# Full Text: NeurotransmitterVariation

> Extracted from `2020_NeurotransmitterVariation.pdf`

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PAPER IN FOREFRONT
Measurement of natural variation of neurotransmitter tissue content
in red harvester ant brains among different colonies
Mimi Shin1 & Daniel A. Friedman2 & Deborah M. Gordon2 & B. Jill Venton1
Received: 4 November 2019 /Revised: 3 December 2019 /Accepted: 13 December 2019
# Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Colonies of the red harvester ant, Pogonomyrmex barbatus, regulate foraging activity based on food availability and local
conditions. Colony variation in foraging behavior is thought to be linked to biogenic amine signaling and metabolism.
Measurements of differences in neurotransmitters have not been made among ant colonies in a natural environment. Here, for
the first time, we quantified tissue content of 4 biogenic amines (dopamine, serotonin, octopamine, and tyramine) in single
forager brains from 9 red harvester ant colonies collected in the field. Capillary electrophoresis coupled with fast-scan cyclic
voltammetry (CE-FSCV) was used to separate and detect the amines in individual ant brains. Low levels of biogenic amines were
detected using field-amplified sample stacking by preparing a single brain tissue sample in acetonitrile and perchloric acid. The
method provides low detection limits: 1 nM for dopamine, 2 nM for serotonin, 5 nM for octopamine, and 4 nM for tyramine.
Overall, the content of dopamine (47 ± 9 pg/brain) was highest, followed by octopamine (36 ± 10 pg/brain), serotonin (20 ± 4 pg/
brain), and tyramine (14 ± 3 pg/brain). Relative standard deviations were high, but there was less variation within a colony than
among colonies, so the neurotransmitter content of each colony might change with environmental conditions. This study
demonstrates that CE-FSCV is a useful method for investigating natural variation in neurotransmitter content in single ant brains
and could be useful for future studies correlating tissue content with colony behavior such as foraging.
Keywords Neurotransmitters tissuecontent . Pogonomyrmex barbatus/redharvester ants . Dopamine . Serotonin . Octopamine .
Tyramine . Capillary electrophoresis/electrophoresis . Fast-scan cyclic voltammetry . Carbon-Fiber microelectrode
Introduction
Colonies of red harvester ants (Pogonomyrmex barbatus) for-
age for seeds in the desert as their food and water source [1, 2].
Differences in collective regulation of foraging activity are
associated with brain gene expression differences, specifically
highlighting biogenic amine neurophysiology [3–5]. The ant
nervous system consists of a large central brain, which medi-
ates the use of olfaction in ant social behavior, with smaller
optic lobes attached to each end [6]. Ants utilize
neurotransmitters, such as dopamine, serotonin, octopamine,
and tyramine, to modulate complex behavior related to ag-
gression, learning, and memory [5, 7, 8]. In ants, exterior
and foraging workers tend to have higher levels of brain do-
pamine than interior or nursing workers, and manipulation of
brain dopamine signaling can influence foraging activity [4,
9]. A recent field study demonstrated that dopamine plays a
central role in regulating foraging activity in harvester ants [4].
Colonies treated with exogenous dopamine increased their
foraging activities but colonies treated with 3-iodotyrosine, a
dopamine biosynthesis inhibitor, decreased their foraging ac-
tivity. Starvation and social context led to changes in biogenic
amine levels in working ants [10]. Therefore, methods to
study neurotransmitter content in the ant brain could offer
valuable information about which neurotransmitters are up-
regulated or downregulated, and how the variation in neuro-
transmitters contributes to behavioral phenotypes.
Separating and detecting neurotransmitters in the ant
brain is difficult because the absolute amount of neuro-
transmitter is so small in each tiny brain. High-
Published in the topical collection featuring Female Role Models in
Analytical Chemistry.
