1. Introduction
Insulin signaling occurs when insulin activates the insulin receptor (IR), this
ultimately results in glucose uptake by the cell. The process is orchestrated by
intracellular signalling, including the phosphoinositide 3-kinase (PI3K) pathway
and the phosphorylation of Akt/protein kinase B [1]. Insulin signaling within the
brain plays important roles in cognition, learning and memory. When insulin
signaling becomes dysregulated, such as in the case of insulin resistance and
long-term compensatory elevations of circulating insulin, an increased risk for
developing cognitive pathologies occur, such as Alzheimer’s disease [2]. The link
between insulin resistance and cognition is shown in rodent studies which use the
Morris Water Maze (MWM), a task used to evaluate spatial learning and memory [3].
Animals that present with insulin resistance have reduced performance in the MWM
[4, 5, 6, 7, 8, 9, 10, 11, 12]. These deficits can be ameliorated by central insulin infusion, which can
increase performance in the memory component of the MWM [13].
Within the central nervous system (CNS), the hippocampus, an area responsible
for memory and learning, is a prominent target for insulin signaling. A high
density of IRs within the dorsal hippocampus suggests a role for insulin in the
formation of spatial memory [14, 15]. In support, CA1 hippocampal insulin
administration enhanced memory consolidation and retrieval in the MWM, which is
considered to specifically test for hippocampal-dependent cognitive deficits.
This suggests that insulin directly regulates memory by acting on the hippocampus
[14, 16]. Using a lentiviral vector to downregulate IRs in the rat hippocampus,
Grillo and colleagues demonstrated that hippocampal insulin resistance results in
deficits in hippocampal synaptic transmission and hippocampal-dependent learning
[17]. Importantly, these deficits were seen to be independent of metabolic or
endocrine imbalances. This study provides important evidence that hippocampal
insulin signaling facilitates neuroplasticity and cognition [17].
Insulin signaling within the CNS declines with age and insulin resistance is a
risk factor for age-related Alzheimer’s disease and cognitive decline [18]. With
aging, a marked decrease in glucose transporters [19], insulin receptors [20],
insulin levels [21] and insulin signaling [22] have all been observed, which has
been linked to age related hippocampal memory impairments [23]. However, central
insulin resistance and aging do not always go hand-in-hand, with factors such as
body composition, lifestyle and exercise having important influences. Notably,
central and peripheral insulin resistance do not always occur concurrently, as
dysregulated CNS insulin signaling can precede, or help initiate, the onset of
peripheral insulin resistance [24].
As well as aging, sex can also influence insulin signaling within the CNS. Male
rats have increased anorexigenic sensitivity to insulin infusion compared to
female rats [25], while acute intranasal insulin in female patients improved
hippocampal-dependent memory, whereas males did not show an enhancement [26].
Another study examined individuals with mild cognitive impairments and
demonstrated that men show an improvement in working memory after intranasal
insulin, but only at a dose twice as high as shown to be effective in women [27].
Some of these sex differences appear to be the result of differential gonadal
hormone levels [28]. Overall, a strong link between intra-hippocampal insulin
signaling and cognitive performance has been identified, however, the specific
neuronal population mediating these effects within the hippocampus has not yet
been identified.
Neuropeptide-Y (NPY), a 36-amino acid peptide, is abundantly expressed
throughout the brain, including the hippocampus [29]. NPY is involved in the
regulation of biological and pathophysiological functions including feeding
behaviours, neuroplasticity, memory and learning [30]. NPY has a modulatory role
in spatial memory and learning as it appears to exercise both stimulatory and
inhibitory effects on memory, contingent on the NPY receptor subtype manipulated,
dose applied, neuroanatomical brain systems involved, temporal step (i.e.,
retrieval, acquisition, retention, consolidation) and memory type [31, 32, 33, 34].
