Long-Term Overconsumption of Sugar Starting at Adolescence Produces Persistent Hyperactivity and Neurocognitive Deficits in Adulthood

Abstract

Sugar has become embedded in modern food and beverages. This has led to overconsumption of sugar in children, adolescents, and adults, with more than 60 countries consuming more than four times (>100 g/person/day) the WHO recommendations (25 g/person/day). Recent evidence suggests that obesity and impulsivity from poor dietary habits leads to further overconsumption of processed food and beverages. The long-term effects on cognitive processes and hyperactivity from sugar overconsumption, beginning at adolescence are not known. Using a well-validated mouse model of sugar consumption, we found that long-term sugar consumption, at a level that significantly augments weight gain, elicits an abnormal hyperlocomotor response to novelty and alters both episodic and spatial memory. Our results are similar to those reported in attention deficit and hyperactivity disorders. The deficits in hippocampal-dependent learning and memory were accompanied by altered hippocampal neurogenesis, with an overall decrease in the proliferation and differentiation of newborn neurons within the dentate gyrus. This suggests that long-term overconsumption of sugar, as that which occurs in the Western Diet might contribute to an increased risk of developing persistent hyperactivity and neurocognitive deficits in adulthood.

Full text

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ORIGINAL RESEARCH
published: 07 June 2021
doi: 10.3389/fnins.2021.670430
Edited by:
Vittorio Calabrese,
University of Catania, Italy
Reviewed by:
Nafisa M. Jadavji,
Midwestern University, United States
Yinghua Yu,
Xuzhou Medical University, China
*Correspondence:
Arnauld Belmer
arnauld.belmer@qut.edu.au
Selena E. Bartlett
selena.bartlett@qut.edu.au
†These authors share senior
authorship
Specialty section:
This article was submitted to
Neuroenergetics, Nutrition and Brain
Health,
a section of the journal
Frontiers in Neuroscience
Received: 22 February 2021
Accepted: 22 April 2021
Published: 07 June 2021
Citation:
Beecher K, Alvarez Cooper I,
Wang J, Walters SB, Chehrehasa F,
Bartlett SE and Belmer A (2021)
Long-Term Overconsumption of
Sugar Starting at Adolescence
Produces Persistent Hyperactivity and
Neurocognitive Deficits in Adulthood.
Front. Neurosci. 15:670430.
doi: 10.3389/fnins.2021.670430
Long-Term Overconsumption of
Sugar Starting at Adolescence
Produces Persistent Hyperactivity
and Neurocognitive Deficits in
Adulthood
Kate Beecher1, Ignatius Alvarez Cooper2, Joshua Wang1, Shaun B. Walters3,
Fatemeh Chehrehasa2†, Selena E. Bartlett1*†and Arnauld Belmer1*†
1Addiction Neuroscience and Obesity Laboratory, School of Clinical Sciences, Translational Research Institute, Faculty of
Health, Queensland University of Technology, Brisbane, QLD, Australia, 2Addiction Neuroscience and Obesity Laboratory,
School of Biomedical Sciences, Translational Research Institute, Faculty of Health, Queensland University of Technology,
Brisbane, QLD, Australia, 3School of Biomedical Sciences, University of Queensland, Brisbane, QLD, Australia
Sugar has become embedded in modern food and beverages. This has led to
overconsumption of sugar in children, adolescents, and adults, with more than
60 countries consuming more than four times (>100 g/person/day) the WHO
recommendations (25 g/person/day). Recent evidence suggests that obesity and
impulsivity from poor dietary habits leads to further overconsumption of processed
food and beverages. The long-term effects on cognitive processes and hyperactivity
from sugar overconsumption, beginning at adolescence are not known. Using a
well-validated mouse model of sugar consumption, we found that long-term sugar
consumption, at a level that significantly augments weight gain, elicits an abnormal
hyperlocomotor response to novelty and alters both episodic and spatial memory. Our
results are similar to those reported in attention deficit and hyperactivity disorders.
The deficits in hippocampal-dependent learning and memory were accompanied by
altered hippocampal neurogenesis, with an overall decrease in the proliferation and
differentiation of newborn neurons within the dentate gyrus. This suggests that long-
term overconsumption of sugar, as that which occurs in the Western Diet might
contribute to an increased risk of developing persistent hyperactivity and neurocognitive
deficits in adulthood.
Keywords: sucrose, hyperactivity, neurocognitive deficits, neurogenesis, adulthood
INTRODUCTION
The concept of “sugar addiction” and the classification of sugar as a substance of abuse are still
debated. There is, however, increasing evidence of overlap in the brain circuitry and molecular
signaling pathways involved in sugar consumption and drug abuse (for recent review see Jacques
et al., 2019). Humans consume sugar and food to regulate homeostatic energy balance, but also
for pleasure and comfort. This hedonistic desire for palatable food is reward-driven and overeating
may result in maladaptive/negative neuroplasticity that overrides homeostatic regulation (Kenny,
2011). In humans, sugar and sweetness can induce dopamine release, reward and craving that are
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Beecher et al. Sucrose Consumption and Neurocognitive Deficits
comparable in magnitude to those induced by addictive drugs,
suggesting that sugar changes brain reward signaling and
circuitry similar to other drugs of abuse (Avena and Hoebel,
2003b;Rada et al., 2005;Lenoir et al., 2007;Klenowski et al., 2016;
Shariff et al., 2016, 2017).
High sugar and/or high fat diets have been shown to
precipitate addiction-like psychiatric phenotypes in a number of
rodent studies (Avena et al., 2009;Avena, 2010;Criscitelli and
Avena, 2016). In rats, intermittent consumption of 10% (w/v)
sucrose or 25% (w/v) glucose solution) elicits hallmark signs
of addictive behavior such as binging, tolerance, craving (Rada
et al., 2005), cross-sensitization (Avena and Hoebel, 2003b) and
symptoms of withdrawal (Colantuoni et al., 2002;Avena et al.,
2008) such as anxiety-(Colantuoni et al., 2002;Avena et al., 2008;
Parylak et al., 2012;Eudave et al., 2018;Gueye et al., 2018;Xu and
Reichelt, 2018) and depressive-like behaviors (Vollmayr et al.,
2004;Iemolo et al., 2012;Harrell et al., 2015;Santos et al., 2018).
