Julia Zalewski
Specific Aims
Leptin is a protein hormone crucially important in the control of feeding behavior.
Secreted from white adipose tissue, leptin acts a signal for body energy stores. The hypothalamus
of the brain is important in neuroendocrine functions and feeding behavior. There is a link
between the hypothalamus and leptin. The hypothalamus has the highest concentration of leptin
receptors in the brain (Caron et al., 2010). Leptin directly affects neurons in specific parts of the
hypothalamus such as the arcuate nucleus (ARH), the ventromedial nucleus (VMH), and the
lateral hypothalamic area (LHA). The ARH is our focus in this proposal as it has been associated
with obesity and contains high levels of leptin activated neurons. Leptin works on two groups of
neurons in ARH. One group, co expresses αMSH, a melanocortin peptide, derived from proopiomelanocortin (POMC) and cocaine and amphetamine-regulated transcript (CART). These
neurons inhibit appetite when activated by leptin. The other group, co expresses the neuropeptide
Y (NPY) and agouti-related peptide (AgRP). These neurons increase appetite and are inhibited
by leptin. It has been proposed that the biological effects are sent through pathways originating
from these two groups of ARH neurons (Bouret and Simerly, 2007).
The formation of the hypothalamus is characterized by developmental processes in three
categories: (1) neurogenesis, (2) the movement of cells to their final target, and (3) the formation
of circuits. There has not been as much focus on embryonic development of neurons, especially
hypothalamic neurons controlled by leptin. Therefore, this study will focus on neurogenesis of
mice that lack leptin (Lepob/Lepob). Of the studies done on neurogenesis, it has been shown that
neurons of the ARH are born at E12-E16 (Bouret and Simerly, 2007). This suggests there is
important critical pre-natal developmental period, which is why changes in the intrauterine
environment may affect hypothalamic neurogenesis. Additionally, there has been an increasing
appreciation that changes in nutrition in leptin levels early in life can cause structural problems
in the hypothalamic feeding circuits. Maternal obesity causes increased leptin levels postnatally
and decreases the hypothalamic response to leptin during the critical period (Bouret, 2010a).
The overall goal of this study is to understand how the development of pathways that are
responsible for controlling body weight and energy balance, are affected by leptin. Our
hypothesis is that embryonic neurogenesis in the hypothalamus is directly affected by leptin.
These studies will help improve our understanding of how changes in the prenatal nutritional
environment can lead to obesity and diabetes.
Aim 1. Determine birth date of hypothalamic neurons in embryos without leptin.
In mice with leptin (WT) we determined the birth dates of hypothalamic neurons that control
energy balance and leptin-activated cells. Now we will test Lepob/Lepob mice using in vitro
assays and immunohistochemistry. We will use BrdU, which is a biomarker of cells that are
dividing, and a neuronal marker to examine neurogenesis in the embryonic hypothalamus of
mice.
Aim 2. Determine if embryonic leptin has the ability to correct neurodevelopmental
and metabolic problems in Lepob/Lepob
Changes in the intrauterine environment can affect hypothalamic neurogenesis, axonal outgrowth
and even mRNA expression. We will inject embryos in Lepob/Lepob mothers with leptin during
the critical period of development for ARH neurons to see if a certain level of leptin can correct
or improve these developmental problems. A combination of techniques will be used to view
how leptin effects ARH projections, mRNA expression and neurogenesis.
Julia Zalewski
Background and Significance
The importance of studying leptin’s effects in the hypothalamus can help further our
understanding of how obesity develops. Leptin is a protein hormone and the DNA sequence for it
was found on the obese (ob) gene (Halaas et al., 1995). Mice that have mutations on both ob
genes (ob/ob) cannot produce leptin and therefore have lots of fat storage as leptin cannot control
the POMC and CART neurons. Previous studies have shown that the loss of autophagy in POMC
neurons causes metabolic defects and causes abnormal hypothalamic axonal projections (Coupe
et al., 2012). Our study will focus on these mice that lack leptin (Lepob/Lepob) in order to
determine the effects that leptin has on neurogenesis in the hypothalamus.
Leptin in the Hypothalamus
The hypothalamus has been the focus of feeding regulation because it is the region of the
brain that contains high numbers of neurons that are responsible for metabolic regulation and
respond to hormonal and nutritional signals. Leptin is one of these hormonal signals that control
neurons in the hypothalamus. These groups of neurons controlled by leptin are found in the
arcuate nucleus (ARH). The ARH contains neurons that coexpress neuropeptide Y (NPY) and
agouti-related peptide (AgRP). These neurons promote feeding and are inhibited by leptin (Fig.