* B. Jill Venton
bjv2n@virginia.edu
1
Department of Chemistry, University of Virginia,
Charlottesville, VA 22904, USA
2
Department of Biology, Stanford University, Palo Alto, CA 94305,
USA
https://doi.org/10.1007/s00216-019-02355-3
/ Published online: 7 January 2020
Analytical and Bioanalytical Chemistry (2020) 412:6167–6175

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performance liquid chromatography (HPLC) has been
used for the separation and quantification of neurotrans-
mitters in ant brains, typically from pooled samples of
multiple brains, in colonies that were housed in the labo-
ratory, or after pharmacological treatment [4, 10–12]. An
alternative method for tissue content analysis in limited
sample volumes is capillary electrophoresis (CE) with
electrochemical detection, [13] which has been used to
determine neurotransmitter tissue content in individual
Drosophila brains [14–18]. Micellar electrokinetic chro-
matography (MEKC) was initially coupled with ampero-
metric detection to measure neurotransmitters and their
metabolites in individual Drosophila brains [17–20].
Additionally, dopamine and octopamine were further de-
tected in the subregions of fly brains [18]. However,
amperometry lacks chemical selectivity thus peak identi-
fication relies on the analyte retention time and requires
the use of internal or external standards. Fast-scan cyclic
voltammetry (FSCV) uses a cyclic voltammogram (CV)
to identify the analyte and has been extensively used to
measure neurotransmitters in animal models [21–24]. The
detector is a carbon-fiber microelectrode (CFME), which
is excellent for neurochemical detection because of its
small size, excellent electrochemical properties, and sen-
sitivity [25–27]. In previous work, CE-FSCV was used to
analyze neurotransmitters, including dopamine, serotonin,
tyramine, octopamine, and histamine, in single larval or
adult Drosophila central nervous systems [14–16]. The
red foraging ant brain is small, approximately 950 μm ×
600 μm × 550 μm (length, width, thickness), a volume of
314 nL, so CE should be a good method to quantify bio-
genic amines in a single ant brain.
Here, we developed CE-FSCV to quantify four major
insect neurotransmitters: dopamine, serotonin,
octopamine, and tyramine, in a single red harvester ant
brain from colonies collected in the field. These are the
first measurements of tissue content of biogenic amines in
single forager brains of ants collected in a natural popu-
lation. Dopamine was the most abundant biogenic amine,
followed by octopamine, serotonin, and tyramine. Tissue
content of biogenic amines in ant brains showed signifi-
cant differences across colonies. The relative standard de-
viation within a colony was lower than when data from all
colonies were pooled, showing that variance is greater
among colonies than within a single colony. Levels of
dopamine did not correlate with the other neurotransmit-
ters, but levels of octopamine did correlate with levels of
tyramine and serotonin. There was a trend towards corre-
lation of behavioral response to exogenous dopamine with
dopamine content; thus, future studies can investigate the
relationship between biogenic amine contents and colony
behavior to understand the neurophysiological basis of the
evolution of collective behavior.
Experimental methods
Chemicals
All chemicals were purchased from Sigma unless stated oth-
erwise. 10 mM of tyramine, octopamine, serotonin, and dopa-
mine were prepared as a standard stock solution in 0.1 M
perchloric acid. Ant brains were dissected in cold citric acid.
A total of 200 mM NaH2PO4 with 1 mM tetraborate (pH 4.5)
was used for the separation buffer and 100 mM NaH2PO4
(pH 6.5) for the detection cell buffer. Any solutions injected
into the CE system were filtered with a 0.2-μm nylon filter
(Fisher, Suwanee, GA, USA).
Ant brain homogenate preparation
Detailed methods for collecting foragers from 9 focal colonies
in natural populations are described in Friedman et al. [4].