In addition, sugar consumption has been shown to increase
reward seeking, impulsivity to feed and compulsivity in rats
willing to endure noxious stimuli such as extreme cold, heat and
foot-shock to procure sugar and highly palatable foods (Cabanac
and Johnson, 1983;Avena et al., 2005;Foo and Mason, 2005;
Oswald et al., 2011). Interestingly, rats are also more resilient
to foot shock punishments when seeking for palatable food
compared to methamphetamine (Krasnova et al., 2014).
Increasing evidence shows that unrestricted consumption
of high-sugar food and beverages within the Western Diet
might be linked to the increased obesity epidemic (Stanhope,
2016;Johnson et al., 2017;Freeman et al., 2018;Yoshida and
Simoes, 2018;Sigala and Stanhope, 2021). A strong association
between attention-deficits/hyperactivity disorders (ADHD) and
overweight/obesity have further been revealed (Altfas, 2002;
Strimas et al., 2008;Cortese et al., 2016;Cortese, 2019).
Taken together, these data suggest that sugar-induced obesity
may participate to the developing pathogenesis of ADHD-
like symptoms in western countries. In children, high sugar
consumption correlates with hyperactivity (Kim and Chang,
2011) and in adults, with inattention and impulsivity (Li et al.,
2020). However, some inconsistencies remain regarding the
potential correlation (Yu et al., 2016;Farsad-Naeimi et al., 2020)
or not (Del-Ponte et al., 2019) with ADHD (Johnson et al.,
2011;Paglia, 2019). In rodents, high-sucrose consumption also
impairs neurocognitive functions such as spatial learning, object
recognition, behavioral inhibition and fear-memory (Kendig,
2014;Reichelt et al., 2015;Kruse et al., 2019;Spoelma and Boakes,
2021). Interestingly, high sucrose intake during pregnancy
elicits ADHD-like behavioral phenotypes in mice offspring, with
increased locomotor activity, reduced attention/learning and
impulsivity (Choi et al., 2015). Anxiety, depression, and cognitive
deficits are strongly associated with impaired hippocampal
neurogenesis in animal models, although evidence for a causative
relationship is often lacking. Indeed, anxiety and spatial memory
deficits elicited by long-term consumption of sucrose are
accompanied by alterations in hippocampal neurogenesis and
physiology (Molteni et al., 2002;Stranahan et al., 2008;van
der Borght et al., 2011;Lemos et al., 2016;Reichelt et al.,
2016). While drugs of abuse such as ethanol are known to
negatively affect neurogenesis, the effect of high levels of sugar
consumption requires further characterization since link between
neurogenesis to anxiety and depression has not been fully
explored (Xu and Reichelt, 2018).
Sucrose became embedded in modern food and beverages, and
the aforementioned studies suggest that sugar overconsumption
satisfies all criteria for the classification of sugar as a drug of
abuse, with its chronic abuse proposed to produce overweight,
locomotor, emotional and cognitive impairments. However, it
remains unclear whether a lifetime of chronic overconsumption
of sucrose, starting at adolescence, affects locomotor behavior,
emotions and cognition through adulthood. Therefore, we used
a mouse model of long-term intake of sucrose to determine
the effects on locomotion, anxiety, memory, and hippocampal
neurogenesis. Our results show for the first time that long-
term consumption of sucrose leads to significant weight gain
and produces persistent hyperactivity and learning impairments,
correlated to reduced hippocampal neurogenesis in adult mice.
These results suggest that long-term sugar intake in the Western
Diet might play a role in the pathogenesis of attention deficits and
hyperactivity-related disorders.
MATERIALS AND METHODS
Animals and Housing
Five-week-old male C57BL/6J mice (ARC, WA, Australia) were
individually housed under reverse-light cycle conditions (lights
off at 9:00 am) in a climate-controlled room with ad libitum
access to food (standard mouse chow) and water. Following
one week of habituation to the housing conditions, mice were
offered sucrose or water during the drinking sessions. All
procedures were approved by The University of Queensland
and The Queensland University of Technology Animal Ethics
Committees under approval QUT/053/18 and complied with
the policies and regulations regarding animal experimentation
and other ethical matters, in accordance with the Queensland
Government Animal Research Act 2001, associated Animal
Care and Protection Regulations (2002 and 2008), as well as
the Australian Code for the Care and Use of Animals for
Scientific Purposes, 8th Edition (National Health and Medical
Research Council, 2013).
Sucrose Consumption
All mice had food and acidified-filtered water available at all
times. The sucrose solution was freshly prepared weekly and was
presented in 50 ml plastic falcon tubes fitted with rubber stoppers
and a 6.35 cm stainless-steel sipper tube with double ball bearings.
Mouse weights were measured daily to calculate the adjusted
g/kg intake of sucrose. Two groups of mice (n= 23 mice/group)
had ad libitum access to 25% sucrose or water for 12 weeks.
Briefly, mice were given access to one bottle of 25% (w/v) sucrose
solution and one bottle of water available at all times, or two
bottles of water (controls) available at all times. Sucrose and
water containing bottles were weighed daily. Two other groups
of mice (n= 23 mice/group) were trained in the in a restricted
access model of sugar consumption (Drinking-In-the-Dark) for
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12 weeks as previously described (Rhodes et al., 2005;Patkar et al.,
2017, 2019;Belmer et al., 2018). Briefly, mice were given access
to one bottle of 25% (w/v) sucrose for a 2 h period Monday to
Friday. Drinking sessions started 3 h into the dark cycle. Sucrose
containing bottles were weighed prior to presentation, as well as
2 h after presentation.
Behavioral Testing
Following 5 weeks of sucrose consumption, one set of animals
undertook five behavioral tests were conducted over 6 weeks
(n= 8 per group; Figure 1A). These behavioral tests were
conducted during dark cycle and were withdrawn from sucrose
for 24 h. Elevated-plus-maze (EPM), a behavioral test used to
observe anxiety-related behavior, was performed in an apparatus
comprising of four arms, 2 open arms and 2 closed arms
(35 cm ×5 cm), elevated 50 cm above the floor. The closed
arms were fenced with 40 cm high walls. The experiment went
for 5 min, with initial mouse placement in the center, facing the
open arm. The number of entries and time spent in each arm
was recorded using ANY-maze tracking software (Stoelting, IL,
United States) (Walf and Frye, 2007;Belmer et al., 2018).