Hippocampal NPY has been associated with spatial learning and memory during the
MWM, as increased levels of NPY mRNA were observed in the dentate gyrus of the
hippocampus following MWM exposure [35]. Further, brains from Alzheimer’s
patients show loss of NPY-positive neurons in the hippocampus [36], while NPY
injections into the dorsal hippocampus increases memory retention in mice [37].
Altogether, these data show that NPY and insulin signaling play important roles
for learning and memory by signaling within the hippocampus.
To date, it is unknown how disruption of insulin signaling in NPY expressing
neurons affects performance in hippocampal dependent cognitive tasks, such as the
MWM. To address this, we utilised the Cre-lox recombination technique in mice to
selectively knock out IRs in NPY expressing neurons
(IR;NPY). Mice were tested in the MWM at 6, 12 and 24
months of age to assess how aging influences behavioural deficits induced by
ablated IRs on NPY neurons.
2. Materials and methods
2.1 Animals
A conditional knockout mouse model was generated to selectively knockout the IR
in NPY expressing neurons (IR;NPY). This mouse model has
been validated in previous work, which functionally demonstrated that IRs were
deleted from NPY neurons [38]. To generate this conditional knockout, Floxed IR
mice (IR) [39] were crossed with NPY mice [40] to generate
double heterozygous mice; IR;NPY. These mice were then
crossed again with IR mice to generate IR;NPYmice. Breeding colonies were maintained by mating IR mice with
NPY;IR mice. All mice were bred on a C57Bl/6J background.
Littermates that lacked the Cre recombinase enzyme (IR) were used as
controls as they express normal IR signaling within NPY-expressing neurons [38].
This mouse line was maintained at Australian BioResources Ltd, Moss Vale, NSW,
Australia, with genotyping also being performed at this facility. For behavioural
studies, 56 IR;NPY (28 females and 28 males) and
59 IR control mice (30 females and 29 males) were
tested at 6 and 12 months of age. Due to age-related health issues of some mice,
52 IR;NPY (26 females and 26 males) and 54 IR
control mice (28 females and 26 males) were tested at 24 months of age. An
additional 20 male mice were used for immunohistochemistry analysis (10
mice/genotype, 6 months of age). Mice were housed two to four per cage (37
23 14 cm) under temperature-controlled conditions (22
2 C) with a 12 hour light-dark cycle (07:00 on–19:00 off).
Upon arrival, mice were handled and allowed to become acclimated to their new
environment. For the duration of the experiment, unless otherwise specified mice
were provided ad libitum access to water and standard laboratory chow
from a home cage dispenser. Body weights and food intakes were measured one week
prior to the commencement of each behavioural testing time point using a manual
averaging balance. Energy intake was calculated based on the quantity of food
consumed and the known caloric density of the standard chow diet.
Experimental procedures were approved by the University of New South Wales
Animal Care and Ethics Committee in accordance with the Australian Code of
Practice and Use of Animals for Scientific Purposes. Nine animals were euthanised
between 12 and 24 months due to health issues (4 female, 5 male). These animals
were included in the data analysis for earlier timepoints.
2.2 Morris Water Maze
The protocol used for training and testing in the MWM is based on established
methods [13, 41]. Mice were trained to use distal spatial cues surrounding the
maze to locate a hidden escape platform situated beneath the surface of the
water. The water was kept at 22 degrees Celsius and rendered opaque by the
addition of a non-toxic tempera powder. On Day 1 the escape platform was colored
with black and white stripes and was raised 5 mm above the water level. 60
seconds was allowed for the mouse to locate the escape platform. Mice were gently
guided onto the platform if they did not locate the platform in the allocated
time. The mouse was allowed to remain on the platform for 15 seconds before being
relocated to its home cage. This procedure was repeated for two trials with a 5
minute inter-trial period. From Days 2–4 the escape platform was positioned at
the centre of the NW quadrant. Each mouse received four trials per day over three
consecutive days with an inter-trial interval of 5 minutes. Each trial involved
the release of the mouse from one of four fixed points (N, S, E, W), the starting
quadrant positions. The starting positions were assigned in random order to
prevent the use of a praxis strategy (using a learned sequence of movements),
rather than a spatial mapping strategy and data from the four daily trials were
averaged each day. Mice were dried and warmed after each training trial. Mice
were allowed 60 seconds to locate the escape platform which was covered in white
tape and submerged 5 mm below the surface. Mice were guided to the platform
location if they failed to locate it in the allocated time. The mouse remained on
the platform for 15 seconds before being placed back into the tank at one of the
other four start positions. This was continued until the mouse had been allowed
to find the submerged platform from all four quadrants. On Day 5 the 90 second
probe trial was performed where the platform was removed. The time spent in the
target quadrant and path length in the probe trial were scored using EthoVision
(Noldus Information Technology, XT v5.1, Wageningen, The Netherlands). The
position of the coordinates and cues were changed during testing at 12 and 24
months of age.