1). The other group of neurons in the ARH coexpress α-melanocyte-stimulating hormone, which
is derived from POMC and CART. These neurons inhibit appetite and are stimulated by leptin
(Fig.1). Both AgRP/NPY and POMC neurons send projections to other parts of the
hypothalamus, such as the paraventricular (PVH) and dorsomedial (DMH) nuclei, and the lateral
hypothalamic area (LHA), where they release peptides to regulate energy balance. Like the
ARH, these regions also play important roles in controlling feeding behavior and energy balance.
Leptin receptors are expressed in various tissue such as the muscles and the gut, but the highest
expression occurs in the ARH. In response to leptin level, the ARH will produce varying levels
of neurotransmitters and neuropeptides that regulate food intake and body weight. It reduces the
effects of NPY and AgRP (feeding stimulant). It promotes α-MSH and CART (appetite
suppressers). The projections to the PVH are particularly important because the nucleus
provides major inputs to brain stem regions that regulate autonomic functions.
Leptin effects the development of these pathways and projections. The density of the
ARH axons in Lepob/Lepob mice innervating the PVH decreases by 10-fold compared to wild
type (WT) (Bouret, 2010a). Additionally, treating Lepob/Lepob neonates with leptin, restores a
normal pattern of the ARH projections; however, the same leptin treatment in adults does not
reverse the abnormal ARH projections seen in Lepob/Lepob (Bouret et al., 2004). These pathways
don’t become fully developed until three weeks after birth (Bouret, 2010b). These results show
that there is a postnatal critical period of development. This is important for our study as these
neurons that projecting to the PVH are born in the ARH and the development of these neurons
and pathways could be affected by changes in the intrauterine environment. Additional studies
have found that there is a second critical period of development, prenatally, which was
discovered through birth dating studies of neurons. Our study will be focusing on this prenatal
critical period of development.
Julia Zalewski
Figure 1. Leptin's actions and effects on the ARH, VMH, LHA. Leptin circulates in the blood stream and binds to
receptors on NPY/AGRP- and -MSH/CART-producing neurons in the ARH to cause a series of responses mediated by
centers downstream including the PVN to control thyroid secretion, feeding behavior, and energy conservation.
Projections are also sent to the VMH and the LHA. (Flier, 2004)
Development of Neurons in the hypothalamus
There have been extensive studies on the postnatal critical period of development in the
hypothalamus but not much has been done on the prenatal period, specifically looking at
neurogenesis. The development of the hypothalamus is important in understanding how neurons
are born and how their projection pathways are set up. Development of the hypothalamus begins
with neurogenesis in the third cerebral ventrical. Here divisions produce cells that will produce
neurons. It is these neurons that travel from their location in the proliferative zone to form the
multiple nuclei and areas that make up the hypothalamus (Bouret, 2010b). The most recent birth
dating study has found that ARH neurons are born between E12-E16, with most born on E12
(Ishii and Bouret, 2012). The birth date of neurons in the ARH is important because this
indicates a critical period where changes in the intrauterine environment can cause
developmental and nutritional consequences for the embryo later in life. Ishii and Bouret’s study
also found that the neurons in the ARH are the first to express POMC mRNA on E12, as well as
NPY cell bodies at E14. Likewise, the number of cells in the ARH is important because this
where anorexigenic neurons, POMC and NPY, are born. The more neurons of one type would
cause a higher chance of either increasing or decreasing feeding behavior. It has been shown that
leptin deficiency results in a loss of cortical neurons born during embryonic life (Bouret et al.,
2004). The effect of no leptin on the number of cells in hypothalamus is what we are looking at
in the first aim.
Neurogenesis in the hypothalamus
One previous study looked at neurogenesis in Lepob/Lepob adult mice. They did not
specifically determine the birth date of neurons in the hypothalamus. However, they found out
that the number of cells in the hypothalamus was decreased in Lepob/Lepob mice (McNay et al.,
2012). Additionally, treatment of hypothalamus with leptin seemed to increase the number of
cells. This is relevant to our study as we now know neurogenesis can occur in Lepob/Lepob adults
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but at a lower rate. Leptin has been hypothesized many times to have a prenatal effect in the
development of neurons. A premature surge of leptin (either prenatally or induced by leptin
supplementation in neonates) alters energy regulation by the hypothalamus and contributes to
weight gain and leptin sensitivity (Bouret, 2010b). This surge could be the cause of the
neurodevelopmental problems. In this study we hypothesize to find similar results as McNay et
al. of neurogenesis in embryos, in that there is increased cells when injecting leptin.