Novelty suppressed feeding (NSF) test measures the novelty
induced anxiety/impulse control by assessing the latency to
approach and eat familiar food in a novel/aversive environment.
Animals were food-deprived for 16 h before being placed in an
open area (L = 40 cm, W = 36 cm, H = 18 cm) with new bedding
and a piece of familiar chow in the middle of the area. Latency to
feed was measured in seconds before the animal eats the food, by
two experimenters blind to the diet (Bevilacqua et al., 2010).
Marble burying (MB) is used to test anxiety and obsessive-
compulsive disorder-like behavior. MB was performed in novel
individual plastic cages (21 cm ×38 cm ×14 cm) containing
5 cm thick sawdust bedding. Ten glass marbles (diameter 10–
12 mm) were evenly spaced in 2 rows of 5 marbles on the bedding.
After 20 min, the number of unburied marbles was averaged
from counting by two experimenters. A marble covered at least
two-third (2/3) of its size by saw dust will be considered as
“buried”(Deacon, 2006;Belmer et al., 2018).
Open-field (OF) test is used to measure exploratory behavior,
general locomotor behavior and anxiety. OF was performed in
an open arena of 30 cm ×30 cm ×40 cm. The floor was
divided into 16 equal squares (7 cm ×7 cm) and a central
region of 10 cm ×10 cm was considered as the center, outside
this central region was considered as the periphery. Mice were
initially placed in one corner, and allowed to explore freely for
10 min. The number of entries and the time spent in the center or
periphery were recorded using the ANY-maze software (Bailey
and Crawley, 2009;Belmer et al., 2018). General locomotor
activity was assessed using the open field apparatus and recorded
using ANY-maze software.
The forced swimming test (FS) is commonly used to test the
efficacy of antidepressants and by extension, to assess depressive-
like behaviors. The FS test was conducted in a cylindrical glass
container measuring 50 cm in height and a diameter of 20 cm.
The immobility time was recorded using ANY-maze tracking
software (Yankelevitch-Yahav et al., 2015).
Memory Assessment
Using a separate group of animals on the same drinking protocol,
two recognition/spatial memory tests were conducted (n= 15 per
group, Figure 3A). After 8 weeks of 25% sucrose consumption,
recognition (episodic) memory was assessed using novel object
recognition (NOR) test. NOR protocol ran over 4 days and was
performed in the open field apparatus. On the first day, mice were
habituated to the open field apparatus for 10 min. The second
and third day, mice were presented with two identical objects for
10 min. No animals were excluded from analysis based on their
object exploration times. On the last day, under 24 h withdrawal
to 25% sucrose solution, one of the two familiar objects was
replaced with a novel object (Figure 3D) and the interactions
with the objects, including metrics such as object exploration time
and latency to reach the novel and familiar objects were recorded
on ANY-maze software (Leger et al., 2013).
The same animals continued to drink 25% sucrose for 2 more
weeks (Week 10) before spatial recognition was measured using
the Morris Water Maze. This test evaluates the animal’s ability to
escape a stressful situation in a large pool of water. The heated
pool (22◦C; 150 cm diameter) was divided into 4 quadrants
with a designated visual cue in each quadrant (Figure 3G).
We used a 4-day protocol (Flores-Ramirez et al., 2019) starting
with a pre-training/habituation day where the location of the
escape platform was introduced with the platform being visible
1 cm above the water surface. Each animal was placed on the
platform for 10 s before being released into the water facing the
platform, less than 10 cm away. Once the platform was reached,
place mouse onto the platform for 10 s then released again at a
greater distance (20 cm). This was repeated for a third time with
the release greater again (30 cm). If platform was not reached,
experimenter gently guided the mouse to the platform and the
animal was released in the water again until the mouse swam
to the platform unaided. On day 2 (training day), the platform
was partially covered by water and odorless non-toxic paint was
added so the platform was not visible. Each animal had 8 trials
of 60 s duration to swim to the platform, being released from
each quadrant while facing the wall twice in a random order.
Between trials the mouse had a 45 s break before the next trial
commenced in a warmed holding cage. On day 3 (testing day),
performed under 24 h withdrawal from 25% sucrose solution,
each animal was released from the furthest quadrant to reach
the platform and were removed from the pool if they did not
reach the platform in 60 s. The time spent to reach platform was
recorded manually having up to 1 min to reach the platform.
Testing videos was recorded using ANY-maze. Day 4 involved
probing the anima’s memory, with the platform being removed
and the amount of time spent in the quadrant or in platform area
recorded using ANY-maze. Heating pads and lamps were used
and readily available across all four days.
Neurogenesis
Following 12 weeks of sucrose drinking, a total of three
intraperitoneal injections of the cell proliferation marker, 5-
ethynyl-20-deoxyuridine (EdU; 50 mg/kg) were administered
over 2 weeks (days 0, 7, and 15) as previously described
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(Belmer et al., 2018;Patkar et al., 2019). This dose has been
reported to label all actively dividing precursors in the mouse
subgranular zone (Mandyam et al., 2007). One week after
the last EdU injection, animals were deeply anesthetized with
sodium pentobarbital (100 mg/kg, Lethobarb, Virbac, Australia)
and transcardially perfused with 4% paraformaldehyde. Brains
were harvested and postfixed in the same fixative overnight
at 4◦C. Thirty micron-thick coronal vibratome sections were
collected and kept floating in ice-cold 0.1M phosphate buffer
saline (PBS). Sections containing the hippocampus were selected
for immunohistochemistry. The sections were permeabilized
in 1% Triton X100, 0.1% Tween20 in PBS for 1 h at room
temperature and incubated with EdU Click-iTTM EdU Alexa
FluorTM 488 Imaging Kit as per supplier recommendation
(Thermo-Fisher Scientific, C10637). When required, antigen
retrieval (0.05% Tween-20 in 10 mM sodium citrate, pH 6, 5–
15 min at 80◦C) was performed. After three washes in PBS,
sections were incubated in blocking solution (2% normal goat
serum, 0.3% Triton X100 and 0.05% Tween-20) for 1 h at
room temperature. The sections were then probed for markers
of each stage of neurogenesis (Figure 3A) and incubated with
primary antibodies, diluted in blocking solution, for 24 h at
room temperature, washed 3 times in blocking solution and
then incubated with corresponding secondary antibodies, for
2 h at room temperature (Supplementary Table 1). When
biotinylated secondaries were used, sections were incubated in
streptavidin-CY3 for 30 min at room temperature. Sections
were mounted in Prolong gold antifade mountant with DAPI
(Thermofisher Scientific).