Foraging ants were collected in the morning of 9/4/2017 at a
long-term study site near Rodeo, New Mexico [28]. Ants were
directly collected into liquid nitrogen and stored in a −80 °C
freezer until dissection. Ant brains were dissected in 50 mM
chilled citric acid using fine tweezers and stored in individual
tubes at −80 °C freezer until the day of the experiment. The
protocol for homogenizing ant brains was adapted from Fang
et al. [14]. For the brain homogenate, a sample vial was pre-
pared using a gel-loading pipette by trimming the tip and then
sealing it with a flame. An isolated frozen ant brain was
thawed and transferred to the sample vial with minimal buffer
which was then replaced with 5 μl of 70% 5 mM perchloric
acid and 30% acetonitrile. The sample vial containing the
buffer and brain was centrifuged at 9000 rpm for 1 min
(Brinkman Instruments, Westbury, NY) at room temperature.
A silver metal wire (28 gauge, 0.325 mm o.d.) was used as a
pestle to break up the brain. The sample was centrifuged again
at 9000 rpm for 2 min and the homogenized sample was
sonicated in a water bath for 10 min (Fisher, Suwanee, GA).
The sonicated sample was centrifuged through a 40-μl aerosol
filter pipette (VWR, PA) to an empty sample vial at
13,000 rpm for 3 min. Electrokinetic injections were made
by placing the end of the separation capillary into this vial.
The standard and brain homogenate samples were kept on ice
in the dark until analysis to minimize degradation of the
samples.
Capillary electrophoresis with fast-scan cyclic
voltammetry
The CE with FSCV detection system was built in-house, and
the procedure was performed as described in Fang et al. [14].
Briefly, a separation capillary (11 μm i.d, 148 μm o.d.,
Polymicro Technologies, Phoenix, AZ) was cut to a length
of 40–42 cm using a capillary column cutter (Scientific
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Instrument Services, Ringoes). The end of the capillary used
for the detection was flame etched, removing about 1 cm of
the polyimide coating to expose the fused silica. The etched
end was glued into a 2-cm long larger capillary (250 μm i.d,
359 μm o.d., Polymicro Technologies, Phoenix, AZ), leaving
the etched region exposed, and the capillary was then inserted
into the CE detection cell (Fig. 1).
To fabricate a carbon-fiber microelectrode, a 30-μm carbon
fiber (World Precision Instruments, Sarasota, FL) was aspirat-
ed into a capillary which was pulled into two electrodes, and
the carbon fiber was trimmed as close as possible to the end of
the capillary glass sheath. The electrode was placed approxi-
mately 10 μm from the end of the separation capillary.
Stainless-steel and chloridized silver wires were attached to
the cell to serve as the CE ground and reference electrode,
respectively. End column detection was employed to mitigate
the impact of high separation voltage on FSCV response. A
total of 15 kV was applied for 15 s for the sample injection,
and 9 kV was applied for the separation, using a DC power
supply (Spellman, Plainview, NY). The cell buffer was slowly
flowed through the cell to prevent analytes from accumulat-
ing. Standard solutions of 50 nM dopamine and octopamine
and 25 nM serotonin and tyramine were first injected for a
calibration run. Then a tissue sample was injected.
For the FSCV detection, we used a Waveneuro potentiostat
to collect data (5 MΩ headstage, Pine Research Instrument,
Durham, NC). A triangular waveform of −0.4 V to 1.3 Vand
back at −0.4 V at 400 V/s was applied to the electrode every
100 ms. HDCV software (provided by R.M. Wightman,
University of North Carolina, Chapel Hill, NC) was used for
data collection and analysis. PCIe-6363 (National
Instruments, Austin, TX) was used to apply FSCV waveform
and to collect data.
Statistical analysis
All statistical analyses were performed using GraphPad Prism
7.02 (GraphPad Software, Inc., La Jolla, CA). Error bars rep-
resent mean ± standard error of the mean.
Results and discussion
Capillary electrophoresis and fast-scan cyclic
voltammetry
The objective of this study was to analyze and compare vari-
ation in 4 neurotransmitters in single brains across 9 colonies
of the red harvester ant (P. barbatus). This is the first study
reporting the quantitative measurements of all major biogenic
amines from foraging ants collected from a natural environ-
ment. CE-FSCV separation had not been used for ant brains
before, but procedures were adapted from Drosophila studies
[14]. The amount of neurotransmitter in each ant brain is
small, so field-amplified sample stacking was employed as a
preconcentration step to inject much of the analyte onto the
capillary in a narrow zone. Field-amplified sample stacking
works by using a lower conductivity sample buffer, here,
acetonitrile/water with perchloric acid to stabilize the amines.
