The effects of long-term honey, sucrose or sugar-free diets on memory and

anxiety in rats
Lynne M. Chepulis
a
, Nicola J. Starkey
b,
, Joseph R. Waas
a
, Peter C. Molan
a
a
Department of Biological Sciences, Waikato University, Hamilton, New Zealand
b
Department of Psychology, Waikato University, Hamilton, New Zealand
a b s t r a c t a r t i c l e i n f o
Article history:
Received 11 September 2008
Received in revised form 5 March 2009
Accepted 9 March 2009
Keywords:
Honey
Sugar
Ageing
Memory
Anxiety
Cognition
Elevated Plus Maze (EPM)
Y maze
Sucrose is considered by many to be detrimental to health, giving rise to deterioration of the body associated
with ageing. This study was undertaken to determine whether replacing sucrose in the diet long-term with
honey that has a high antioxidant content could decrease deterioration in brain function during ageing.
Forty-ve 2-month old Sprague Dawley rats were fed ad libitum for 52 weeks on a powdered diet that was
either sugar-free or contained 7.9% sucrose or 10% honey (which is the equivalent amount of sugar). Anxiety
levels were assessed using an Elevated Plus Maze, whilst a Y maze and an Object Recognition task were used
to assess memory. Locomotor activity was also measured using an Open Field task to ensure that differences
in activity levels did not bias results in the other tasks. Anxiety generally decreased overall from 3 to
12 months, but the honey-fed rats showed signicantly less anxiety at all stages of ageing compared with
those fed sucrose. Honey-fed animals also displayed better spatial memory throughout the 12-month period:
at 9 and 12 months a signicantly greater proportion of honey-fed rats recognised the novel arm as the
unvisited arm of the maze compared to rats on a sugar-free or sucrose-based diet. No signicant differences
among groups were observed in the Object Recognition task, and there appeared to be no differences in
locomotor activity among groups at either 6 or 12 months. In conclusion, it appears that consumption of
honey may reduce anxiety and improve spatial memory in middle age.
2009 Elsevier Inc. All rights reserved.
1. Introduction
Ageing is associated with a slow deterioration of cognitive
performance [1,2], with decits in learning and memory occurring
from middle age onwards [35]. At the biochemical level, ageing
results in an imbalance between oxidative stress and naturally
occurring antioxidant compensatory mechanisms [6,7], leading to a
progressive increase in the steady state concentration of oxidatively-
modied DNA and proteins in the brain [810]. Indeed, increased
oxidative damage is related to reductions in learning [1114] and
memory [13,15,16] and increased anxiety [17,18].
Diet may play an important part in this process as higher glycemic
index (GI) ingredients (such as the simple sugars, sucrose andglucose)
can be detrimental to health because of prolonged or elevated
postprandial hyperglycemia [19]. Elevated blood glucose levels are
associated with the formation of glycated proteins and advanced
glycation endproducts (AGEs) which form spontaneously in pro-
oxidant environments [20]. Once formed, AGEs exacerbate the
development and progression of several chronic disorders (e.g.
hypertension, vascular disease, diabetes and atherosclerosis (reviewed
by [21]).
Consumption of antioxidant compounds can attenuate oxidative
damage [8,22], improve cognitive performance in animals [8,2325]
and slow the deterioration in memory and learning in middle- and
older-age humans [8,12,15,16,2629]. In addition to this, antioxidants
may also play a role in anxiety as evidenced by their ability to reduce
anxiety-like behavior in mice [30] and rats [22,31,32]. Furthermore,
anxiolytic treatment with citalopram decreases the elevated antiox-
idant enzyme levels observed in social phobia, and selective serotonin
reuptake inhibitors (SSRIs) are able suppress free radical formation
[33].
Honey is a naturally occurring source of antioxidants [3436] and
it also contains a mix of simple and complex sugars, vitamins,
minerals, acids and enzymes [37]. Honey has a lower GI than sucrose
[38], with some types of honey having a much lower GI [19], which
may lead to less prolonged postprandial hyperglycemia and thus less
free radical generation. Indeed, rats fed a honey-based diet, showed
less weight gain after short- and long-term feeding [39,40], and
improved HDL cholesterol and blood sugar levels after long-term
feeding, compared to those on a sucrose-based diet [40].
A meta-analysis of more than 20 studies indicated that increased
rates of mortality and morbidity are associated with high blood
glucose levels and high GI diets in both diabetic and non-diabetic
Physiology & Behavior 97 (2009) 359368
Corresponding author. Department of Psychology, Faculty of Arts and Social
Sciences, The University of Waikato, Private Bag 3105, Hamilton, NewZealand. Tel.: +64
7 8562889x6472; fax: +64 7 8585132.
E-mail address: nstarkey@waikato.ac.nz (N.J. Starkey).
0031-9384/$ see front matter 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.physbeh.2009.03.001
Contents lists available at ScienceDirect
Physiology & Behavior
j our nal homepage: www. el sevi er. com/ l ocat e/ phb
patients [41]. Thus, replacing sucrose in the diet with honey may
decrease the deterioration in health associated with ageing. The aimof
this study was to assess anxiety and memory in rats during long-term
feeding of a honey, sucrose or sugar-free diet, and to determine
whether replacing sugar in the diet with honey had any impact on
anxiety or memory.
2. Materials
2.1. Experimental animals, housing and procedure
Forty-ve male Sprague Dawley rats, approximately 8 weeks at the
start of the trial, were sourced from the small animal research facility
at AgResearch, Hamilton, New Zealand. The trial was carried out in an
animal facility at Waikato University, Hamilton, New Zealand, with
animals housed individually in standard rat cages with plastic bottoms
and metal grid tops (452530 cm high). The holding room was
maintained at 221 C with a 12 h light/dark cycle (lights off
0700 h). A 1012 cm long piece of 90 mm PVC tubing was placed into
each cage. Additional environmental enrichments (including small
plastic containers, pegs, sticks and pieces of doweling) were routinely
added to the cages. Food jars containing the appropriate diet were
placed into the respective cages and new diet added to the jars every
two days so that food was available ad libitum. Water was freely
available, and replaced twice weekly. This study was approved by the
Waikato University Animal Ethics Committee.
