
M glutamate, conditions which produced
the greatest magnitude shifts in computed global power relative to the water control, a strong
negative correlation was observed between trial order and gamma power [r = -.73, p <.05;
Fig 8. Glutamate Response. Global power (1.5Hz– 40Hz) within the left parahippocampal gyrus as a function of
concentration of glutamate. A significant increase in mean global power after Bonferonni correction (α= .006) is
indicated.
doi:10.1371/journal.pone.0167231.g008
Fig 9. Concentration Dependence: Glutamate. Gamma (30Hz– 40Hz) power within the left parahippocampal
gyrus plotted as a function of concentration of the injected material.
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rho = -.71, p <.05 (Fig 10)]. This relationship indicates that gamma power linearly decreased
as a function of time upon injections of the same concentrations of glutamate which tran-
siently increased gamma power activity. This relationship was not observed when plotting trial
order with gamma power independent of selective concentration bands (p>.05). In other
words, the negative relationship was only maintained when narrow-band concentrations
which induced maximal responses relative to control conditions were selected.
Two minor right hemispheric effects were noted. First, an increase in delta power (1.5Hz–
4Hz) within the right parahippocampal gyrus was noted upon injection of 1 mM (M = 401.97,
SEM = 14.67) glutamate relative to the sham condition (M = 351.70, SEM = 9.27), t(4) = -2.90,
p<.05, r
2
= .68. When considering a computed global average of spectral power differences
the effect size associated with significant differences between 1 mM and the sham condition
increased to 73%. Second, an increase in gamma power (30Hz– 40Hz) within the right hippo-
campal body was noted upon surface injections of 10 nM (M = 1.63, SEM = .07) glutamate rel-
ative to the sham condition (M = 1.27, SEM = .09) [t(4) = 3.32, p <.05, r
2
= .73].
Having identified the “peak” concentration of 10
7
M, which optimally induced gamma
power increases within the left parahippocampal gyrus, a series of trials were completed in
order to plot the time-course of glutamate power over 20 minutes (Fig 11). An ANOVA
revealed significant differences in gamma power over time, F(5, 17) = 4.25, p <.05, η
2
= .64.
No other frequency band was affected (p>.05). Post-hoc tests revealed two homogeneous sub-
sets with the 1 minute post-injection condition loading separately from all other time condi-
tions. The increase in power was equivalent to ~ 0.7 μVHz
-1
on average.
Surface Injections of Ketamine
Dose-dependent curves of high-frequency PDs identified within the left parahippocampal
gyrus occurred from exposure to a narrow band of molar concentrations of glutamate. We
Fig 10. Repeated Exposure: Glutamate. Gamma (30Hz– 40Hz) power within the left parahippocampal gyrus for
trials involving surface injections of 10
8
to 10
6
M glutamate as a function of trial order, or implicitly, time.
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investigated the potential mechanisms governing the effects. A focused experimental proce-
dure was designed whereby left hemispheric hippocampal bodies and parahippocampal gyri
were exposed to various concentration of ketamine (S4 File), an N-methyl-D-aspartate
(NMDA) receptor antagonist [14]. It was hypothesized that decreases in high-frequency PDs
within the parahippocampus but not the hippocampus would result if the operating mecha-
nism was common to that which was underlying the glutamate effects.
A one-way ANOVA selecting for the parahippocampal gyrus revealed that beta1 PD differ-
ences from the pre-injection period (i.e., a 30 second period immediately preceding the injec-
tion) to the post-injection period (i.e., a 30 second period immediately following the injection)
differed as a function of concentration of ketamine [F(4,14) = 4.09, p <.05, η
2
= .62 (Fig
12A)]. Homogeneous subsets revealed the primary source of variance was a difference between
the 1 nM condition and the water control (p <.05). Post-hoc t-tests confirmed this difference
marked by a decrease in power after injection [t(4) = 3.63, p <.05, r
2
= .77]. A proportionally
similar decrease was also noted when using an average of beta1 and gamma activity [t(4) =
2.86, p <.05, r
2
= .67 (Fig 12B)]. Concentration effects were not noted for the hippocampal
body across any spectral power band (p>.05). That high-frequency activity was enhanced by
glutamate and suppressed by ketamine suggests a common site of action and reduces the prob-
ability that the effects were simple artifacts of injections. That both of these phenomena were
observed within the parahippocampal gyrus but not the hippocampus indicates internal
consistency.