This method is convenient because it allows one to dilute the
ant brain samples in slightly larger volumes (5 μl). For FSCV
detection, a standard waveform was applied to the CFME
scanning from −0.4 V to 1.3 V and back to −0.4 V at
400 V/s every 100 ms, which can detect all the compounds
of interest [14]. Serotonin, octopamine, and tyramine form
side products that can foul electrodes, and thus analyte-
specific waveforms have been developed [29, 30]. However,
fouling from side products is negligible in this study because
the amount of these neurotransmitters available in a fly brain
is small and thus byproduct formation is minimal. CE exper-
iments with standards were run before and after the tissue
samples, and sensitivity for the standards was the same for
pre- and post-experiment calibration runs. This standard
waveform has been previously used for detection of these
biogenic amines [14, 16].
Analysis of tissue content of biogenic amines
in a single ant brain
Figure 2 shows representative examples of CE-FSCV sep-
arations of biogenic amines in a standard solution and from
a single ant brain. The standard solution contains 50 nM
dopamine and octopamine and 25 nM serotonin and tyra-
mine. Each biogenic amine has a distinguishable cyclic
voltammogram (CV) which helps identify the analytes
(Fig. 2a, top). Dopamine and serotonin have one oxidation
and one reduction peak in their CVs at approximately
0.6 V and −0.2 V for dopamine, and 0.5 V and 0.1 V for
serotonin. However, serotonin also has an additional peak
both on the forward and backward scan due to the forma-
tion of a side product [31]. Tyramine exhibits a primary
oxidation peak at 1.1 V, a secondary oxidation peak at
0.6 V, and reduction peak at −0.2 V [29]. While these peak
potentials are similar to octopamine, due to their similar
Fig. 1 Image of capillary electrophoresis detection cell. A reference
electrode (RE) and ground wire are positioned inside of a detection cell.
A carbon-fiber microelectrode (CFME), used for detection, is placed
opposite of separation capillary, approximately 10 μm away
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chemical structures, they are clearly separated using CE
(Fig. 2a, center). Because all the neurotransmitters have
oxidation peaks around 0.6 V, a current vs time trace at
this potential is used to visualize all the peaks in one trace,
as in a traditional electropherogram. However, it is better
to visualize all the data in a color plot (Fig. 2a, bottom)
which represents potential on the y-axis, time on the x-
axis, and current in false color. The color plot, CVs, and
electropherogram are used to identify the separated
analytes from potential interferents. We estimate the limit
of detection (LOD, S/N = 3) was 1 nM for dopamine, 2 nM
for serotonin, 5 nM for octopamine, and 4 nM for tyra-
mine, similar to previously published values [14].