2.3 Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)
The GTT and ITT were conducted in mice following the completion of behavioural
testing at 24 months of age. GTT: Following a 4-h fast, the tip of the tail was
cut (~1 mm) and baseline glucose measured (~5 L)
(Accu-Chec; Roche Diagnostics, IN, USA) and 50 L blood collected for insulin
measurement by ELISA (Crystal Chem, IL, USA). Mice were injected
intraperitoneally with a glucose solution (~200 L/mouse; 1
g/kg). Blood glucose was assessed again at 15, 30, 60 and 120 minutes post
injection and blood collected for insulin was evaluated again at 15 minutes. ITT:
For the ITT, an insulin bolus (1 U/1 kg body weight) was administered via
intraperitoneal injection. Blood glucose was assessed from 5 uL blood at 15, 30,
45 and 60 minutes post injection.
2.4 Immunohistochemistry
10 experimentally naive mice per genotype were injected with insulin (20 IU/kg,
i.p) or saline (i.p; 5 animals per treatment group). After 20 minutes mice were
anaesthetized with an overdose injection of sodium pentobarbitone (120 mg/kg,
i.p), and the brains were fixed by perfusion with 0.9% saline followed by ice
cold 4% paraformaldehyde made in 0.1M phosphate buffered saline (PBS) (pH 7.4).
The brains were immediately removed and post-fixed in 4% paraformaldehyde
overnight at 4 C and then in 30% sucrose solution in phosphate buffer
overnight. Coronal slices of 40 m thickness were collected and
stored at –20 C in cryoprotectant (25% ethylene glycol, 25%
glycerol, 50% distilled water). Three sections per animal were used for
immunohistochemistry. Sections were washed with PBS + 0.1% Tween 20, incubated
in sodium citrate antigen retrieval buffer (10 mM, pH 6.0, 70 C, 10
minutes) and blocked for 1 hour using 5% normal goat serum (Vector labs, S-1000,
Burlingame, CA, USA), 0.1% triton x-100 and 0.1% bovine serum albumin (BSA) in
PBS. Immediately following this, sections were incubated overnight at room
temperature with the primary antibody, which was rabbit anti-phospho-Akt (1:2000
dilution, Cell Signaling Technology, 4060S, Danvers, MA, USA). Phosphorylated Akt
(p-Akt) is a marker of insulin signaling pathway activation [42, 43]. After three
washes in PBS + 0.1% Tween20, sections were incubated overnight at room
temperature with a fluorescent secondary antibody (1:1000 Goat anti-Rabbit IgG (H
+ L) secondary antibody, Alexa Fluor® 488 conjugate ThermoFisher
Scientific, A-11034, NSW, Australia). All antibodies were diluted in antibody
solution (5% normal goat serum, 0.1% Triton X-100, 0.1% BSA, in PBS). Sections
were washed once in PBS + 0.1% Tween20 before being
counterstained in DAPI solution (5 mg/mL, ThermoFisher Scientific, D3571, NSW, Australia) for 5
minutes. Sections then were washed again in PBS + 0.1% Tween20, mounted onto
0.1% gelatinized glass slides and coverslipped with ProLong Diamond antifade
(ThermoFisher Scientific, P36961, NSW, Australia). Sections were visualized for
p-Akt within the brain nuclei of interest which were defined according to the
mouse brain atlas (Franklin and Paxinos [44]), using an Olympus FV1200 confocal
microscope (Olympus, Tokyo, Japan). Using ImageJ software (version 1.47, LOCI,
University of Wisconsin, Madison, Wisconsin, USA), the number of fluorescently
labelled p-Akt cells in the dentate gyrus were counted by one experimenter,
blinded, for all animals. The mean cell counts for each treatment group were then
made into a percentage relative to mean cell counts from the control treatment
group (IR + saline group).