A non-parametric correlation was identified between low-frequency (delta to alpha) but
not high-frequency (beta1 to gamma) SPD differences within the left parahippocampal gyrus
from pre- to post-injection periods and the molar concentration of ketamine administered
(rho = .60, p <.05). These differences were primarily due to an underlying positive correlation
between delta PD differences from pre- to post-injection periods and molar concentrations of
ketamine, rho = .56, p <.05 (Fig 13). The adjacent hippocampal body did not demonstrate
any statistically significant relationships between expressed power density differences from
pre- to post-injection periods and drug concentration (p>.05). Fig 14 demonstrates the non-
relationship observed between delta-band PD differences from pre- to post- injection periods
Fig 11. Time Dependence: Glutamate. Gamma (30Hz– 40Hz) power within the left parahippocampal gyrus as a
function of time elapsed from the point of surface injections of 100 nM (10
7
M) glutamate.
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and molar concentration for the hippocampal body (rho = -.10, p = .73) which was in stark
contrast to that which is displayed in Fig 13 for the parahippocampal gyrus.
In order to substantiate the potential receptor-mediated mechanisms governing increases
in high-frequency microvolt fluctuations associated with NMDA receptor agonist glutamate
and similar decreases associated with NMDA receptor antagonist ketamine, mixed solutions
were generated. Left parahippocampi were exposed to surface injections of water, a mixed
Fig 12. Ketamine Response. Beta1 (14Hz– 20Hz) SPD differences from the pre-injection period to the post-
injection period for left hemispheric hippocampal bodies (HB) and parahippocampal gyri (PHG) exposed to various
concentrations of ketamine (A). High frequency PDs computed from an average of beta1 (14Hz– 20Hz) and gamma
(30Hz– 40Hz) SPD differences from the pre-injection period to the post-injection period for left hemispheric
hippocampal bodies (HB) and parahippocampal gyri (PHG) exposed to 1nM ketamine compared to sham injection
(B). Significant differences are indicated (p <.05).
doi:10.1371/journal.pone.0167231.g012
Fig 13. Concentration Dependence: Ketamine. Non-parametric correlation between delta (1.5Hz– 4Hz) SPD
differences from the pre-injection period to the post-injection period and molar concentration of ketamine for the left
parahippocampal gyrus. A significant correlation was identified (rho = .60, p <.05).
doi:10.1371/journal.pone.0167231.g013
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solution of 10
7
M glutamate and 10
9
M ketamine (i.e. the optimal concentrations which
modulated high-frequency microvolt fluctuations), or a mixed solution of 10
5
M glutamate
and 10
7
M ketamine which served as an alternative control (S5 File). No significant differ-
ences between injection conditions were noted for beta1, beta2, and gamma PDs (p>.05). Fig
15 demonstrates the mutual nullification of glutamate- and ketamine-mediated high-fre-
quency effects within the left parahippocampal gyrus.
Glutamate-Induced Microvolt Fluctuations and Coupled Photon
Emissions
Within a darkened environment, injections of 100 nM glutamate applied to the surface of coro-
nal sections placed in the darkened environment produced increased post-injection (M = 4.60,
SEM = .45) beta2 PDs relative to the pre-injection (M = 3.10, SEM = .31) period for the left
parahippocampal gyrus [t(6) = 2.73, p <.05, r
2
= .55 (Fig 16A)] (S6 File). This response was not
noted for the right parahippocampal gyrus, nor was it observed when water was applied to the
tissue (Fig 16B). The mean photon raw count when the glutamate was applied over the left para-
hippocampal region was 241.2 per 20 ms and 225.3 per 20 ms when water was applied. The dif-
ference (16 photons) per second (50 Hz sampling) was 800 counts. Assuming a typical peak
range photon for the equipment to be associated with 4.2210
19
J the increased photon flux
density from the tissue after glutamate was applied compared to when only water was applied
would have been 3.3810
16
Watts (Joules per s). Because the aperture of the PMT was about
2.2510
4
m
2
, the photon flux power density increase would have been 1.510
12
Wm
-2
.