Figure 2b shows an example separation from a single ant
brain. As shown in the electropherogram (Fig. 2b, center), all
four neurotransmitters in the tissue sample were detected and
well resolved. The brain tissue sample is more complex, con-
taining other electroactive species that are not found in the
standard solution. For example, the color plot shows an extra
peak below the serotonin oxidation peaks in the potential win-
dow between 0.4 Vand 0.5 V. This extra peak for serotonin is
less intense but also appears in the standard solution and elutes
after the primary oxidation peak, indicating it is likely the
byproduct of serotonin oxidation. Similarly, for octopamine,
extra peaks are visible in the potential window between 0.4 V
and 0.5 V (on the forward scan) and 1.2 V and 1.1 V (on the
back scan), below and above the octopamine oxidation peak,
respectively. These extra peaks could be due to the formation
of side products or another electroactive compound that
coeluted. Previously, HPLC with amperometry detection
was used for Drosophila brain tissue analysis and an uniden-
tified analyte coeluted with octopamine, resulting in the over-
estimation of octopamine signal [32]. Here, FSCV provides a
better analysis of co-eluting interferent due to the color plot
that shows the response at all voltages and the electrophero-
gram extracted at the specific potential (0.6 V) bypassing the
interferent signals. Other than these possible interferent sig-
nals, the migration order and time of each neurotransmitter are
Fig. 2 Representative CE-FSCV data for (a) standard (50 nM dopamine
and octopamine and 25 nM serotonin and tyramine) and (b) single red
foraging ant brain sample from colony D30. Brain was prepared in 5 μl of
70% 5 mM HClO4:30% acetonitrile. Standard solution was injected prior
to the tissue sample on the same capillary. As shown in the electrophero-
gram, current versus time plot extracted at 0.6 V, neurotransmitters were
well separated and eluted in order of (1) tyramine (TYR), (2) serotonin (5-
HT), (3) octopamine (OCT), and (4) dopamine (DA). Each eluted
neurotransmitter is identified by its cyclic voltammogram (inset). The
color plot has time on x-axis, potential on y-axis, and current in false color
enabling to visualize each separated analyte. The carbon-fiber microelec-
trode was calibrated with a standard solution and a calibration factor was
used to convert current response in the tissue sample into concentrations
(and the amounts per brain): 17 nM (15 pg/brain) tyramine, 31 nM (33 pg/
brain) serotonin, 34 nM (32 pg/brain) octopamine, 59 nM (56 pg/brain)
dopamine
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the same as those from the standard sample and the color plot
shows no other signals.
To quantify the concentration of neurotransmitters in the
brain sample, the oxidation current of each analyte was con-
verted to concentration using the calibration factor from the
standard sample analysis. The example sample (Fig. 3b), from
colony D30, had 17 nM tyramine, 31 nM serotonin, 34 nM
octopamine, and 59 nM dopamine. Since each sample was
prepared with one brain in 5 μl solution, the concentration
can be back calculated to the amount of each neurotransmitter
in pg per brain. For this ant brain, the amount (pg/brain) was
15 for tyramine, 33 for serotonin, 32 for octopamine, and 56
for dopamine.
Comparisons of biogenic amines in different red
harvester ant colonies
Red harvester ants from 9 colonies were collected in the field
as described in Friedman et al. [4]. Figure 3 shows the data
from each individual ant graphed by neurotransmitter and col-
ony. Data are organized for dopamine in order of the most
dopamine content per colony to the least, and then the same
colony order is used for the other biogenic amines. Overall,
the most abundant biogenic amine content in forager brains
(n = 53 from 9 colonies) was dopamine (47 ± 9 pg/brain),
followed by octopamine (36 ± 10 pg/brain), serotonin (20 ±
4 pg/brain), and tyramine (14 ± 3 pg/brain). One-way
ANOVA shows the significant main effect of colony on tissue
content of each neurotransmitter: for dopamine, F(8, 45) = 7.05,
p < 0.0001; for serotonin, F(8, 45) = 3.199, p = 0.0058; for
octopamine, F(8, 44) = 4.676, p = 0.0003; for tyramine, F(8,
44) = 3.003, p = 0.0089. One interesting observation from this
data is that colony D24 has a higher tissue content of every
neurotransmitter than other colonies. For instance, D24 has a
significantly higher tissue content compared with other colo-
nies except D29 and D33 for both dopamine and serotonin
and D27 only for serotonin. Similarly, D24 has greater
octopamine and tyramine tissue content than all other colonies
except D33, D25, and D19 for tyramine.
Additionally, we tested for correlations between average
values of neurotransmitters within each colony. Interestingly,
dopamine content was not correlated with tissue content of
serotonin (Pearson, r2 = 0.02, p = 0.71), octopamine (r2 =
0.16, p = 0.29), or tyramine (r2 = 0.004, p = 0.86). However,
octopamine content was strongly correlated with serotonin
(r2 = 0.80, p = 0.0011) and tyramine (r2 = 0.69 p = 0.0057),
and tyramine was correlated with serotonin (r2 = 0.66, p =
0.0076).