Animals remained in these cages throughout the study apart from
behavioural testing and when assessing food intake. All animals were
weighed every 12 weeks, and every 2 months food intake was
assessed over the period of a week (including at the start of the trial).
Rats were randomly assigned upon receipt to receive one of three
diets containing either: no sugar, sucrose or honey (n=15 per group).
Animals were tested on the Elevated Plus Maze (EPM) and Y maze
after receiving the experimental diets for 3, 6, 9, and 12 months, in the
Open Field after 6 and 12 months, and in the Object Recognition Test
after 12 months. The tests carried out at each time point were
conducted a week apart, except for the Object Recognition Test, which
was conducted immediately after the Open Field Test at 12 months.
This test was only conducted at one time point (12 months) due to
time constraints at earlier stages of the study. As a greater number of
tests were conducted at 12 months, only 10 rats per diet were assessed
at this time point. Further details regarding the behavioural tests are
presented below. On completion of behavioural testing, animals were
sacriced by cervical dislocation and blood samples were obtained to
assess blood sugar and lipid levels (these data are reported elsewhere,
[40]).
2.2. Experimental diets
A beech forest (Nothofagus solandri) honeydew honey with a high
antioxidant content (TEAC=3.1 mmol/l) was chosen for use in this
study. The honey was comprised of the following sugars (approximate
values g/kg): sucrose (0.5g/kg), fructose (40.9g/kg), glucose (30.1g/kg),
maltose (3.4 g/kg) and maltodextrin (5.1 g/kg). Three experimental diets
werepreparedtocontainnosugar (diet #1), 7.9%sucrose(diet #2) or 10%
honey (diet # 3) as described previously [40]. All diets were prepared to
contain a minimum of 5% water. An additional 21 ml of water was also
added to each kilogramof the non-honey diets to account for the fact that
the honey contained 21% water (measured using a refractometer).
In order to make the results more applicable to humans, the diets
were prepared such that they approximated the composition of a
typical New Zealand diet. Based upon data from the 1997 National
Nutrition Survey [42], the diets were formulated so that of 100% total
energy, 1517% came from protein, 3537% came from fat and 4547%
came fromcarbohydrate. In addition, skim milk powder (Fonterra Co-
Operative Group Ltd, NewZealand) was added to the diets at a level of
8% of the total daily kilojoule intake to simulate the typical daily intake
of milk or dairy products (350400 ml, assuming a total kJ intake of
11,00012,000/day). A low GI starch product (amylose) rather than
standard high GI starch was used in the sugar-free diet as a
replacement for the sucrose/honey.
In an attempt to simulate the oxidative damage that occurs in
humans (on a typical New Zealand diet), the diets were prepared
using pre-used cooking oil (vegetable oil) rather than virgin oil as the
source of fat. The cooking oil was sourced from various commercial
kitchens in Palmerston North, New Zealand and well mixed prior to
inclusion in the diets.
Diets were prepared monthly and were kept in the dark at 4 C or
18 C for the duration of the study. Standard rodent vitamin and
mineral mixes were prepared as described previously [40]. The
composition of the diets is given in Table 1.
2.3. Behavioural studies
The behavioural tests were carried out in a separate room
(temperature approximately 22 C). With the exception of the Y
maze, which required two phases of testing, all tests were carried out
between the hours of 08001200 h. As the rats were kept under a
reversed light/dark cycle, behavioural testing was conducted during
the dark phase. The light level within the test room was at a
maximum of 40 lux, to allow the experimenter to clean the apparatus
and to transfer animals to / from their home cage. Animals were
habituated to the light level for half an hour in the housing room
prior to testing.
Unless specied otherwise, animals were tested in a randomorder.
All test sessions were recorded on videotape for subsequent analysis
using a computer-assisted scoring program (Hindsight Software
Version 1.5, designed by Scott Weiss, University of Leeds). The trained
observer remained blind to the treatment group of the rats until
scoring was completed.
Further details about each behavioural test are provided below.
The type of memory tasks we could use were limited, as we needed
to avoid those that required restricted feeding or food rewards as
this may have interfered with the long-term effects of the different
diets.
Table 1
Composition of the experimental diets (g/kg).
Ingredient Sugar-free Sucrose Honey
Skim milk powder
a
95 95 95
Casein
b
120 120 120
Used oil 160 160 160
Amylose
c
79
Sucrose
d
79
Honey 100
Cellulose
e
50 50 50
Modied mineral mix
f
50 50 50
Sugar-free vitamin mix
g
5 5 5
Starch 365 365 365
Water (ml) 76 76 55
a
Fonterra Co-Operative Group Ltd, New Zealand.
b
80 Mesh, New Zealand Milk Products.
c
Davis Trading Company, Palmerston North, New Zealand.
d
Sigma Chemical Company.
e
Avicel PH102, Commercial Minerals Ltd, Auckland, New Zealand.
f
A mixture supplying: (g kg
1
diet) Ca 4.96, Cl 7.79, Mg 1.06, P 3.81, K 5.24, Na 1.97;
(mg kg
1
diet) Cr 1.97, Cu 10.7, Fe 424, Mn 78.0, Zn 48.2; (g kg
1
diet) Co 29.0, Mo 152,
Se 151.
g
A mixture supplying: (mg kg
1
diet) retinol acetate 5.0, DL--tocopherol acetate
200.0, menadione 3.0, thiamin hydrochloride 5.0, riboavin 7.0, pyridoxine
hydrochloride 8.0, D-panothenic acid 20.0, folic acid 2.0, nicotinic acid 20.0, D-biotin
1.0, myo-inositol 200.0, choline chloride 1500; (g kg
1
diet) ergocalciferol 25.0,
cyanocobalamin 50.0.