2.5 Statistical analysis
The study employed a 2 3 between/within subjects design with the
between level being genotype (IR, IR;NPY) and
the within factor was age (6 months, 12 months, 24 months old). Data were
analyzed using Statistica 12.0 (Dell Software, NSW, Australia) and is presented
as means with standard errors. Data were first tested for normality and repeated
measures (body weight and food intake) and 2-way between group ANOVAs (Behavioral
tests) were followed by Tukey’s honest significance difference (HSD) test for
post-hoc analysis when a significant interaction effect was observed. Differences
were accepted as statistically significant at p 0.05.
Immunohistochemical analysis was performed using a 1-way ANOVA followed by
Tukey’s HSD test.
3. Results
3.1 Body weight and food intake
Male IR;NPY weighed significantly more than Male
IR mice at 6 months
(p 0.05) but not at 12 or 24 months of
age. There were no weight differences between genotypes in female mice at any
age. Increased body weight was observed in both male and female mice with aging
from 6 months to 12 and 24 months (Fig. 1A,
p 0.05). There was a main effect of
sex on food intake, with male mice consuming more than female mice (Fig. 1B,
p 0.05).
Fig. 1.
Body weight and energy intake. (A)
IR;NPY and littermate control mice (IR) gained
weight over time. Male IR;NPY mice displayed significantly
increased body weight compared to male IR mice at 6 months of age. No
differences in body weight between genotypes were observed at 12 or 24 months of
age. Female mice did not show any body weight differences between genotypes at
any time points. (B) Female mice displayed overall lower energy intake than male
mice, however energy intake at 6, 12, or 24 months did not differ between
genotypes. Values are expressed as mean SEM. * = interaction effect
between IR and IR;NPY genotypes, p
0.05. = main effect of time, p 0.05. Analysed by 2-way ANOVA
followed by Tukey’s honest significance difference (HSD) test. 6 and 12 months, n = 59 IR (30 F and 29 M) and 56
IR;NPY (28 F and 28 M). 24 months, n = 54 IR
(28 F and 26 M) and 52 IR;NPY (26 F and 26 M).
3.2 Morris Water Maze
There were no significant differences observed between male and female mice
across genotype and time points during MWM testing, therefore the data for both
sexes have been combined for all of the following results. Both
IR;NPY and IR genotypes demonstrated
similar escape latencies on Day 1 with improvements observed from the first to
the second Visible Platform Trial at 6 and 12 months of age (Fig. 2A;
p 0.05). Similarly, both groups began
to reach the platform faster over the hidden platform training days at all ages
(Fig. 2B; p 0.05), indicating that
there were no learning performance impairments across the groups.
Fig. 2.