Imaging and Analysis
Four coronal sections of whole dentate gyri per animals
were imaged on the Leica DMi8 SP8 Laser Point Scanning
confocal microscope using a 40 ×objective (NA 0.85), x0.5
numerical zoom and 0.5 z-step. Consecutive sections were
used across all staining groups. Images were deconvolved
using Huygens professional v16.10 (Scientific Volume
Imaging, Netherlands) and converted in.tif for subsequent
quantification in Neurolucida 360 (MBF Bioscience). Early stages
of neurogenesis were counted: stage 1: EdU+/GFAP+/Nestin−;
stage 2: EdU+/Nestin+/GFAP−and stage 3: EdU+/DCX+as
well as glial cell types: astrocytes (EdU+/GFAP+), microglia
(EdU+/IBA-1+) and oligodendrocytes (EdU+/Olig 2+), see
Figure 4A. Quantification was performed by an experimenter
blind to the treatment, averaged per animal and plot as
mean ±SEM for each group. Density of counted cells was
normalized to the volume of granular cell layer sampled in each
group as previous described in Belmer et al. (2018) and Patkar
et al. (2019).
Statistics
Comparisons between groups were statistically analyzed using
t-test, one-way or two-way ANOVA, as appropriate, followed by
a Bonferroni-multiple comparison post hoc test using GraphPad
Prism 8 (Graph Pad Software Co., CA, United States). Pvalues
<0.05 were considered significant. All values are expressed as
the mean ±SEM.
RESULTS
Long-Term Sucrose Intake Increases
Weight Gain
We assessed the effects of long-term unrestricted access to
sucrose on body weight over 12 weeks (60 exposures days).
After 12 weeks of access to 25% sucrose, mice exhibited
stable levels of sucrose intake around 80–90 g/kg/day (mean
82.2 ±0.9 g/kg/24 h indicated by the red line; Figure 1B).
A significant increase in overall weight was observed, starting
around 4 weeks and increasing throughout the 12 weeks of
exposure (Figure 1C, Mixed effect repeated measure two-way
ANOVA, F(58,1276 )= 9.265, p= 0.0001) until reaching 10.6%
overweight (Bonferroni multiple comparison: 33.22 ±0.85 g vs
30.03 ±0.59 g, p<0.0002) compared to water controls.
Long-Term Sucrose Intake Does Not
Produce Anxiety- and Depressive-Like
Behavior
Sugar-withdrawn rats consistently exhibit both anxious and
depressive-like symptoms (Vollmayr et al., 2004;Avena et al.,
2008;Iemolo et al., 2012;Parylak et al., 2012;Harrell et al.,
2015;Eudave et al., 2018;Santos et al., 2018). One study in
C57BL/6 J mice consuming high levels of 10% sucrose (around
72 g/kg/24 h) for 4 weeks, found increased anxiety- (EPM) and
depressive-like behavior (tail suspension test) after one week of
withdrawal (Kim et al., 2018). To evaluate the effect of higher
level of sugar intake (around 85 g/kg/24 h) after 6 weeks of
exposure on emotional behavior, we assessed withdrawal-induced
anxious and depressive-like behaviors in the EPM, MB, OF, and
FS tests, 24 h after the last drinking session of the week. The
number of open arm entries within 5 min was similar in the EPM,
between sucrose and water control animals suggesting no change
in anxiety-related behavior (Figure 1D, ns, p= 0.5690, t-test).
The MB test also yielded a similar result with water- and sugar-
exposed animals burying a similar amount of marbles (Figure 1E,
ns, p= 0.9062, t-test). The OF test showed no difference in
the number of entries into the center between sucrose and
water exposed animals, hence confirming the absence of anxiety-
like behavior following long-term unrestricted access to sucrose
(Figure 1F, ns, p= 0.3583, t-test). No change in immobility time
in the FS was evident in sucrose consuming animals suggesting
no depressive-like symptoms (Figure 1G, ns, p= 0.6654, t-test).
Long-Term Sucrose Intake Produces
Hyperactivity/Hyperlocomotion
There is growing evidence showing that addiction and substance
dependence strongly rely on increased arousal, hyperactivity,
impulsivity and compulsion following cessation (Crews and
Boettiger, 2009). To understand how sucrose overconsumption
affects hyperactivity and compulsion, we assessed general
locomotor activity using OF. To evaluate impulse control we
used the NSF, conflict based anxiety test that has previously been
shown to reflect impulsivity-like behavior (Bevilacqua et al., 2010;
Angoa-Pérez et al., 2014;Piggott et al., 2020).
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FIGURE 1 | Long-term sucrose consumption increases mouse weights without altering emotional behavior. (A) Experimental design of the anxiety-related,
depression-related, general locomotor activity and impulse control behavioral testing in elevated-plus-maze (EPM), novelty suppressed feeding (NSF), marble burying
(MB), open field (OF) and forced swimming test (FS). Animals consumed 25% sucrose for 5 weeks prior to each behavioral testing and continued to be exposed to
sucrose for a total of 12 weeks. Behavioral tests were initially conducted 2 weeks (2 wk) then continued every 5 days (5d) after 24 h of sugar withdrawal. Animals
were assigned into two groups: sugar-withdrawn animals and water control. (B) Mice exhibit stable levels of long-term sucrose intake of 82.2 ±0.9 g/kg indicated
by the red line. (C) There was a significant difference in the weights of animals consuming sucrose, compared to water control, starting after 4 weeks, and continuing
throughout the 12 weeks. Data are presented as mean ±SEM; n= 8 mice/group, t-test, ***p<0.001. (D–F) Long-term sucrose consumption does not alter
anxiety-related behavior as seen by no change in the number of entries in the open-arm of the EPM (D), number of marbles buried (E) and number of entries in the
center in the open field (F) compared to water controls. Long-term sucrose consumption does not induce depressive-like symptoms as seen by no differences in the
immobility time in the forced swimming test (G) compared to water controls. Data are presented as mean ±SEM; n= 8 mice/group.