Figure 4 shows the same data as Fig. 3, but the four neu-
rotransmitters are grouped for each colony to observe the var-
iation of tissue content within each colony. A two-way
ANOVA shows a significant variation of neurotransmitter
Fig. 3 Comparison of natural
variations of (a) dopamine, (b)
serotonin, (c) octopamine, and (d)
tyramine levels (pg/brain) in
different red harvester ant
colonies. x-axis gives the different
colony ID in order from the
highest dopamine content to the
lowest. y-axis shows the tissue
content of biogenic amines in the
individual ant brain (pg/brain).
Points are individual ants, with
the bar being the average and
error bar the SEM. The data were
analyzed using one-way
ANOVA. There is a main effect of
colony on biogenic amine tissue
content; dopamine (F(8, 45) =
7.073, p < 0.0001), serotonin (F(8,
45) = 3.199, p = 0.0058),
octopamine (F(8, 44) = 4.676, p =
0.0003), and tyramine (F(8, 44) =
3.003, p = 0.0089). Post-test
differences between colonies are
marked. * p < 0.05, ** p < 0.01,
*** p < 0.0001, Turkey’s multiple
comparisons test
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tissue content within a colony (F (3,177) = 19.62, p < 0.0001),
especially in D24, D29, D33, and D26. In D24, the amount of
dopamine was significantly higher than both serotonin (p =
0.0002, n = 7) and tyramine (p < 0.0001, n = 7), and
octopamine was significantly higher than serotonin (p <
0.0001, n = 7) and tyramine (p < 0.0001, n = 7). Similarly, col-
onies D29 and D33 had higher dopamine content than seroto-
nin and tyramine (D29: serotonin p < 0.05 and tyramine p <
0.0001, n = 6; D33: serotonin and tyramine p < 0.05, n = 6). In
D26, dopamine was significantly more than tyramine
(p < 0.05, n = 6). In general, dopamine was the most abundant
biogenic amine in every colony except D24 and D30, which
had slightly higher octopamine levels. Tyramine was general-
ly lowest except for colony D19, which had lower serotonin
content. In each colony, tyramine content was lower than
octopamine content. Tyramine is the synthetic precursor to
octopamine; thus, higher octopamine could lead to lower ty-
ramine levels. This inverse relationship was also clearly
shown in different life stages of Drosophila [16].
In previous studies of other ant species, the dopamine tis-
sue content was significantly higher than other biogenic
amines, similar to red foraging ants here [33, 34]. Similar
patterns were observed in other insects, including
Drosophila larvae, where dopamine concentrations were
highest and tyramine concentrations the lowest, while tyra-
mine was higher than octopamine in adult brains [16]. In hon-
eybees, as in the ants reported here, dopamine tissue content in
the brain was found to be highest followed by octopamine,
serotonin, and tyramine. [35]. In higher order species, mam-
mals also have higher dopamine tissue content than serotonin
content [13, 36]. Thus, the patterns observed here for ant col-
onies are similar to those observed in other species.
The relative standard deviations (RSD) within and among
colonies were examined to understand variance. Table 1
shows the RSD for each colony for each neurotransmitter,
the average of those values, as well as the overall RSD when
the individual values of all the colonies are pooled together.
Variances within a colony in the level of each neurotransmitter
were lower than the variance for all samples pooled, indicating
that differences among colonies are larger than variation with-
in colonies. Colony variance for each neurotransmitter (53%
for dopamine, 72% for serotonin, 63% for octopamine, and
65% for tyramine) was lower than overall RSDs (71% for
dopamine, 90% for serotonin, 125% for octopamine, and
126% for tyramine). Although D24 has significantly larger
tissue content of each neurotransmitter than other colonies,
the overall dopamine RSD when D24 is removed (68%) is
still larger than the mean RSD by colony. This analysis shows
that there is more variance in neurotransmitter content among
colonies than within a single colony. Therefore, there may be
environmental factors like food availability, heat, or humidity
promoting the variation in neurotransmitters between colonies
that contribute to the larger variance.