Morris Water Maze performance. Mean escape latencies were
collected for each trial day to assess performance over time. Time spent in the
target quadrant was measured to assess hippocampal-dependent memory. (A) Mean
escape latencies did not differ between IR;NPY and
however, were significantly reduced on Day 2 for mice aged 6 and 12 months. At 24
months of age, no significant difference in mean escape latency was observed
between Day 1 and Day 2. (B) Both IR;NPYand IR groups reached the platform faster across the hidden
platform days at 6, 12 and 24 months of age. (C) Both groups swam similar path
lengths in the target quadrant during the probe trials. At 24 months, a decline
in path length was exhibited in both IR;NPY and
IR mice when compared over time to 6 and 12 month time points. (D) At
6 and 12 months of age, IR;NPY mice spent significantly
less time in the target quadrants compared to IR control mice. At 24
months of age, no differences between genotypes was apparent. A main effect of
age was seen at 24 months of age, with IR;NPY mice spending
less time in the target quadrant compared to performance at 6 or 12 months of
age. Values are expressed as mean SEM. * = interaction effect between
IR and IR;NPY genotypes, p 0.05.
= main effect of time, p 0.05. Analysed by 2-way ANOVA
followed by Tukey’s honest significance difference (HSD) test. 6 and 12 months, n
= 59 IR and 56 IR;NPY. 24 months, n = 54
IR and 52 IR;NPY.
During the probe test, both groups swam similar path lengths until 24 months
when both groups had reduced path length (Fig. 2C;
p 0.05). These data suggest that
differences between genotypes were not due to sensorimotor or motivational
deficits. IR;NPY mice spent less time in the target
quadrant compared with control (IR) mice at 6 and 12 months. By 24
months both groups had reduced spatial performance with no differences between
genotypes (Fig. 2D; p 0.05).
3.3 Glucose tolerance and insulin sensitivity
To rule out differences in peripheral glucose metabolism in
IR;NPY mice, glucose tolerance and insulin sensitivity were
examined. Both groups displayed a similar reduction in blood glucose after
peripheral glucose injection (p 0.05, Fig. 3B) and
peripheral insulin injection (p 0.05, Fig. 3A).
Similarly, both genotypes displayed similar insulin release following glucose
injection (p 0.05, Fig. 3C).
Fig. 3.
Blood glucose and insulin. (A)
IR;NPY and IR mice produced similar glucose
tolerance after peripheral glucose injection over 120 minutes. (B) Both groups
displayed similar insulin sensitivity after insulin injection over 60 minutes.
(C) Plasma insulin levels were not significantly different between
IR;NPY and IR mice at baseline or 15 minutes
after glucose injection. Values are expressed as mean SEM. * = p 0.05; by 2-way ANOVA followed by Tukey’s honest significance difference (HSD)
test. N = 59 IR and 56 IR;NPY.
3.4 Hippocampal p-Akt
Peripheral insulin injection (20 IU/kg), compared to an i.p injection of saline,
resulted in an increase of phosphorylated Akt in the dentate gyrus of the
hippocampus in IR animals (Fig. 4A,B). Phosphorylation of Akt was
reduced in IR;NPY mice (Fig. 4C,D), indicating decreased
insulin action in the dentate gyrus of knockout animals (Fig. 4E).
Fig. 4.
Representative photomicrographs showing phosphorylated-Akt (green) and DAPI (blue) in the dentate gyrus of the hippocampus. 6 month old IR;NPY or IR mice were given a peripheral saline or insulin injection and brain slices were then prepared to examine phosphorylated-Akt (p-Akt). (A) The degree of hippocampal p-Akt in the IR + saline group was used as a control. (B) P-Akt was significantly increased in IR + insulin group, indicating insulin activity. (C) As expected, IR;NPY + saline group displayed p-Akt similar to the IR + saline group. (D) IR;NPY + insulin group did not display the expected increase in p-Akt. (E) p-Akt immunofluorescent counts were compared between treatment groups as a percentage of control levels (IR + saline) of activity. Values are expressed as mean SEM. * = p 0.05; by 1-way ANOVA followed by Tukey’s honest significance difference (HSD) test. Scale bars represent 200 m and 50 m (insets). N = 5/group (10 IR and 10 IR;NPY).