360 L.M. Chepulis et al. / Physiology & Behavior 97 (2009) 359368
2.4. Elevated Plus Maze (EPM)
The elevated plus maze was used to assess anxiety-like behaviour.
The EPM was designed as reported previously [43,44] such that the
maze consisted of two open arms that were 5010 cm wide at right
angles to two closed arms that were 501040 cm high, with a
central area that was 10 cm square. The maze was made out of dark
grey PVC plastic and painted with matt black paint on the insides of
the closed arms and the top of the open arms. White lines marked out
the centre area so that it was clearly visible on the videotape when the
animal moved into a new area of the maze. The maze was elevated to
50 cm above the ground.
At the start of the test session, each animal was transferred from
the housing room to the experimental room in its home cage. The
animal was then placed in the centre square of the EPM, facing
towards an open arm and left to explore the EPM for 5 min before it
was returned to its home cage and the animal housing room. The maze
was fully cleaned with mild cage cleaning detergent and wiped dry
between animals. Animals that fell from the EPM were returned to
their home cage and excluded from subsequent analyses.
Measures taken included traditional measures, based on location,
and ethological measures such as, exploratory behaviour and risk
assessment [43,4548]. More specically, the length of time and
number of entries into the open arm, center square and closed arm
were recorded (an animal was dened as being in an open or closed
arm when all four paws were in the arm). Exploratory behaviour
included the frequency of rearing in each area of the maze. In addition,
two risk assessment behaviours were scored: head dips (looking over
the edge of the maze) and head peeps (rat in closed arm looking
around the corner of the wall into the open arm). Head dips were
differentiated into those occurring from the relative safety of the
closed armor centre square (protected head dips) and those occurring
in the open arms (unprotected head dips) [46,47].
2.5. Y maze
The Y maze was used to assess spatial memory (see [49,50]). The
maze consisted of three arms that were 5016 cm long, 32 cm high
and 120 apart. The maze was made out of grey PVC plastic and
painted matt black on the inside. A layer of mixed soiled bedding was
placed in the Y maze to mask any olfactory cues from previous
animals. A removable door made from the same PVC plastic as the
maze was used to block access to one arm as required. Spatial cues
were afxed to the end of each arm (cardboard 1515 cm with an
image of either, a black triangle, a patterned circle or a patterned
square) and the same cues were xed to the wall of the testing room
above the arms of the maze.
Y maze testing consisted of two phases. During phase 1 the animal
was placed into the end of one arm (facing the centre) and allowed
access to that arm(the STARTarm) and one other (the OTHER arm) for
10 min. The third arm(the NOVEL arm) was blocked by the removable
door. Choice of which arm was start, other and novel was rotated
with each new animal. The rat was then removed from the maze and
returned to its home cage in the housing room for an inter-trial
duration of 4 h, as previous research has shown that Sprague Dawley
rats demonstrate no recognition memory after 6 h but that
recognition memory occurs for time periods of 4 h or less [49,50].
The shavings in the Y maze were mixed thoroughly, and the walls of
the maze wiped down with mild detergent after every animal. After
4 h, the rat was placed back into the start arm of the maze and given
free access to all three arms for 5 min (phase 2).
The scoring of this test focused on phase 2. In particular, the rst
arm entered was noted and the frequency of entries into each arm
every minute for the duration of the test session was recorded. Entry
into an arm was dened as placement of all 4 paws at least 5 cm into
the arm. Animals were excluded fromthe analyses if they did not leave
the start arm for the duration of the test session, or if they spent N75%
of their time in the centre area.
2.6. Open Field task
The Open Field task was used to obtain a separate measure of
locomotor activity [5153]. The arena (10010050 cm high) was
made from large Perspex sheets covered in black polythene, with a
thick layer of soiled bedding covering the oor. The animal was placed
in the centre of the Open Field and given 5 min to explore (they were
not habituated to the test arena). Following this, at the six month test
session, the animal was returned to the holding room, but at the
12 month test session, the rat then completed the Object Recognition
task which is described below.
The Open Field task was scored as follows: A sheet of clear plastic
was overlaid on the image of the arena on the monitor used for
scoring, and the area divided into a 55 grid, each section of the grid
being equivalent to a 2020 cm portion of the open eld (this area
being greater than the head to rump length of the rats). Using this
grid, the arena was divided into three zones: centre, middle and
outside. The number of line crosses and the location (centre, middle
and outside) of the rat was recorded throughout the test session.
From this the following behavioural measures were calculated: dura-
tion in centre, middle and outside zones, frequency of entries to each
zone, number of line crosses within the middle and outside zones,
and the total number of movements (including zone entries and line
crosses).
2.7. Object Recognition task
The Object Recognition task used the Open Field arena [54,55] and
due to time constraints, only 10 animals (randomly selected) from
each diet group were assessed.
The 5 min exposure to the arena in the Open Field test (see above)
was used as the habituation phase of this task. After this, the animal
was placed back into its home cage for 3 min, and two identical white
cylindrical plastic objects (objects 1 and 2; 22 cm9 cm) were placed
in the arena, each 10 cm away from a corner (two corners of one side
of the arena were used). The animal was then placed back into the
centre of the arena, and given 3 min to explore the 2 objects
(acquisition phase). Following this, the animal was again placed back
into its home cage for a 3-min retention interval. Subsequently, for the
retention phase, the animal was placed back into the centre of the
arena and given 3 min to explore a familiar object (an identical copy of
those used in the acquisition phase) and a novel object (an orange
spray bottle lled with water with its trigger disabled). The rat was
then returned to its home cage. The location (left or right) of the novel
object was alternated with each newanimal and all four objects were
wiped down with mild cleaning solutions between each test. The
soiled bedding in the arena was mixed around between animals to
mask any olfactory cues.