The regression equation relating the change in power density for the beta2 band to the num-
bers of photons was 0.0394 multiplied by the number of photons (plus 4.314, the constant). This
means that for an increase of every ~10 photons per s over the left parahippocampal region
Fig 14. Null Hippocampal Effect: Ketamine. Non-parametric correlation between delta (1.5Hz– 4Hz) SPD
differences from the pre-injection period to the post-injection period and molar concentration of ketamine for the left
hippocampal body. No significant correlation was identified (p>.05).
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after glutamate application the corresponding beta2 band power increased by 0.39 μV
2
Hz
-1
.
When applied over the 10 Hz increment of beta2 (20 to 30 Hz) this would be 3.9 μV
2
or about
2μV.
A bivariate non-parametric correlational analysis was performed with beta2 PDs and raw
photon counts obtained 10 cm over the tissue within the vertical plane. Spearman rho values
indicating the relationship between beta2 and raw photon counts were examined for hemi-
sphere-, compound-, and time-dependent differences. An ANOVA revealed a three-way inter-
action of these factors [F(1,31) = 8.38, p = .008, η= .23]. The primary source of variance was
identified as a difference in rho values associated with the pre-injection period (M = -.44,
SEM = .18) and the post-injection period (M = .30, SEM = .20) for the left parahippocampal
gyrus [t(6) = 2.61, p <.05, r
2
= .53]. Fig 17 demonstrates this difference which was not noted
for the right parahippocampal gyrus. Alternative PD-photon relationships (e.g. delta PD-pho-
ton, theta PD-photon, etc.) were not observed (p>.05).
Signal Complexity and Frequency Discrimination: Heschl’s Gyrus
HFDs were calculated for each transformed segment of EEG data, including the baseline
condition, using MatLab. The results of Kolmogorov-Smirnov tests for normalcy were signifi-
cant, thus non-parametric methodologies were employed. Significant effects for hemisphere
(Mann-Whitney U, p = 0.017) and waveform (Mann-Whitney U, p = 0.008), but not location
of sensor were found and directed further analyses. Selecting for each individual hemisphere
and waveform condition revealed significant differences in the HFDs of the EEG data recorded
during the right sine wave condition (KW χ
2
(9) = 17.927, p = 0.036). Subsequent post-hoc anal-
yses revealed three groups (average HFDs = 1.38, 1.40, 1.42) where the HFD for 20 Hz (1.425)
was largest and different from all other conditions, including baseline (1.398) (Fig 18).
Fig 15. Glutamate-Ketamine Response. Beta1 (14Hz– 20Hz), beta2 (20Hz– 30Hz), gamma (30Hz– 40Hz) PDs
within the left parahippocampal gyrus exposed to surface injections of water (Water), 10
7
M glutamate and 10
9
M
ketamine (10
7
G + 10
9
K), or 10
5
M glutamate and 10
7
M ketamine (10
5
G + 10
7
K). No significant differences
were observed (p>.05).
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ANOVAs revealed that delta (η
2
= .30), theta (η
2
= .28), and beta2 (η
2
= .28) PDs differed as
a function of frequency for square-wave stimuli presented to the medial aspect of HG within
left hemispheric sagittal sections (p <.05) (S7 File). Differences were not identified as a func-
tion of the frequency of the stimulus for all combinations of factors within the right hemi-
sphere, for presentations to the antero-lateral aspect of HG within the left hemisphere, or
for sine wave signals (p>.05). It was strictly a square-wave effect. Applying the Bonferonni
method, a corrected alpha level (α= .016) was selected as a conservative threshold beyond
which differences were considered significant. Frequency-dependent differences in theta band
spectral power (4.0 Hz– 7.5 Hz) remained significant after Bonferonni correction (p <.016).
Differences within delta and beta2 bands did not meet this threshold. The primary sources of
variance were identified to be differences between 20Hz (M = 89.52, SEM = 5.88) and 5,000Hz
(M = 64.60, SEM = 4.01), 20 Hz (M = 89.52, SEM = 5.88) and 20,000 Hz (M = 65.02, SEM =
2.64), as well as 100Hz (M = 77.97, SEM = 2.12) and 20,000 Hz (M = 65.02, SEM = 2.64) which
are visualized in Fig 19A. The effect sizes (r
2
) associated with each significant difference were
.55, .59, and .59 respectively. In contrast, Fig 19B demonstrates the overlap between frequency-
responses within the right hemisphere.