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Sucrose consuming mice displayed a higher general
locomotor activity as observed by an increase in total distance
traveled (Figures 2B,C) compared to water control animals
(Figures 2A,C,p<0.0001, t-test). This hyperlocomotion
was accompanied by hyperactivity as evidenced by increased
average speed in center of the open-field apparatus (Figure 2D,
p= 0.0008, t-test), total average speed (Figure 2E,p<0.0001,
t-test) in the OF apparatus, and reduced latency to feed in the
NSF, compared to water controls (Figure 2F,p= 0.0005, t-test).
Combined with hyperactivity, this lack of novelty-induced
feeding inhibition could be interpreted as a reduction of impulse
control to anxiogenic environment and further suggests that
chronic sucrose consumption produces both hyperactivity and
impulsivity (Bevilacqua et al., 2010;Angoa-Pérez et al., 2014;
Piggott et al., 2020).
Long-Term Sucrose Intake Alters Both
Episodic and Spatial Memory
Since hyperactivity correlates with memory impairments (Ortega
et al., 2020), we assessed the consequences of chronic sucrose
consumption and subsequent hyperactivity on learning and two
types of memory, episodic and spatial, using the NOR and MWM
tests, respectively. Although sucrose consuming mice learn to
discriminate between old and new objects, the proportion of
interaction time with the novel object compared to old object was
significantly lower than water control mice at 5 min (p= 0.048)
and 10 min (p= 0.028) following the presentation of the objects
(Figure 3B, Two-way ANOVA, F (1,84)= 10.35, p= 0.0018),
hence showing a significant reduction of the area under curve
(Figure 3C,t-test, p= 0.0008).
In the MWM, sucrose and water drinking mice showed similar
latency to reach a visible platform, confirming that, albeit sucrose
mice being slightly overweight compared to water controls,
there was no alteration in learning or their swimming behavior
(Figure 3E,t-test, p= 0.63). However, when the platform
was hidden, sucrose-consuming mice took longer than water
consuming mice to reach the platform area (Figure 3F,t-test,
p= 0.025). These results suggest that chronic overconsumption of
sucrose alters both episodic and spatial memory without affecting
the learning process.
Long-Term Sucrose Intake Decreases
Hippocampal Cell Proliferation and
Neurogenesis
Anxiety, depression, and cognitive deficits are strongly associated
with alteration in hippocampal neurogenesis. Although, sucrose
did not elicit anxiety- or depression-like behavior, we assessed
whether sucrose-induced deficits in memorization was associated
with changes in hippocampal neurogenesis (Figure 4A). Mice
chronically consuming a highsucrose diet showed a reduction in
the overall density of dentate gyrus proliferating cells (EdU+)
compared to water controls (Figure 4B,p= 0.0332, t-test). This
decrease in cell proliferation was likely mediated by decreased
neurogenesis, as evidenced by a reduction in the density of both
putative stem cells (stage 1: EdU+/GFAP+/Nestin−;Figure 4C,
p= 0.0127, t-test) and neuronal progenitors (newborn neurons,
stage 2: EdU+/GFAP−/Nestin+;Figure 4D,p= 0.0106, t-test)
compared to water controls, suggesting that continuous sucrose
intake alters the transition or differentiation of progenitors
into the proliferating phase. No change within differentiated
neuroblasts was observed (stage 3; EdU+/DCX+;p= 0.2309,
t-test, not shown) suggesting sucrose consumption principally
affects the earlier stages of neurogenesis.
Restricting the Availability of Sucrose
Dampens the Neurocognitive Deficits
The WHO’s guidelines recommend restricting the availability of
sugar in the current diet and advocate a four-time reduction
of the daily intake of sugar. Therefore, we investigated the
consequences of restricting access to sucrose solution to only
2 h/day, on weight gain, locomotion, emotion, cognition,
and neurogenesis. Mice showed a daily intake of sucrose of
20.9 ±0.3 g/kg (Figure 5A), about 4 times less than when access
was unrestricted (Figure 5B,t-test, p<0.0001). This lower
daily intake was associated with no change in weight gain over
12 weeks of exposure, compared to water controls (Figure 5C,
Two-way ANOVA, F (1, 11) = 0.3511, p= 0.5655).
We then evaluated if restricting access to sucrose alters
anxiety- and depression-related behavior in the same behavioral
tests. Restricting the access to sucrose to 2 h per day did not elicit
any change in anxiety-like behavior, as evidenced by no difference
in the number of open-arm entries in the EPM (Figure 5D,
t-test, p= 0.95), no difference in the number of marbles buried
(Figure 5E,t-test, p= 0.69) and no difference in the number
of entries in the center of the OF (Figure 5F,t-test, p= 0.11).
No difference was observed in the immobility time in the FS
(Figure 5G,t-test, p= 0.51) suggesting that restricting access to
sucrose does not elicit depressive-like behavior.
Interestingly, restricting access to sucrose still elicited
hyperactivity as shown by significant increases in the total
distance traveled (Figure 5H,t-test, p<0.0001), the speed
in the center (Figure 5I,t-test, p= 0.0002) and total speed
(Figure 5J,t-test, p<0.0001) in the OF, compared to water
control animals. However, there was no change in the latency to
feed compared to water controls (Figure 5K,t-test, p= 0.37).
These results suggest that restricting sucrose availability and
reducing overall daily intake does not affect the inhibitory control
to resist food, despite promoting hyperactivity. This increased
hyperactivity with no deficits in control to resist food was not
accompanied by any alteration in memory, as evidenced by no
change in the proportion of time interacting with a novel object
(Figure 5L,t-test, p= 0.97) or the latency to reach the platform
area in the MWM (Figure 5M,t-test, p= 0.10). This absence of
memory deficits was accompanied by no change in hippocampal
neurogenesis, however, an increased oligodendrogenesis was
observed in the dentate gyrus (Supplementary Figure 1).