Variation of neurotransmitter levels among colonies could
be correlated with differences in expression or activity of syn-
thesis or metabolic enzymes. Dopamine is biologically de-
rived from L-tyrosine, which is converted to L-DOPA, by the
enzyme tyrosine hydroxylase [37]. L-tyrosine is also the pre-
cursor for tyramine and octopamine, as they are synthesized
through tyrosine decarboxylase to tyramine and then through
tyramine-β-hydroxylase to octopamine [38]. Therefore, if
there is upregulation of L-tyrosine, levels of dopamine, tyra-
mine, and octopamine are expected to rise. For example, here
we found that the colony with the highest content for dopa-
mine, also had high content for octopamine and tyramine,
suggesting that L-tyrosine might have been high. However,
dopamine tissue content is not significantly correlated to that
of octopamine and tyramine. In comparison, octopamine and
tyramine contents were highly correlated, likely due to their
shared synthesis pathway. Serotonin, on the other hand, has a
completely separate synthesis pathway with tryptophan as the
synthetic precursor. However, serotonin is significantly corre-
lated with octopamine and tyramine content across colonies,
suggesting that colonies may vary in amino acids intake, like
L-tyrosine and tryptophan, or in the expression of key en-
zymes that cause the broad upregulation of synthesis
pathways.
Differences in neurotransmitter content across colonies
may also be due to differences in the brain metabolism of
biogenic amines. The main difference in metabolism between
Fig. 4 Tissue content of neurotransmitters within each colony. There was
a significant variation of tissue content within a colony (F(3,177) = 19.62,
p < 0.0001, two-way ANOVA) and across the colonies (F(8, 177) = 14.6, p
< 0.0001). Significant differences in neurotransmitters were shown in
D24, D29, D33, and D26. In D24, dopamine was higher than serotonin
(p = 0.0002, Tukey’s multiple comparison test) and tyramine (p < 0.0001)
and octopamine was higher than serotonin (p < 0.0001) and tyramine
(p < 0.0001). In D29, dopamine was higher than serotonin (p < 0.05)
and tyramine (p < 0.001). D33 had higher dopamine content compared
with serotonin, octopamine, and tyramine (p < 0.05) whereas dopamine
was only higher than tyramine in D26 (p < 0.05)
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insects and mammals is that monoamine oxidase, the main
metabolic enzyme in mammals, is not present in insects
[39]. The primary dopamine metabolic pathway in insects
occurs through N-acetylation by the enzyme, arylalkylamine
N-acetyltransferase (aaNAT), which converts dopamine to N-
acetyl dopamine (NADA). NADA is taken up either by glial
cells in the brain or cuticle in sclerotization [40]. Dopamine
can also be metabolized by Ebony to β-alanyl-dopamine
(BADA), which is directly converted back to dopamine by
Tan, BADA hydrolase [23]. The aaNAT enzyme is involved
in the metabolic pathway of all the biogenic amines, thus their
tissue contents would be expected to change similarly if
aaNAT activity was affected.
A previous study demonstrated that dopamine plays a cen-
tral role in regulating foraging activity in the harvester ants [4].
Ants treated with dopamine at a dose that increased brain
dopamine titer increased their foraging activity compared to
control ants treated with buffer. Here, there was a trend toward
a significant correlation between dopamine tissue content and
the changes in foraging trips after exogenous dopamine treat-
ment (Fig. 5). Colonies with higher dopamine levels have less
increase in foraging activity after dopamine treatment, which
implies exogenous dopamine treatment could be ineffective at
promoting foraging activity if endogenous dopamine levels
are already elevated (Fig. 5, Pearson’s r2 = 0.55, p < 0.05).