A discriminant analysis revealed that wide-band PDs (1.5Hz– 40Hz) derived from the pos-
tero-medial aspect of HG within the left hemisphere upon stimulation by square-wave 20Hz
and 20,000Hz signals successfully classified 100% of cases in a corrected model [(n = 12), Λ=
.09, χ
2
(6) = 16.64, p <.01, canonical R
2
= .95]. No other combination of factors, including
those selecting for the antero-lateral aspect of the same gyrus, could reproduce a successful
classification of cases when attempting to discriminate 20Hz and 20,000Hz (p>.05). The same
Fig 16. Glutamate Response in a Darkened Environment. Beta2 (20Hz– 30Hz) PDs for periods of pre- and post-
injection for right (dark) and left (light) parahippocampal gyri exposed to 1 mL surface injections of 100 nM
glutamate (A) and water (B). A significant difference from pre-to-post-injection periods for the left parahippocampal
gyrus was revealed (p <.05).
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confluence of factors (i.e. postero-medial, left hemisphere, square waves) generated 100% clas-
sification of cases when discriminating 20Hz and 5,000Hz, [Λ= .10, χ
2
(6) = 16.27, p <.05,
canonical R
2
= .95]. Together, these results suggest that the posterior and medial aspects of HG
within the left hemisphere but not the adjacent anterior and lateral components maintain fre-
quency discrimination capacities long after death in EFA-fixed human brain tissue–a property
unobserved within analogous areas of the right hemisphere.
Discussion
One of the most important perspectives afforded by the pursuit of knowledge through system-
atic and scientific methods is to assume nothing. Axioms, self-evident truths, and (most fre-
quently) designation by decree of authority or unchallenged faith in traditions have often been
major impedances to the types of discoveries that lead to shifts in paradigms and a more accu-
rate or at least a different perspective of the human condition. As neuroscientists we have been
taught or have assumed that the fixed human brain is an unresponsive mass of organic residual
that has replaced what was once a vital, complex structure that served as the physical substrate
for thought, consciousness, and awareness. The results of the present experiments strongly
suggest we should at least re-appraise the total validity of that assumption.
Histological analyses indicated that there was general neuronal conservation that is discern-
able by routine light microscopy (Fig 1). Although neuronal (soma) Nissl-dominant stains do
not discern the integrity of the fields of dendrites or the fidelity of their spines, we have found
in unpublished studies with rat brains that those fixed in EFA for protracted periods (years)
and later processed through modified Fox-Golgi (zinc chromate) methods exhibited some
remarkable integrity of dendritic-spine processes. EFA had been selected based upon
Fig 17. Glutamate Induced Microvolt-Photon Pairings. Non-parametric correlations (Spearman rho values)
between beta2 (20Hz– 30Hz) PDs within and raw photon counts over the left (light) and right (dark)
parahippocampal gyri for the 25 seconds preceding (Pre) and proceeding (Post) injections of 100 nM glutamate
applied to the surface of the coronal sections. Significant differences in non-parametric correlations from pre-to-
post-injection periods is noted for the left parahippocampal gyrus (p <.05).
doi:10.1371/journal.pone.0167231.g017
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experimental comparisons as the primary mode of long-term fixation in our laboratory many
years ago [15] regardless of the initial post-mortem immersion (for human brains), because of
the cytological detail it retained, its compatibility with a multitude of different stain types, and
more recently because of its capacity to express immunochemical properties following specific
“rejuvenating” pre-treatments.
If some proportion of the living microstructure remains with the potential to be activated,
then electrophysiological patterns similar to those in the living brain should be elicited by
physiologically-appropriate concentrations of classic neurotransmitters that would influence
primarily only particular frequency bands. We selected the parahippocampal region as the pri-
mary focus because of the central role of this structure in human cognitive phenomena. First,
it is the primary locus for the initial representation of experience (“memory”) as indicated by
the marked decrement in this capacity following loss or lesions such as the cases of HM [16]
and RB [17]. Secondly, as demonstrated by the precision of Pierre Gloor’s [18] micro- and
macro-anatomical analyses this region directly accesses and reciprocally receives input and
output respectively from the entire cerebral cortical manifold. Third, this region, particularly
in the right hemisphere, is remarkably sensitive in the living state [19] as well as the fixed state
[20] to ambient geomagnetic activity to which all human beings are usually immersed.