The latency to approach the objects, number of visits to each object
and the total time spent exploring each object were recorded during
the acquisition and retention phases. A rat was considered to be
exploring or visiting an object when its nose was either touching or
b2 cm away from the object, but not if sitting on it [54,55].
2.8. Statistical analyses
Due to the range of behavioural tests conducted, various analyses
were used and are explained in more detail at the beginning of each
section. In the interests of clarity, the results section reports the
statistically signicant univariate ANOVA results only (not the overall
MANOVA results). All analyses were performed using SPSS version
14.0. Statistical data is reported as MeanSE, and a result was
deemed to be statistically signicant if pb0.05.
361 L.M. Chepulis et al. / Physiology & Behavior 97 (2009) 359368
3. Results
For reasons unrelated to diet, four rats had to be excluded fromthe
study, two fromthe sugar-free group and one fromeach of the sucrose
and honey groups.
3.1. Food intake and body mass
Food intake was assessed over a week every two months (see [40]
for further details). Thus, over the course of the study, food intake was
assessed seven times. A mixed 3 (diet: sugar-free, sucrose, honey)7
(time: food intake at 0, 2, 4, 6, 8 10, 12 months) ANOVAwas conducted
to determine if food intake differed among the rats on each of the
three diets. These data are summarized in Fig. 1. This analysis revealed
a signicant main effect of time (F(6, 228)=13.43, pb0.01), with food
intake increasing during the rst four months of the study (Fig. 1).
However, there was no signicant difference in food intake among the
three diets, nor was there a signicant timediet interaction. These
analyses were repeated using the total kilojoule intake, resulting in
similar ndings (not reported).
To examine the effects of the three diets on body mass, a mixed 3
(diet: sugar-free, sucrose, honey)5 (time: body mass at 0, 3, 6, 9, and
12 months) ANCOVA was conducted, with food intake as a
covariate (see Fig. 2). This analysis revealed a signicant main effect
of diet (F(2,37)=8.89, pb0.01) and a signicant diettime interaction
(F(8,148)=4.72, pb0.01), but no main effect of time (F(4,148)=1.91,
pb0.05). To investigate the interaction further, one way between
group ANCOVAs were conducted at each time point, followed by
Bonferroni correctedpost hoc tests. As canbe seeninFig. 2, at the start of
the study there were nosignicant differences among the groups, but by
3 months sucrose-fed rats weighed signicantly more than those on the
sugar-free diet (pb0.01). Similarly, at six months the sucrose-fed rats
were signicantly heavier than those fed either honey (pb0.05) or the
sugar-free diet (pb0.01). This trend continued to the end of the study
with the sucrose-fed rats remaining signicantly heavier than those on
either of the other diets (sugar-free pb0.01; honey pb0.05). The mean
percentage weight gain over the period of the study was 100%, 103% and
130% for rats fed the sugar-free, honey and sucrose-based diets
respectively.
Thus, the sucrose-fed rats gained signicantly more weight than
either of the other two groups whilst consuming a similar quantity of
food.
3.2. Elevated Plus Maze
Because of the large size of animals at the completion of the study
(mean weights=600700 g) the rats had difculty tting into or
turning round in the closed arms of the EPM at the 12-month testing
session. For this reason, only data from the 3, 6 and 9 month testing
sessions were included in the statistical analyses. These data are
summarized in Table 2, Figs. 3 and 4.
To investigate the effect of diet on the rats' behaviour on the EPM, a
3 (time: 3, 6, 9 months)3 (diet: sugar-free, sucrose, honey) mixed
MANOVA was conducted. Post hoc analyses were conducted using a
Bonferroni correction.
The univariate ANOVAs revealed that, irrespective of diet, the
percentage of the time spent in the open arm changed signicantly
withtime (F(2, 64)=6.1, pb0.01), signicantly decreasing at 6 months
compared to 3 months (pb0.05), but increasing signicantly at
9 months compared to 6 months (pb0.01). In addition, the total
number of rears decreased signicantly with time (F(2,64)=23.5;
pb0.01). Rearing at 3 months occurred signicantly more often than
at 6 or 9 months (both pb0.01). In contrast, the frequency of
unprotected head-dips increased (F(2,64)=3.6, pb0.05) between 6
and 9 months (pb0.01), which probably reects the increased time on
the open arms.
A signicant main effect of diet (collapsed across the three test
sessions) was observed for the percentage of time spent on the open
armof the EPM(F(2, 32)=4.8, pb0.05), with honey-fed rats spending
a signicantly greater proportion of the test time (twice as much) on
the open arms compared to those fed sucrose or the sugar-free diet
(both pb0.05; see Fig. 3).
With regard to the interaction between diet and time, the
duration of time spent in the open arm of the EPM approached
signicance (F(4, 64)=2.24, p=0.07). As can be seen in Fig. 4, one
way between-group ANOVA's conrmed that there was no signicant
difference among the diet groups with respect to open arm duration
at 3 months or 6 months but by 9 months (F(2,33)=3.8, pb0.05) the
rats fed the honey-based diet spent twice as long in the open arms of
the EPM compared to those on the other diets (pb0.05).