DISCUSSION
Sugar became embedded in the food and beverage chains,
leading to overconsumption in children and adolescents (Han
and Powell, 2013;Dereñ et al., 2019). It is therefore important to
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FIGURE 2 | Long-term sucrose consumption produces hyperactivity and reduces the control to resist food. Tracking plot of water- (A) and sucrose-consuming mice
(B) illustrating that long-term sucrose consumption increases the total distances traveled in the open field (C), the speed in the center (D) and total speed (E) in the
open-field compared to water controls. Long-term sucrose consumption also reduced the latency to feed suggesting reduced control to resist food (F). Data are
presented as mean ±SEM; n= 8 mice/group. ***p<0.001, ****p<0.0001.
investigate how long-term intake of sugar, starting at adolescence,
leads to long-term effects into adulthood. The present study
shows there are deleterious effects of long-term sugar intake, on
weight gain, hyperactivity, impulsivity, and deficits in memory
and hippocampal neurogenesis.
While the WHO recommends that the amount of sugar in
sugar-sweetened be reduced by four-fold to decrease the risk of
childhood overweight and obesity (WHO, 2019), the impact of
sugar intake on the rise in obesity rates is still debated (Alexander
Bentley et al., 2020). Indeed, overall sugar consumption has
dropped since the mid-1990’s whereas the obesity rate has
continuously increased. It has been proposed that this rise
in obesity could result from a delayed effect of excess sugar,
suggesting that adult obesity could be driven by high sugar intake
over a life span (Alexander Bentley et al., 2020). There are many
ways that a decrease in overall sugar consumption and an increase
in obesity rates can be viewed. Sugar has hidden properties,
activates the hypothalamus, inhibits ghrelin and leptin, leading to
over-eating other types of foods (Jacques et al., 2019). It has been
shown that both obesity and sweet-taste can be passed on through
epigenetic modification (Öst et al., 2014;Donkin et al., 2016;
Ling and Rönn, 2019). This means that once obesity or sugar
preference has been established, it can be passed on in families
for up to 3 generations going forward.
In line with this, we observed that unrestricted access to sugar,
beginning at adolescence, only starts to affect weight gain into
adulthood, after 4–5 weeks of sucrose consumption. Although
rodent models of diet-induced obesity use body weight as a
measure of obesity, the effects of high-sugar diets on body weight
are inconsistent (Jurdak et al., 2008;Jurdak and Kanarek, 2009).
While we observed a significant increase in weight gain in sucrose
consuming animals, we cannot comment on the potential link
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FIGURE 3 | Long-term sucrose consumption alters episodic and spatial memory. (A) Experimental design of the testing of episodic and spatial memory in the
novel-object recognition (NOR) and Morris-watermaze (MWM) tests. Animals consumed 25% sucrose for 8 weeks prior to behavioral testing and continued to be
exposed to sucrose for a total of 12 weeks. Memory tests were conducted 3 weeks apart. Animals were assigned into two groups: sugar-withdrawn animals and
water controls. (B–D) Long-term sucrose drinking animals interact less with a novel object across the duration of the test, compared to water controls (B), as
confirmed by a reduction in the area-under-curve (AUC) (C). The 2 objects used alternatively in the novel-object-recognition test, which are discernible in shape, size
and color, are pictured in panel (D).(E–G) Long-term sucrose consuming mice showed no alteration in their learning and swimming behavior to reach a visible
platform (E), however, they showed an increased latency to reach the zone where the platform was previously placed (F) compared to water controls. Schematic
drawing of the MWM apparatus used is depicted in panel (G). Data are presented as mean ±SEM; n= 8 mice/group. *p<0.05, ***p<0.001.
between obesity and long-term unrestricted intake of sugar.
Further investigation of metabolic marker expression, such as
adiposity, insulin resistance, leptin, adiponectin, will be needed
to elucidate how long-term sucrose consumption predisposes to
obesity. Interestingly, we found that a 4-times reduction of daily
sucrose intake is able to prevent sugar-induced increase in weight
gain, supporting the WHO’s guideline to reduce the impact of
sugar on the rise of obesity rate.
We showed that long-term sucrose consumption did not
produce any anxiety- or depression-related behavior, although
previous studies have shown that acute and chronic withdrawal
from sucrose can induce anxious and depressive like behaviors
(Colantuoni et al., 2002;Avena et al., 2008;Iemolo et al., 2012;
Parylak et al., 2012;Harrell et al., 2015;Eudave et al., 2018;
Kim et al., 2018;Santos et al., 2018). Anxiety- and depression-
like behaviors are likely present if withdrawal follows extensive
periods of sucrose consumption. Short-term exposure to sucrose
(<1 month) does not lead to increased anxiety-like behavior in
rats (Parylak et al., 2012) while longer exposure (>1 month)
results in increased anxiety-like behavior 24 h after withdrawal
(Colantuoni et al., 2002;Avena et al., 2008). Interestingly, short-
term unrestricted access to 10% sucrose induced depression-
and anxiety-like behavior after one week withdrawal (Kim et al.,
2018). Since the aforementioned studies used different models of
sucrose consumption with concentrations of sucrose solutions
ranging from 7.9 to 35%, we cannot rule out an absence
of anxiety-like behavior in our study due to methodological
or interspecies differences. In addition, many studies incited
a binge-like patterns of sugar (10% sucrose or 25% glucose)
consumption by food-depriving the animals for 12 h before
the sucrose drinking sessions. The food deprivation/restriction
introduced in these studies may have changed motivational
states and reward seeking behavior, adding another level of
psychological stress (Toth and Gardiner, 2000). Strikingly,
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FIGURE 4 | Long-term sucrose consumption reduces the early phases of hippocampal neurogenesis. (A) Stages of neurogenesis in the dentate gyrus of the
hippocampus. In stage 1 (proliferation phase; in yellow), the newly generated cells express glial fibrillary acidic protein (GFAP) and are putative progenitor/stem cells