During the late 19
th
century portions of the hippocampal region were argued by anti-Dar-
winian debaters as the unique feature that discriminated human brains (and presumably the
special nature of this species) from other primates [21]. It has been known for decades that
temporal lobectomies as a treatment for intractable epilepsy eliminated the psychotrophic and
hallucinogenic effects of LSD [22]. The central role of the parahippocampal region and its
Fig 18. Fractal Dimensions by Frequency: Heschl’s Gyrus. Results of the Higuchi Fractal Dimension (HFD)
Kruskall-Wallis test. Groups as revealed by post-hoc Mann-Whitney U tests are indicated by shading of bars, with
the exception of the baseline (error bars = SEM).
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decreased connectivity from the retrosplenial cortex during hallucinatory experiences induced
by LSD [23] also highlights the potentially unique feature of this structure. Carhart-Harris et al
[23] found that the functional disconnectivity was strongly correlated with the rating of ego
“dissolution” that was inferred to reflect the importance of this circuit to maintain the sense of
self. Tagliazucchi et al. [24], employing a slightly different approach, noted that the LSD effects
enhanced global between-module interactions within those regions rich in 5-HT
2a
receptors.
Within the fixed dead human brain increases in theta power within the right hippocampal
body was observed after application of 100 nM and 100 μM concentrations of serotonin.
Unlike the living brain the serotonin immediately and directly apposed the tissue upon appli-
cation and was not diluted by either the multiple blood-brain barriers or the catabolising envi-
ronment of enzymes. The double peak suggests two receptor subtypes that are consistent with
those reported within the hippocampus [25]. The elicitation of gamma power within an even
Fig 19. Frequency Discrimination. Theta (4Hz– 7.5Hz) PDs within the postero-medial (Medial; light) and antero-
lateral (Lateral; dark) aspect of Heschl’s Gyrus within the left (A) and right (B) hemispheres as a function of square-
wave signals with frequencies ranging between 20Hz to 20, 000 Hz. Significant differences after correction (α=
.005) are indicated.
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smaller concentration (10 nm) from the right parahippocampus region suggests an intrinsic
separation of some remaining infrastructure that differentiates 4–7 Hz and 30–40 Hz patterns.
This is important simultaneity in light of the common observation that gamma ripples are
superimposed upon the massive theta activity within this region [2628]. This intrinsic associ-
ation has been argued by Bear [29] to be a primary electrophysiological correlate by which
consciousness and awareness are coupled to memory. That this structure in the right but not
the left hemisphere displayed the effect indicates the responses were specific and that there
may be some particular residual within the right hemisphere. Rouleau and Persinger [12] on
the bases of similar results have suggested that the implications of the massive historical data
base of surgical stimulation of patients and the interpretation of the etiology of their colourful
experiences might be reconsidered.
Glutamate is considered the major excitatory neurotransmitter of the brain and is a major
correlate of the processes that contribute to long-term potentiation (LTP) which are the first
phases of memory consolidation [30]. The peak power density within the gamma range over
the left parahippocampal region also suggested that some residual of two receptor subtypes
remained with affinities in the nanoMolar and milliMolar range. The increase in power within
the gamma range after the applications of these two concentrations was between 0.3 and
0.5 μV
2
Hz
-1
which is within the range of shifts in cerebral cortical activity that we have mea-
sured to be associated with consciousness and specific tasks [31,32]. The laterality of the effect
was clearly indicated. In contrast the right parahippocampal region displayed power increases
that were primarily evident across the gross band of activity; this occurred for the milliMolar
range. That the effects were dynamic and not passive was indicated by the transience of the peak
response (Fig 9) and the gradual “habituation” or diminishment with repeated trials (Fig 10).
Glutamate has been shown to induce biophotonic activities [33] in neural circuits. Several
authors have suggested that biophoton patterns may be central to neural information process-
ing and decoding that may depend upon quantum brain mechanisms [3436]. The left para-
hippocampal gyrus responded significantly to surface applications of 100 nM of glutamate
solutions by increasing the power spectra within the 20 to 30 Hz range by about 1.5 μV
2
Hz
-1
compared to the previous baseline conditions while in the darkened environment. This was
not observed for the right equivalent region. There were also moderately strong correlations
between the numbers of photons emitted after the injection (but not before) and the power
density for 30 to 40 Hz, the gamma range but not for other PD frequency bands.