Examining the three diet groups separately, one-way repeated-
measures ANOVAs revealed signicant alterations in the time spent
on the open arms over the three test sessions for all three groups
(sugar-free: F(2,20)=6.79, pb0.01; sucrose F(2,22)=4.64, pb0.05;
honey F(2,26)=20.52, pb0.01). Openarmtime increased signicantly
Fig. 1. The effect of diet composition on food intake of rats. Data are presented as mean
food intake (SE) of rats fed diets that were either sugar-free, or contained 7.9% sucrose
or 10% honey. All diets were formulated to contain equivalent amounts of honey/sugar/
amylose and water (the honey was 21% water).
Fig. 2. The effect of diet composition on the weight gain of rats. Data are presented as
mean weight (SE) of rats fed diets that were either sugar-free, or contained 7.9%
sucrose or 10% honey. All diets were formulated to contain equivalent amounts of
honey/sugar/amylose and water (the honey was 21% water). indicates sucrose-fed
rats were signicantly heavier than sugar-free rats at that time point (pb0.01),
indicates sucrose-fed rats were signicantly heavier than honey-fed rats at that time
point (pb0.05).
362 L.M. Chepulis et al. / Physiology & Behavior 97 (2009) 359368
between3 and6 months (pb0.05) and3 and9 months (pb0.01) inrats
fed the sugar-free diet, and between 3 and 9 months (pb0.05) in those
fed sucrose. Similarly, honey-fed rats showed a signicant increase in
open arm time between 3 and 6 months (pb0.01), 6 and 9 months
(pb0.01) and 3 and 9 months (pb0.01).
Thus, over time, the rats showed a decrease in rearing. Following
longer term consumption of the different diets, the rats on the honey-
based diet spent signicantly more time on the open arm of the EPM
compared to sugar-free or sucrose-fed rats. There were no signicant
differences between the rats on the sugar-free or sucrose diets.
3.3. Y maze
Initially, Chi Square Tests were used to compare the number of rats
initially entering either the novel or other arm for each of the three
diets at each time point (3, 6, 9, 12 months; see Table 3). Analyses
revealed that a signicantly greater proportion of honey-fed rats
chose to explore the novel arm rst rather than the other arm during
the 3 (
2
(1)=11.3), 6 (
2
(1)=9.3) and 9 (
2
(1)=8.1) month
testing sessions (all pb0.01), with the 12 month data also nearing
signicance (p=0.06). In contrast, the proportion of sucrose-fed rats
entering the novel arm compared to the other arm was signicantly
greater only at the 3 month testing session (
2
(1)=7.1, pb0.01),
with no differences being apparent for the remaining testing sessions.
For rats on the sugar-free diet, a signicantly greater proportion entered
the novel arm as their arm of rst choice during the 3- (
2
(1)=7.1;
pb0.01) and 6-month (
2
(1)=4.6; pb0.05) test sessions only.
Further Chi Square analyses were conducted to compare the
percentage of rats entering the novel arm rst on each of the three
diets. At three months there were no signicant differences in the
proportion of animals entering the novel arm on each of the three
diets. In contrast, at six months, signicantly more honey-fed rats
entered the novel armrst compared to those on a sucrose-based diet
(
2
(2)=8.24, pb0.05). At nine months this trend continued with
more honey-fed rats (approached signicance) entering the novel
arm rst compared to either of the other groups (
2
(2)=4.92,
Table 2
The behaviour of rats on the EPM after receiving sugar free, sucrose or honey-based diets for 3, 6 and 9 months.
Behaviour 3 months 6 months 9 months
Sugar-free Sucrose Honey Total Sugar-free Sucrose Honey Total Sugar-free Sucrose Honey Total
% Open
entries
12.503.0 15.382.9 16.343.6 14.841.8 13.683.9 9.212.8 20.53.5 14.62.1 22.562.6 19.073.9 32.824.8 25.312.6
% Open
duration
6.952.2 9.802.2 13.163.3 10.051.6 6.062.2 4.131.7 8.771.9 6.381.1+ 10.883.2 8.282.2 19.073.0 13.011.77$$
% Closed
duration
64.448.4 65.033.1 63.146.1 64.183.5 53.576.9 59.547.4 54.446.6 55.823.9 61.387.3 66.085.05 43.677.0 56.464.0
Rear (f) 3.620.8 5.380.9 5.711.2 4.930.6 1.920.5 1.460.4 1.290.43 1.550.25++ 1.550.4 1.380.4 1.500.5 1.470.3++
Protected
head dip (f)
1.230.5 2.310.5 0.790.2 1.420.3 1.920.5 1.30.5 1.930.4 1.730.3 2.180.6 2.310.5 3.000.5 2.530.3
Unprotected
head dip (f)
0.920.4 1.620.42 2.141.1 1.580.4 0.770.3 0.770.5 1.290.30 0.950.22 1.20.4 1.540.5 3.430.8 2.160.4$
Head peep (f) 8.921.5 9.230.9 9.291.0 9.150.7 5.461.2 6.691.6 6.51.3 6.20.8 7.911.3 9.770.9 7.501.3 8.390.7
Data are presented as MeanSE. f =frequency. + pb0.05, ++ pb0.01 compared to 3 months; $=pb0.05, $$=pb0.01 compared to 6 months.
Fig. 3. The effect of diet composition on the percentage of the test time spent in the
closed and open arms of the EPM (collapsed across the three, six and nine month test
sessions). The data presented are the mean percent of total test time (SE) on the open
and closed arms over the three test sessions (3, 6 and 9 months). Rats were fed a diet
that was either sugar-free or contained 7.9% sucrose or 10% honey (honey is 21% water)
and tested on the EPM following 3, 6 and 9 months on these diets. =pb0.05.
Fig. 4. The effect of diet composition and test session (time spent on the diet) on
duration in the open arm of the EPM. Data are presented as meanSE. Rats were fed a
diet that was either sugar-free or contained 7.9% sucrose or 10% honey (honey is 21%
water). =pb0.05, compared to sucrose at same time point; + pb0.05, ++ pb0.01
compared rats on the same diet at 3 months; $=pb0.05, $$=pb0.01 compared to rats
on the same diet at 6 months.