4. Discussion
In the present study, we aimed to determine if the loss of IRs in NPY-expressing
neurons negatively affected cognitive performance in the MWM, a robust measure of
hippocampal-dependent memory performance. Because cognitive performance can be
affected by aging, amongst other factors, we tested mice at 6, 12 and 24 months
of age. We then examined p-Akt as a measure of insulin signaling in our knockout
IR;NPY mouse model. This mouse model has been validated
previously and is a robust measure of IR deletion in NPY neurons [38]. This was
demonstrated by the authors upon ICV infusion of insulin which caused downstream
activation of p-AKT in IR mice but not IR;NPYmice. NPY cells were genetically tagged with a mCherry marker which showed
extensive overlap with p-Akt upon ICV insulin infusion in IR mice
[38].
Consistent with previous research, male IR;NPY mice had
significantly increased body weight compared with IR control mice
[38]. This increased body weight may be due to two reasons; a loss of insulin
signaling leading to an upregulation of the orexogenic NPY [45] and reduced
energy expenditure in IR;NPY mice [38]. In contrast to
previous work, no difference in energy intake was observed between genotypes,
however, this could be due to differences in study design [38]. Interestingly,
differences in body weight by genotype were only observed in male mice at 6
months of age, suggesting that disrupted homeostatic signalling in
IR;NPY mice is reduced in later life. It is unclear why
female mice did not show similar body weight differences between genotypes,
however it is known that sex plays a role in insulin-dependent regulation of
energy homeostasis [25]. Despite sex-differences in body weight at 6 months old,
there were no sex differences observed in either genotype during MWM performance,
hence the data for male and female were combined. This is in contrast to both
rodent and human studies showing that sensitivity to ICV insulin infusion [25] or
intranasal insulin [27] affects males and females behavior differently. While
this is interesting, our data clearly show that disruption to endogenous insulin
signaling is not significantly affected by sex, at least during
hippocampal-dependent memory tasks.
In the MWM, IR;NPY mice spent less time in the target
quadrant compared to IR control mice during the probe trial at 6 and
12 months of age. Importantly, these differences were not a consequence of
reduced motivation or locomotor abilities of mice, as all mice showed similar
escape latencies during learning of the MWM task. Knockout of IRs on NPY neurons
did not affect spatial learning during the hidden platform days, therefore this
mouse model has specific deficits of spatial memory retrieval during a
hippocampal-dependent memory task. These data are consistent with a previous
study which found that insulin-resistant mice present with hippocampal memory
impairments but intact spatial learning on the MWM [13]. Moreover,
intrahippocampal insulin administration improves spatial memory performance in
the probe test [13, 46]. Importantly, while these studies show that exogenously
administered intra-hippocampal insulin significantly improves cognitive
functioning, our results demonstrate that endogenous IR signaling has similarly
vital roles in maintaining appropriate cognitive functioning. In line with our
work, disrupted or ablated IR signaling is correlated with neuroinflammation and
cognitive deficits, including reduced spatial memory acquisition [47, 48], while
administration of intra-hippocampal PI3K inhibitors impaired memory retrieval
[49].
Through the use of our conditional IR;NPY knockout mouse
model, we conclusively determined that insulin’s actions in promoting hippocampal
function are, at least in part, mediated through NPY cells. The hippocampus is
rich in NPY neurons expressing IRs [50, 51, 52, 53] and is also crucial in spatial
navigation and memory formation [54, 55]. NPY signalling can enhance stem cell
proliferation and neurogenesis via the Y1 receptor within the dentate gyrus [56]
and it is possible that insulin signaling may potentiate NPY neuronal activity
within the hippocampus to support learning and memory. While these findings do
not exclude the contribution of other cell types in mediating
hippocampal-dependent cognition, they do clearly indicate that IRs on
NPY-expressing cells are an instrumental part of a spatial memory circuit.