located in the subgranular zone (SGZ). The cells in stage 2 (differentiation phase; in green) will lose their GFAP and start expressing Nestin. This determines their fate
in the neuronal lineage. Stage 1 progenitors not only give rise to newborn neurons, they can also turn into glial cells. Glial cells [astrocyte and oligodendrocyte
progenitor cell (OPC)] can convert back into newborn neurons here in stage 2 and vice-versa. In stage 3, the immature neurons express doublecortin (DCX; in cobalt
blue) and have started to migrate into the granule cell layer (GCL) of the dentate gyrus. As the neuron matures, it will send its dendrites toward the molecular layer
(ML) of the dentate gyrus and extend their axonal projections toward the hippocampal CA3 pyramidal cell layer and will start losing their DCX and start expressing
postmitotic neuronal marker NeuN and calretinin (stage 4; magenta). As the neuron establishes synaptic contacts from the entorhinal cortex and it sends output to
the CA3 and hilus regions of the hippocampus the neuron is classified as in stage 5 (in cherry red). Stage 5 neurons start expressing calbindin and continue
(Continued)
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FIGURE 4 | Continued
expressing NeuN. Unlike the astrocytes and oligodendrocytes that are derived from the neuroectoderm, microglia (in cyan blue) are neuroglia derived by embryonic
mesoderm. Abbreviations used: GFAP, glial fibrillary acidic protein; OPC, oligodendrocyte progenitor cell; Olig2, oligodendrocyte lineage transcription factor 2; IBA-1,
ionized calcium binding adaptor molecule 1; DCX, doublecortin; NeuN, Fox-3, Rbfox3, or Hexaribonucleotide Binding Protein-3; SGZ, subgranular zone; GCL,
granule cell layer; ML, molecular layer [original drawing, created using Biorender, adapted from Lucassen et al. (2010)]. (B) Long-term sugar consumption reduced
the density of EdU positive cells (green) in the dentate gyrus of the hippocampus. (C) There was a reduction in the density of EdU+(green)/GFAP+(red)
immunoreactive cells indicating a reduction in stage 1 (putative stem cells) neurogenesis. (D) A reduction was also observed in the number of EdU+(green)/Nestin+
(red)-immunoreactive cells indicating a reduction in stage 2 (neuronal progenitors). All images are colocalized with DAPI (blue). Data are presented as mean ±SEM;
n= 8 mice/group. *p<0.05. Representative image scale bar is 100 µm and close up representative image scale bar is 10 µm.
longer sucrose exposure (>3 months) showed no significant
effects on anxiety- and depression-like behavior in rats (Cao
et al., 2007;Chepulis et al., 2009), hence suggesting that long
term (i.e., greater than 1 month but less than 3 months)
consumption of sucrose increases anxiety-like behavior, followed
by a return to baseline levels of anxiety- and depression-related
behavior at 3 months.
Overall, we observed no anxious or depressive behavior in
mice after 6–10 weeks of sucrose consumption. The reliability
of our findings is bolstered by the fact that we used two
different models, restricted and unrestricted. Perhaps sugar
does not produce the same emotional deficits between species.
An explanation could be due to differences metabolism, with
a well-described faster metabolism in mice compared to rats
(Radermacher and Haouzi, 2013). It may also be possible
that higher concentrations of sucrose (greater than 10%)
recruits different/additional neural circuits, involving other
behavioral deficits, such as hyperactivity and/or impulsivity
that mask the behavioral inhibition produced by anxiogenics
cues/environments.
Our results showed that long-term sucrose overconsumption
increases basal locomotor activity, which could be interpreted
as hyperactivity. There are limited studies examining the effect
of sucrose on locomotor activity, with, to the best of our
knowledge, only one study reporting no change in locomotor
activity in rats (Avena and Hoebel, 2003a). Our observation
of sugar-induced hyperactivity prompted the investigation of
the effect of long-term sugar consumption on impulse control
and inhibitory control to resist food. Interestingly, long-
term sucrose consumption reduced impulse control in the
novelty-suppressed feeding, and this was not observed when
sucrose access was restricted. Although novelty-suppressed
feeding test is primarily to assess anxiety-like behaviors,
studies have shown that a reduced latency to feed could be
correlated with augmented food seeking and increased meal
size (Biddinger et al., 2020), increased hunger and reduced-
feeding control after fasting (Burghardt et al., 2016), and
together with hyperlocomotion, increased motor impulsivity
(Bevilacqua et al., 2010;Angoa-Pérez et al., 2014;Piggott
et al., 2020). This suggests that the reduced latency to feed we
observed after long-term sucrose intake could be the result of
impulsivity. However, there remains disparity in the literature
regarding sugar’s effect on impulsivity in rats with studies
suggesting sugar does not (Stein et al., 2015;Wong et al.,
2017) and others supporting sugar does produce impulsivity
(Steele et al., 2017). Therefore, further investigation is required
to identify the mechanism underlying the effect of sugar
on locomotor and impulsive behavior in mice. This could
be explored further using delay discounting test (temporal
discounting) or 5-choice serial reaction time task (visual
attentional processes and impulse control) (Reichelt et al., 2015,
2016;Lemos et al., 2016).
Excessive sucrose consumption in adolescent rats has been
associated with deficits in spatial memory or object recognition
memory (Reichelt et al., 2015, 2016;Lemos et al., 2016), and
this could be principally mediated by the fructose component of
sucrose (Hsu et al., 2015). However, the link between memory
deficits and changes in hippocampal neurogenesis following
long-term sucrose consumption has been relatively unexplored.
In our study, reduction in hippocampal neurogenesis was only
observed when memory deficits were observed, for example,
when access to sucrose was unrestricted.
Indeed, no change in hippocampal neurogenesis was observed
when sucrose access was restricted, and memory not affected. Our
results further suggest that unrestricted sucrose consumption
likely affects neurogenesis by reducing cell proliferation,
generation of putative stem cell and survival/maturation of
newborn neurons.