363 L.M. Chepulis et al. / Physiology & Behavior 97 (2009) 359368
p=0.08). However at 12 months there were no signicant differences
among the three groups.
Following this, analyses were conducted to determine if the
frequency of entries into and duration of time in each arm (start,
other, novel) differed among the three dietary groups and/or across
the four test sessions. Preliminary analyses indicated that exploration
was greatest in the rst few minutes overall arm entries decreased
signicantly over the rst 3 min in all test sessions (pb0.05). Thus,
subsequent analyses focused on data from the rst 3 min of phase 2 of
the Y maze test when exploration was at its greatest (as suggested by
[49,56]).
Amixed3 (arm: start, other, novel)4 (session: 3, 6, 9, 12 months)3
(diet: sugar-free, sucrose, honey) MANOVA was conducted for the
frequency and duration of arm entries. This analysis revealed signicant
maineffects for thefrequencyof entries tothedifferent arms of theYmaze
(F(2,48)=30.56, pb0.01), but not for duration. Overall, the mean
number of entries into the start arm (2.780.12) was higher than into
the other arm(1.850.14) or novel arm(2.320.10, bothpb0.01), and
rats entered the novel arm signicantly more often than the other arm
(pb0.01).
Both the frequency of arm entries (F(3,72)=6.14, pb0.01) and the
duration of time (F(3,72)=4.95, pb0.01) spent in the arms differed
across the four tests sessions. Rats made signicantly fewer arm
entries in the last session (1.840.15) compared to the three earlier
tests (2.740.17 (3 months), 2.320.18 (6 months) and 2.350.13
(9 months; all pb0.05). The mean amount of time the rats spent in the
arms decreased at six months (42.231.82) compared to three
months (47.721.83; pb0.05), but increased between 6 and
12 months (45.180.97; pb0.05). This is a reection of the alteration
in the amount of time the rats spent in the centre part of the Y maze.
The diet the rats consumed also produced a signicant difference
in the frequency of arm entries (F(2,24)=5.18, pb0.05). The honey-
fed rats showed signicantly fewer mean armentries compared to the
sucrose-fed rats (sugar-free 2.240.16, sucrose 2.740.17, honey
1.970.18; pb0.05). The univariate ANOVAs did not reveal any
signicant interaction effects.
Thus, in the Y maze, at 3 months a signicantly greater proportion
of rats entered the novel arm rst in all diet groups. At six months a
signicantly greater proportion of rats from the sugar-free and honey
groups entered the novel arm rst, whilst at 6 and 12 months the
distinction between the novel and other armwas only apparent in the
honey-fed rats. In addition, overall the honey-fed rats made
signicantly fewer arm entries compared to the sucrose-fed rats.
3.4. Open Field
A 2 (time: 6 months, 12 months)3 (diet: sugar-free, sucrose,
honey) mixed MANOVA was conducted on the data from the Open
Field. These data are presented in Table 4. There were no signicant
differences in open eld exploration among the three diet groups at
either time point. The univariate ANOVAs indicated that irrespective
of diet, animals generally exhibited less locomotor activity at the
12 month session compared with the 6-month session. These data are
summarized in Fig. 5 (collapsed across diet). The mean number of visits
to the middle (F(1,21)=8.92, pb0.01) and outer zones (F(1,21)=7.33,
pb0.05) of the arena was signicantly higher at the 6-month testing
Table 3
Armof rst choice for rats in Phase 2 of the Y maze after receiving sugar-free, sucrose or
honey-based diets for 3, 6, 9, or 12 months.
Sugar-free Sucrose Honey
Novel Other Novel Other Novel Other
3 months
a
86 14 86 14 93 7
6 months
b
79 21 57 43 92

8
9 months
a
64 36 64 36 87

13
12 months
c
78 22 60 40 80 20
Data are presented as percentage of animals.
pb0.05, pb0.01 observed n signicantly Nexpected n (expected n 50%).

pb0.05 versus proportion of sucrose-fed rats choosing the novel arm rst.

pb0.10 versus proportion of sucrose- and sugar-free rats choosing the novel arm rst.
One animal from the honey-fed group (6 months) and one animal from the sugar-free
group (12 months) were excluded from the analysis as they did not enter either the
other or novel arm after leaving the start arm.
a
n=14, 14 and 15 for animals fed the sugar-free, sucrose and honey-based diets.
b
n=14, 14 and 13 for animals fed the sugar-free, sucrose and honey-based diets.
c
n=9, 10 and 10 for animals fed the sugar-free, sucrose and honey-based diets.
Table 4
Exploratory behaviour of rats in an open eld after receiving sugar-free, sucrose or honey-based diets for 6 and 12 months.
6 months 12 months
Sugar-free Sucrose Honey Sugar-free Sucrose Honey
Centre square Duration (s) 3.450.98 1.320.47 2.521.57 1.190.32 2.511.01 2.120.63
Frequency 2.140.40 1.330.24 1.750.49 1.290.29 1.440.24 1.130.13
Middle zone Duration (s) 18.812.89 16.213.65 15.054.40 16.095.52 28.0713.41 18.116.32
Frequency 7.141.42 5.440.85 5.501.40 4.000.95 3.330.85 3.130.97
Line cross 5.431.13 3.001.03 5.381.8 3.861.63 1.780.85 2.631.12
Outer zone Duration (s) 277.513.23 276.648.10 282.435.37 282.724.97 269.1014.16 279.776.83
Frequency 6.001.13 5.112.42 4.881.14 3.710.71 3.000.71 3.001.02
Line cross 56.2912.23 58.229.64 66.634.81 42.296.13 24.006.26 30.873.18
All zones Total moves 77.0015.65 73.119.89 84.136.88 55.148.88 33.566.27 40.754.40
Data are presented as MeanSEM.