Interestingly, during the probe test, no difference between
IR;NPY and IR controls were observed in
the time spent in the target quadrant at 24 months of age. Together, both groups
also scored significantly lower when compared to their probe trial at 6 and 12
months of age. Similarly, both groups of mice swam similar path lengths at 6
and 12 months of age, however at 24 months both groups of aged mice exhibited
decreased path length in the target quadrant, consistent with a floor in
performance. These results may be explained due to age-related effects on insulin
sensitivity and cognition. Consistent with these findings, other work has found
that while young rats show improved performance on the MWM after insulin
infusion, aged rats do not show sensitivity to the effects of insulin [46]. This
could be due to an age-related decline in IR- immunoreactivity within
the hippocampus [57]. Aged animals also exhibit upregulated pro-inflammatory
cytokines such as IL-1, TNF- and IL-6 in the hippocampus,
which have been shown to directly impair insulin receptor activity and signaling
[58]. These pro-inflammatory cytokines may also contribute to age-related
hippocampal insulin resistance [59]. Therefore, our aged IR mice may
have cognitive decline equivalent to the effect seen with loss of IR on NPY cells
due to aged-related hippocampal insulin insensitivity. To support our MWM data,
we identified that IR;NPY mice exhibit reduced p-Akt in the
dentate gyrus of the hippocampus at 6 months of age, suggesting that these mice
exhibit down-regulated intra-hippocampal IR signaling. Reduced p-Akt is also seen
in states of central insulin resistance [60], which has been implicated with the
cognitive decline and cognitive pathologies seen in Alzheimer’s disease [61].
There are some limitations in the present study. This work focuses on the role
of IR on NPY cells within the hippocampus, however NPY is expressed elsewhere in
the brain, such as the hypothalamus [62]. Nevertheless, the present study uses
the MWM as a test model, which is known to specifically test hippocampal-based
spatial learning and memory [3]. Another consideration is that p-Akt signaling
immunohistochemical analysis between IR;NPY mice and
IR mice was only undertaken at 6 months of age. Considering both
genotypes of mice exhibited decreased path length and time spent in the target
quadrant at 24 months of age, it would be interesting to determine if this memory
deficit correlates with impaired p-Akt signaling in both
IR;NPY mice and IR mice.
5. Conclusions
The present study has shown that mice with a tissue specific knockout of IRs in
NPY expressing neurons (IR;NPY) demonstrated impaired
performance in the probe trial of the MWM compared with control mice at both 6
and 12 months, supporting previous studies that have shown IRs play crucial roles
in spatial memory. Importantly, we present novel evidence that these effects are
mediated by NPY-expressing cells. Interestingly, no difference between genotypes
was observed in aged (24 months old) mice. Further research is required to
determine age-related physiological changes in IR signaling which modulate
spatial memory and learning. Together, this data provides valuable insights into
how IR signaling in discrete regions affects cognition, which could have
important implications for pathologies related to insulin resistance, such as
obesity and Alzheimer’s disease.
Abbreviations
CNS, Central Nervous System; GTT, Glucose Tolerance Test; IR, Insulin Receptor;
ITT, Insulin Tolerance Test; MWM, Morris Water Maze; NPY, Neuropeptide Y.
Author contributions
DB and HH designed the research study. EG, JT, JG and KA performed the research
and analysed the data. EG, CM, DB, NR and LZ wrote and reviewed the manuscript.
All authors contributed to editorial changes in the manuscript. All authors read
and approved the final manuscript.
Ethics approval and consent to participate
Animals were raised and handled at Australian BioResources Ltd, Moss Vale, NSW,
Australia, with genotyping also being performed at this facility. Animals were
transferred to University of New South Wales for approved experiments.
Experimental procedures were approved by the University of New South Wales Animal
Care and Ethics Committee in accordance with the Australian Code of Practice and
Use of Animals for Scientific Purposes (ACEC 16/21A).
Acknowledgment
We thank the anonymous reviewers for the constructive criticism of this article.
Funding
This work was supported by the Australian Research Council (DE160100088 and
DP170100063) and a Ramaciotti Foundation Establishment Grant.
Conflict of interest
The authors declare no conflict of interest.