However, this effect is largely driven by the one colony with
7large amounts of dopamine (colony D24) and so future
studies are needed to understand the relationships be-
tween ant behavior and biogenic amine content. For
example, biogenic amines could be correlated with col-
ony behaviors such as foraging or as they change with
environmental conditions such as humidity. A future
study might investigate metabolites along with biogenic
amines to determine if metabolism changes are correlat-
ed with foraging behavior among colonies in the field.
Conclusions
Capillary electrophoresis with fast-scan cyclic voltammetry
(CE-FSCV) was used to separate and quantify dopamine, se-
rotonin, octopamine, and tyramine in single harvester ant
brains. This is the first time quantifying biogenic amines in 9
different ant colonies that were obtained in the field. Levels of
neurotransmitters are about 10–100 pg of each neurotransmit-
ter per brain, with more dopamine than serotonin. There was
significant variation among colonies in each biogenic amine
were observed across colonies, with larger differences be-
tween than within colonies. Colonies that had higher dopa-
mine content were less responsive to foraging activity in re-
sponse to dopamine treatment. Future studies could investi-
gate the effect of environmental conditions, like humidity, on
each neurotransmitter within and among colonies.
Furthermore, CE-FSCV could be used to examine natural var-
iations of neurotransmission among colonies by analyzing
metabolites.
Funding information This research was funded by NIH R01MH085159
to the Venton Lab and a grant from the Stanford Neurosciences Institute
to the Gordon lab.
Data availability The data supports the finding of this study are available
in figshare [DOI: https://doi.org/10.6084/m9.figshare.10023080].
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of
interest.
Fig. 5 Comparison of dopamine content in each colony to exogenous
dopamine treatment response. (a) Left and right y-axis represent DA
content (pg/brain, n = 6 or 7 in each colony, black squares) in a single
forager brain and foraging activity in response to orally administrated
dopamine (3 mg/ml, blue dots) from 9 different colonies, respectively.
The foraging activity behavior data is reprinted from Friedman et al. [4].
Colonies with less dopamine content available in the brain were more
responsive to exogenous dopamine treatment. (b) Correlation of
dopamine tissue content and response to exogenous dopamine. There
was a significant correlation (Pearson’s r2 = 0.55, p < 0.05)
Table 1 Relative standard
deviations (RSDs) of measured
neurotransmitter in each colony
RSD (%)
D24
D29
D33
D26
D25
D19
D30
D36
D27
Average
colony
RSD
Overall
RSD (not
by colony)
Dopamine
25
54
53
43
84
30
70
81
39
53
71
Serotonin
57
77
95
82
136
24
75
71
32
72
90
Octopamine
77
77
48
82
67
25
74
75
39
63
125
Tyramine
77
67
100
61
69
44
105
27
43
66
106
6173
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## Page 8

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## Page 9

Mimi Shin is a postdoctoral re-
searcher in the Department of
Chemistry at the University of
Virginia. She received her Ph.D.
from the University of Kansas in
2016. Her research interest is in
developing various analytical
techniques, using electrochemis-
try and imaging, to characterize
neurotransmitter release in the
brain of various animal models
from insects to rodents.
Daniel Ari Friedman is currently
a postdoctoral researcher in
Entomology at the University of
California, Davis. He received
his Ph.D. from Stanford
University in 2019, advised by
Professor Deborah Gordon, in
the Department of Biology. He is
interested in the evolution of ge-
netics, physiology, and colony
traits in eusocial insects.
Deborah M. Gordon is Professor
in the Department of Biology at
Stanford University. Her lab
group studies the collective regu-
lation of behavior in ants and how
it functions ecologically.
B. Jill Venton is Professor and
Chair of the Department of
Chemistry at the University of
Virginia. She is also affiliated
with the Neuroscience Graduate
Program and Brain Institute. Her
research interests are in develop-
ing new electrochemical and sep-
arations techniques for measure-
ments of neurotransmitters in tis-
sue.
6175
Measurement of natural variation of neurotransmitter tissue content in red harvester ant brains among...


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*Extraction method: pymupdf*