Fig. 5. Exploratory behaviour of rats in the open eld at six and twelve months (data
collapsed across diet groups). Data represent Mean frequency (SE) irrespective of the
diet the rat consumed. Clear bars=6 months, hatched bars=12 months. *=pb0.05,
**=pb0.01 compared to six months.
364 L.M. Chepulis et al. / Physiology & Behavior 97 (2009) 359368
session compared to the 12-month session. In addition, the mean
number of line crosses in the outside zone of the arena decreased by half
(F(1,21)=32.60, pb0.01), and the mean number of total moves in the
arena (including zone entries and crosses inside a zone) decreased by
4050% (F(1,21)=41.59, pb0.01) by the end of the second 6 months of
the study.
Thus, diet did not appear to alter locomotor activity in the open
eld, however as the animals aged, activity generally decreased.
3.5. Object Recognition task
The analyses for the Acquisition and Retention Phase were carried
out separately. Firstly, in the Acquisition Phase, when animals were
presented with two identical objects, a 2 (object: 1, 2 (identical))3
(diet: sugar-free, sucrose, honey) mixed MANOVA was conducted for
the frequency and duration of object exploration. Neither the duration
nor the frequency of exploration of each object differed signicantly.
Furthermore, there were no signicant differences in object explora-
tion among the three diet groups, and no signicant interaction. These
data are summarized in Fig. 6A. In addition to this, analyses were also
conducted to examine the latency to explore the two objects across
the three diet groups. However there were no signicant effects.
Similar analyses were carried out for the Retention Phase (where
one of the original objects was replaced with an identical copy and one
was replaced with a novel object) and these data are summarised in
Fig. 6B. Irrespective of diet, the novel object was visited signicantly
more frequently (F(1,22)=15.8; pb0.01) and explored for a sig-
nicantly longer period of time (F(1,22)=41.8; pb0.01) compared to
the familiar object. Inadditionthere was a signicant difference among
the three diets for the frequency of object exploration (F(2,22)=3.68,
pb0.05) but not for duration. This was due to the honey-fed rats
exploring the objects signicantly less frequently than rats on the
sucrose or sugar-free diets (both pb0.05). However, there was no
signicant object diet interaction. Reecting these ndings, the
latency to visit the identical copy and the novel object differed
signicantly overall, the time the novel object was rst explored being
approximately a third of that of object 3 (16.4 vs 48.4 s, F(1,17)=12.9;
pb0.01). However, there was no signicant effect of diet and no
signicant object diet interaction.
Thus, rats on all three diets explored the novel object signicantly
more compared to the other object and overall the honey-fed rats
explored the objects less frequently than the other two groups.
4. Discussion
Overall, this study revealed several interesting differences follow-
ing the long term consumption of sugar-free, honey or sucrose-based
diets. Firstly, the sucrose-fed rats gained signicantly more weight
than either of the other two groups whilst consuming a similar
quantity (and kilojoule level) of food. The differential weight gain may
be due to differences in the GI of the sucrose and honey, with honey
having a lower GI. In support of this, honey- and sugar-free-fed rats
showed reduced glycated hemoglobin levels (a measure of blood
glucose) compared with those fed sucrose after 12 months [40].
However, as fructose metabolism elicits different hormonal responses
compared to glucose [57], it is unlikely that differences in GI can fully
explain these ndings. Furthermore, as sucrose/honey/amylose
comprised only 10% of the diet, we do not know if this was sufcient
to alter the GI of the diet overall. Indeed, unpublished data from our
laboratory indicate that rats fed 60% honey show less weight gain
compared to those fed a similar amount of mixed sugars (as in honey)
or sucrose, suggesting that some other bioactivity of the honey plays a
part. This may be the insulinmimetic effects of the hydrogen peroxide
produced by the honey [39], but it is unclear if hydrogen peroxide
reaches sufcient levels in vivo to elicit such a response. Alternatively,
differences in weight gain may be a result of the high antioxidant
content of the honey, as rats fed green tea showed increased
thermogenesis in brown adipose tissue [58]. Thus, further research
is needed to fully explain these ndings.
Data from the behavioural tests revealed changes over time, which
were unrelated to the animals' diet. In the EPM the proportion of the
test time spent on the open arm increased, indicative of a decrease in
anxiety. In the Y maze and the Open Field, activity / exploration
decreased over time. One of the limitations of the current study is
repeated testing, which makes it difcult to separate out the effects of
ageing from prior experience in the test environments. We were
unable to nd published studies that have examined the effects of
repeated testing at three month intervals however the behavioural
effects of a short (24 h1 week) inter-test interval are well
Fig. 6. The effect of three diet composition on duration (A) and frequency (B) of object
exploration in the Object Recognition task. Data are displayed as MeanSEM. Clear
bars=Sugar-free; Stripes=Sucrose; Hatched=Honey. Object 1 and Object 2 were
identical and presented rst, Familiar Object was a copy of the previous objects and was
paired with a Novel Object. * =pb0.05, **=pb0.01 compared to Familiar Object
overall. $=pb0.05 compared to sucrose and sugar-free diets.
365 L.M. Chepulis et al. / Physiology & Behavior 97 (2009) 359368
documented in the EPM. Early studies indicated that repeated testing
led to no change in open arm exploration (e.g. [45,59]). However,
there is nowmounting evidence to suggest that repeated testing leads
to increased avoidance of the open arms in both rats and mice (e.g.