Reduced cell proliferation followed by reduced production
of neuronal progenitors suggest reduced neurogenesis/turn over
(Cisternas et al., 2015). This result is in accordance with previous
ethanol studies in rodents suggesting sugar consumption is also
intervening at the G1 phase of the cell cycle, changing the
number of cells entering the S phase (Belmer et al., 2018;Patkar
et al., 2019). Interestingly, we did not see an effect on neuroblast
differentiation, suggesting the reduction in the initial number of
putative stem cells able to dedifferentiate and proliferate. This
could be due to the generation of pluripotent transit-amplifying
progenitor cells [TAPs (Potten and Loeffler, 1990)] that remain
in quiescence for long periods before differentiation (Doetsch
et al., 2002). It is possible that sugar reduces the number of TAPs
resulting in the changes observed here. Early-stage neurogenic
deficits have not been observed previously. Our results found
a reduction in putative stem cell and newborn neurons with
no change in differentiated neuroblasts. This absence of effect
of sugar on neuroblast differentiation has also been reported
in rats with unrestricted access to sucrose, therefore confirming
a degree of interspecies similarity. Another explanation of
the reduction in proliferating cells is cell death. Unrestricted
consumption of sugar has been shown to increase apoptosis
(TUNEL) suggesting our reduction in proliferating cells could
be due to neuronal death as a result of sugar overconsumption
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FIGURE 5 | Restricting access to sucrose consumption results in an absence of increased weight gain with no emotional and cognitive alterations. (A) Mice exhibit
stable levels of long-term sucrose intake of 20.9 ±0.3 g/kg indicated by the red line. This level of sucrose intake is four-fold lower than when sucrose access was
unrestricted (B) and was not associated with any changes in weight gain (C), or anxiety-related behavior as seen by no change in the number of entries in the
open-arm of the EPM (D), the number of marbles buried (E) and the number of entries in the center in the open field (F) compared to water controls. Restricting
long-term sucrose consumption did not induce depressive-like symptoms as seen by no differences in the immobility time in the forced swimming test (G) compared
to water controls. Restricted sucrose consumption increases the total distances traveled in the open field (H), the speed in the center (I) and total speed (J) in the
open-field, however, there was no change in the latency to feed (K), the time spent with a novel object (L) and the latency to reach the zone where the platform was
previously placed (M), compared to water controls. Data are presented as mean ±SEM; n= 8 mice/group. ***p<0.001, ****p<0.0001.
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Beecher et al. Sucrose Consumption and Neurocognitive Deficits
(van der Borght et al., 2011). Neuronal maturation and survival
have been reported to be reduced in the dentate gyrus of the
hippocampus after sugar consumption (van der Borght et al.,
2011;Cisternas et al., 2015). However, it is possible their protocol
of cell proliferation labeling (BrdU injection two weeks prior) has
misevaluated neuronal maturation and survival as the maturation
process of neurons can take up 1–2 months in rodents (Zhao
et al., 2008).
A limitation of this investigation is that the results may
not be applicable to female models of sugar overconsumption,
as only male mice were tested. C57/Bl6 male mice have both
increased overall activity and heightened anxiety when compared
to female mice particularly in maternal separation and chronic
stress models (Romeo et al., 2003;Veenema et al., 2007;Niwa
et al., 2011), meaning that subtle differences in the behavior of
male mice are more easily detected compared to female mice.
Female sex hormones also influence appetitive signaling in the
brain (Gao et al., 2007;Santiago et al., 2016), which therefore
increases the methodological complexity of including female
mice in this study, as all data would need to be normalized
to the estrous cycle. Females are widely underrepresented
in preclinical models of addiction (Beery and Zucker, 2011;
Shansky, 2019), with a majority of studies in this field conducted
exclusively on male mice. This is a major ongoing issue with
neuroscience research, and future investigations should explore
if similar behavioral consequences of sugar consumption are
present in female rodents. However, we believe the results of
this study provide foundational knowledge that can be extended
upon to benefit addiction and obesity generally. Together, our
study demonstrates that excessive sugar consumption starting
at adolescence elicits profound locomotor and memory deficits
in adulthood, that may mimic the hyperactivity and cognitive
dysfunctions observed in attention deficits and hyperactivity-like
disorders. More interestingly, our results reveal that restricting
sugar consumption intake, as recommended by the WHO, might
be effective in limiting the negative consequences of sugar on
obesity, and locomotor and cognitive impairments.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The animal study was reviewed and approved by The University
of Queensland and The Queensland University of Technology
Animal Ethics Committees under approval QUT/053/18.
AUTHOR CONTRIBUTIONS
KB, AB, and SEB were responsible for the study concept and
design. KB and IAC carried out the drinking experiments. KB,
AB, and IAC performed behavioral animal experiments, analyzed
the data and interpreted the findings. KB designed and performed
the immunohistochemistry experiments and acquired the images
with the technical advice of SBW at SBMS facility at The
University of Queensland. KB and AB drafted the manuscript and
drafted the figures. KB and JW drafted the drawing in Figure 3.
SEB, IAC, FC, JW, and AB reviewed and edited the manuscript.
All authors have critically reviewed the content and approved
final version for submission.
FUNDING
This work was supported by National Health and Medical
Research Council (NHMRC) (GNT1146417) to SEB.
ACKNOWLEDGMENTS
We are thankful to PACE animal facility manager Lisa Foster
and her staff Miranda Sleath, Rachel Smith, and Annie Villalta-
Burgett for the exquisite care of our animals. We are grateful
to the imaging facility of the Translational Research Institute,
the facility manager Sandrine Roy and the microscopy officer
Ali Ju, and the School of Biomedical Sciences at University of
Queensland, facility manager Shaun B. Walters for the extensive
use of resources.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnins.
2021.670430/full#supplementary-material
Supplementary Figure 1 | Restricting access to sucrose consumption augments
hippocampal oligodendrogenesis. (A,B) Restricted sugar consumption increased
the density of EdU positive cells (green) in the dentate gyrus of the hippocampus.
(C,D) There was no change in the density of stage 1 putative stem cells
[EdU+/GFAP+,(C)] or stage 3 immature neurons [EdU+/DCX+,(D)] suggesting
no change in neurogenesis, however, it appears that the increased density of
EdU+proliferative cells was mediated by an increased density of oligodendrocytes
progenitors [EdU+(green)/Olig+(magenta), (E,F)]. (G) There was no difference in
the density of proliferative microglia in the dentate gyrus, following restricted sugar
consumption, compared to water controls. All images are colocalized with DAPI
(blue). Representative image scale bar is 100 µm and close up representative
image scale bar is 10 µm.
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