The mean numbers of photons per s was equivalent to a photon flux density of about 10
12
Wm
-2
which is the same order of magnitude as those generated in rat hippocampal slices when
coupled to theta activity [37]. This flux density is the same order of magnitude that was mea-
sured from the right hemispheres (at the level of the temporal lobe) when people sitting in very
dark rooms engaged in vivid imagination about white light compared to mundane thoughts
[38]. Finally, the presence of a temporal discrepancy between the left and right temporal lobes
for the spectral flux density of photon emissions while human beings sat with their eyes closed
in a dark room has been measured for this magnitude [39]. In other words, by simply applying
glutamate at concentrations typically encountered within living brain tissue photons were emit-
ted from human tissue that had been fixed in EFA for decades. The flux densities were compara-
ble to that associated with specific cognitions generated by the living brain.
The physical bases to “consciousness” and cognition with the implication of a more ubiqui-
tous property that may occur throughout the universe would be consistent with the philosophy
of Spinzoa [40] and the concept of Ernst Mach [41] that the behaviour of any part of the uni-
verse (“cosmos”) is determined by all of its parts. Similar, more recent approaches have been
expanded and quantified by Hameroff and Penrose [42] and Persinger and St-Pierre [43]. We
have operated upon the assumption that either gravity or electromagnetism–or both as these
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PLOS ONE | DOI:10.1371/journal.pone.0167231 December 1, 2016 21 / 26
are not mutually exclusive–represent physical candidates which could satisfy these parameters.
Our approach has favoured the photon [43] and therefore electromagnetism. The photon may
be the fundamental process that relates complex phenomena over large distances of space and
time and would be unimpeded by restrictions of speed assuming non-local photon-photon
interactions. If this were valid, then an integrating factor must be present such as the common-
ality of the most dominant constituent, the hydrogen atom and the neutral hydrogen line of
1.42 GHz [44]. It may be relevant (but also potentially spurious) that the average spectral
power density produced by the application of glutamate (~210
12
Wm
-2
or kgs
-3
) divided by
the change in microvoltage associated with that application (~210
6
V) results in 10
6
Am
-2
.
Applied across the area of the PMT aperture that would be the equivalent of 10
10
A associated
with the application of the glutamate compared to water. When this current is divided by the
unit charge value of 10
19
As, the residual frequency is 10
9
Hz or GHz which is well within the
range of the neutral hydrogen line. In the absence of a strong hypothetical mechanism which
explains how long-deceased biological material could systematically emit photons, this conver-
gence of numbers should be further considered even if with caution.
Systematic injections of different concentrations of two “psychotropic” compounds, nico-
tine and ketamine, also showed natural, living brain-like responses in terms of both latency
and concentration. Again there were anisotropic hemispheric responses within the regions of
interest. Enhanced theta power associated with nicotine would be consistent with the mem-
ory-enhancing capacity of this cholinergic-stimulating compound [45]. It may be relevant that
nicotinamide adenine dinucleotide (NAD), which contains the molecular structure nicotinic
acid, is a major source of electrons in living biochemical systems. The purine component of
that molecule is synthesized from glutamate, aspartate and glycine. Tryptophan is the precur-
sor of the nicotinamide moiety of NAD and NADP and contributes to the creation of nicotinic
acid. From this perspective the similarity of the theta-band enhancement for the right hippo-
campal regions for both serotonin and nicotine would be expected. These patterns suggest the
possibility that a residual of the intrinsic signatures that reflected the complex biochemical
reactions within brain tissue may still be present in fixed post-mortem tissue and might be
“reactivated”. That the same sites and frequencies were either enhanced by glutamate or sup-
pressed by ketamine at realistic physiological dosages would support this possibility.