[43,46,6064]). Ageing has similar effects with older rats (12
24 months) showing increased anxiety related behaviour compared
to younger rats (10 weeks) [6467]. Clearly, neither of these
explanations offer support for the current ndings. However, one
other consequence of our longitudinal study was that, over time, the
rats became more habituated to handling, which can decrease anxiety
in the EPM [68]. Thus, increased handling may have counteracted the
commonly reported behavioural effects associated with repeated
testing and ageing in the EPM.
Unlike the EPM, the Y maze was changed for each test session
(spatial cues were moved, the start armwas rotated, different sawdust
placed on the oor), and therefore retained a degree of novelty. The
decreased exploration observed in the Y maze may be due to habitua-
tion to the test situation, but as this also occurred in the open eld, this
is unlikely and suggests it may be a consequence of ageing. Indeed,
exploratory behaviour decreases withage invarious tests including the
social interaction test [69], open eld [70,71] and the EPM [72].
On the EPM, irrespective of the age of the animal, honey-fed rats
showed decreased anxiety-like behaviour (increase in the percentage
of the test time spent on the open arms) compared with those fed
either sucrose or sugar-free diets. Furthermore, there was some
evidence that the longer the rats remained on the honey-based diet,
the greater the reduction in anxiety. However, there were no differ-
ences between the rats on sugar-free or sucrose diets. With regard to
spatial memory, honey-fed rats performed better than those fed
sucrose or a sugar-free diet in the Y maze task (a greater proportion of
the honey-fed rats entered the novel arm rst), suggesting that these
animals did not showthe same reduction in spatial memory exhibited
by rats fed the other two diets. The sucrose-fed rats performed most
poorly on this task, only recognizing the novel armat the three month
test period. In contrast, no signicant differences among diet groups
were observed in the Object Recognition task, indicating that different
diets did not alter recognition memory.
It is possible that the antioxidant content of the honey may have
contributed to the decreased anxiety and improved spatial memory of
these rats. This is in keeping with studies which have demonstrated
that high levels of oxidative stress result in increased anxiety [18,73]
and that certain antioxidant compounds (including rosmarinic acid,
vaccinum berries, zingicomb and chlorogenic acid) can decrease
anxiety in rats/mice in the EPM [18,3032]. In addition, dietary anti-
oxidants improved cognitive performance in clinical studies [26,27],
and antioxidant intake has been correlated with improved memory
scores [7476] when ingested at levels similar to those used here [77].
Honey has a range of other bioactive properties including antiviral,
antiinammatory [78], pain reducing [79], immunostimulatory, pre-
biotic [80] and insulinmimetic effects [78]. In addition, honey may
contain compounds that exhibit hormonal and/or neurotransmitter
activities as it acts as if it contains acetylcholine and choline esters
[81]. Thus, this type of activity may play a role in its actions and
certainly warrants further investigation.
Rats on the sugar-free and sucrose diets showed a similar behav-
ioural prole on the EPM, supporting previous ndings that ad lib
sucrose-based diets do not increase anxiety in rats [82]. Nonetheless,
the sucrose-based diet led to a signicant increase (5%) in oxidative
damage (assessed by measuring the amount of advanced glycation
end products present in aortic collagen samples) compared to either
the sugar-free or honey-fed rats [83]. However, whether this differ-
ence is sufcient to be clinically meaningful is currently unknown.
In the Y maze, a signicantly greater proportion of honey-fed rats
entered the novel arm rst compared to those on the other diets but
typically in the Y maze, rats demonstrating good spatial memory also
spend longer in the novel arm during the rst few minutes of the test.
However, with one exception, no differences were observed for either
the number of visits to the novel arm, nor the duration of time spent
exploring that arm of the maze. This may be due to the long inter-trial
interval which could be manipulated in future studies.
There were no differences among diet groups for animals
performing the Object Recognition task, although this differs from
other published work with dietary antioxidants (e.g. [84,85]). This
may be due to the age of the rats, as at ~14 months of age, they were
perhaps not old enough to display a decline in recognition memory.
Alternatively, the inter-trial interval of the Object Recognition task
may have been too short thus making the task too easy and not
sufciently sensitive to detect the relatively subtle differences in
memory among the dietary groups.
In conclusion, it appears that long-term feeding of honey, sucrose
and a sugar-free diet may have some effects on anxiety and spatial
memory in rats, with honey-fed rats exhibiting less reduction in
spatial memory and decreased anxiety at the completion of the study.
Interestingly, rats fed the sugar-free diet generally performed less well
than the honey-fed rats, suggesting that the better performance seen
in honey-fed rats was not due solely to a lower dietary GI content but
involved other components in the honey, possibly antioxidants.
There are several limitations with the current study. Firstly, as
previously noted, it was difcult to separate the effects of age and
habituation due to repeated behavioural testing. However, as all
animals underwent the same standard protocol the behavioural
effects of the different diets could be determined. This approach was
taken for ethical reasons (it reduced the number of animals we used)
and for nancial reasons. Nonetheless, the current ndings justify
future studies with separate groups of rats, in which biochemical
endpoints (e.g. blood lipid, cholesterol) could also be determined.
Secondly, the rats entered this study before they were fully grown.
Thus, a study carried out in mature rats (raised on standard chow) fed
over a similar time period would clarify if the diets have similar effects
in adults. Finally, in order to make these ndings more applicable to
humans, future studies could examine how much honey needs to be
consumed to produce these effects.
In spite of these limitations, this study does suggest that cognitive
decline associated with ageing may be offset by long-term improve-
ments to diet in earlier life. Furthermore, these ndings raise many
more questions regarding how honey produces these effects, how
much honey needs to be consumed and over what time period.
Acknowledgements
This work was funded by Fonterra Brands Ltd, New Zealand as a
part of a PhD program. We would also like to thank Thea Eytan, Bruce
Patty & Rik Broadhurst for their assistance with this study.
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