Whereas electrophysiological studies are regularly conducted with still-living tissue
explants, there are a few notable methodological differences between the aforementioned and
what we have presented. First, tissue preparations, whether measured by single electrodes or
multi-electrode arrays, are usually no thicker than 1 mm where slices of ~400 μm are typical
[46]. Second, measurements of tissue preparations such as those of hippocampal slices are typi-
cally conducted within 24 hours of decapitation and within a nutrient-rich medium which is
supplemented in various ways to inhibit rapid tissue deterioration [46,47]. During this period,
and despite mitigation efforts, a significant proportion of the cells usually die as inferred by
staining procedures [47]. The tissue explants are normally maintained at physiological temper-
ature and immobilized to reduce mechanically-induced damage. Our specimens are chemi-
cally fixed, much thicker (>1cm), older, maintained at room temperature, and not
supplemented in any way. It is therefore curious that in both cases, fluctuations in electric
potential differences can be observed. Multi-electrode array recordings of tissue explants are
known to register spike values of up to 600 μV, though the typical range of fluctuations are
within 10–100 μV [46]. Our measurements of post-mortem, fixed tissue have revealed typical
fluctuations within 1–80 μV with some high-magnitude transients [12]. In this respect, our
measurements are consistent with those observed by others.
Finally, the persistence of essential microstructure was evident by the remaining signal
complexity and frequency discrimination that was still apparent within the transverse
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temporal gyrus. A remarkable frequency dependence for maximum responsivity according to
our measures occurred at the lower boundary of the threshold for hearing in the human brain.
Compared to baseline measurements, the largest discrepancy occurred around 20 Hz. As
recently reviewed by Persinger [48] this is the classic transition between infrasound and regu-
lar sound discernment by the human brain. What is less known is that human auditory system
does respond to <20 Hz sound (mechanical vibrations). However, these regions of the system
are less expansive and have few afferents to regions of the cortices involved with awareness.
This structural substrate appears to remain after death in appropriately fixed brains.
Merker [49] presented an argument that gamma synchrony, rather than representing a cog-
nitively-significant correlate, is more likely an indicator of generic infrastructural control at
the level of the tissue. That is, the cognitive correlate of cortical gamma synchrony is really just
a necessary co-occurrence rather than a central operator of cognitive states. If one assumes
that the brain is “dead” and therefore categorically can’t be conscious, Merker’s [49] interpre-
tation could hold true as we’ve observed a degree of gamma activations which could be indica-
tive of synchrony. However, as self-report methods which require sensory inputs and motor
outputs are unavailable to the post-mortem specimens, consciousness and cognitive states can-
not be measured without inference by electroencephalography. Therefore, the assumption of
an absence of consciousness would be based upon an absence of evidence. From this perspec-
tive, if Merker’s interpretation is incorrect and gamma synchrony is in fact cognitively-signifi-
cant beyond mere activation, the post-mortem brain which displays subtle cortical oscillations,
particularly within the theta and gamma bands as demonstrated here, could express some
capacity for cognitive activation.
Supporting Information
S1 File. Nicotine Trials. This file contains data used in the analysis involving post-mortem tis-
sue exposed to injections of nicotine.
(XLSX)
S2 File. 5-HTP Trials. This file contains data used in the analysis involving post-mortem tis-
sue exposed to injections of 5-HTP.
(XLSX)
S3 File. Glutamate. This file contains data used in the analysis involving post-mortem tissue
exposed to injections of glutamate.
(XLSX)
S4 File. Ketamine Trials. This file contains data used in the analysis involving post-mortem
tissue exposed to injections of ketamine.
(XLSX)
S5 File. Glutamate with Ketamine Trials. This file contains data used in the analysis involv-
ing post-mortem tissue exposed to injections of glutamate with ketamine.
(XLSX)
S6 File. Photon Data. This file contains data used in the analysis involving photon counts
paired to injections of glutamate.
(XLSX)
S7 File. Heschl’s Gyrus. This file contains data used in the analysis involving post-mortem tis-
sue exposed to electrical stimuli directed to the transverse temporal gyri.
(XLSX)
When Is the Human Brain Dead?
PLOS ONE | DOI:10.1371/journal.pone.0167231 December 1, 2016 23 / 26
Author Contributions
Conceptualization: NR NM LT JC MP.
Data curation: NR NM LT JC MP.
Formal analysis: NR LT MP.
Investigation: NR NM LT JC MP.
Methodology: NR NM JC MP.
Project administration: NR MP.
Resources: NR NM LT JC MP.
Supervision: MP.
Validation: NR NM LT JC MP.
Visualization: NR MP.
Writing – original draft: NR MP.
Writing – review & editing: NR MP.
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