Maternal nutrition
It has been determined that the intrauterine environment is important in the prenatal
development and changes to it can cause problems in the development of feeding circuits.
Therefore, maternal nutrition and hormone levels will affect the development of their embryos;
specifically in this study the development of the hypothalamic neurons. Maternal over-nutrition
and under-nutrition have implementations on the development of the embryos. Maternal overnutrition affects leptin sensitivity as demonstrated by the increased levels of leptin-induced
phosphorylation of the signal activator of transcription 3 (pSTAT3, intracellular signaling
pathway of LepRb) (Bouret, 2009). Likewise, maternal obesity causes increased leptin levels
post-natally and decreases the hypothalamic response to leptin during the critical period (Caron
et al., 2010). Additionally, AgRP projections are abnormal. Other studies have focused on
maternal under-nutrition. Studies have shown that offspring born to undernourished mothers
show a similar trait as the pups born to obese mothers, that they are more prone to obesity
throughout their life. However, pups born to underfed mothers have reduced leptin levels during
the postal period (Bouret, 2010c). The specific diet fed to mothers also has effects on their
offspring. Proteins in the maternal diet are important for the development of the hypothalamus as
mice from a mother fed a low-protein diet have the same metabolic and hypothalamic effects as
mice born to underfed moms (Coupe et al., 2010). It has been difficult to determine the factor
that causes these metabolic and neurodevelopmental effects observed in the offspring. However,
leptin remains the prime suspect. As such, it is important to determine the effect leptin has on the
hypothalamus. Our study will go beyond what has been done and use the Lepob/Lepob mice and
leptin injections in the undernourished mother to see if it saves neurogenesis and the metabolic
effects in the hypothalamus.
Significance
Understanding how the hypothalamus functions, when neurons are born and what
neurons are controlled by leptin, can help lead to the possible discovery to preventing obesity
and type II diabetes. The number of cases of obesity and type II diabetes have increased
dramatically over the past 20 years. Roughly 35% of Americans are obese today and rates
continue to rise. With increased obesity rates comes an increase in research of the biological
factors of obesity. This field will continue to develop if people continue eating the way they are
today.
We believe that in a world where the size of a hamburger has tripled from 4 oz to 12 oz
since the 1950’s (Activity, 2006), many people will want to know the effects of leptin and how it
effects our ability to manage weight. Our study could have many implications for human weight
management and genetic treatments, as mice models can provide valuable insights in to the
human biological processes. Mice genetically prone to develop diet-induced obesity are well
suited, because their frequency to become overweight shares various features with human
obesity, such as multiple genes that are responsible for the trait (Bouret, 2010c). Much
Julia Zalewski
information is still needed before we can apply these findings to humans, such as advancing the
techniques used in these experiments. The crucial aspect of this study is to determine if leptin
does indeed act directly upon the hypothalamus to influence neurogenesis.
Research Plan
Prior to our previous work on neurogenesis in the hypothalamus, not much was known on
the birth date of leptin-activated neurons in the hypothalamus. Our results from our most recent
study find that neurons in wild type (WT) mice were born between E12-E16 with a peak at E12
(Ishii and Bouret, 2012). We also observed leptin activated cells using BrdU staining with cFos
immunohistochemisrty. These leptin activated cells were also born at E12. Therefore, the next
step is to determine what role the mother’s environment plays in neurogenesis and the
development of the hypothalamus.
Goals: To understand how the development of pathways that are responsible for controlling
body weight and energy balance, are affected by leptin.
Aim 1: Determine birth date of hypothalamic neurons in embryos without leptin
Aim 2: Determine if embryonic leptin has the ability to correct neurodevelopmental and
metabolic problems in Lepob/Lepob
General approach
Our general approach is to use immunochemistry techniques with leptin injections to
decide out how leptin is acting in the hypothalamus of mice without leptin to affect their feeding
behavior and energy balance. These experiments will use pregnant Lepob/Lepob mice that have
been genetically modified to not have the gene for leptin. We have obtained these mice from a
trustworthy lab. Our plan is to use this line of mice for all our experiments.
Aim 1: Determine birth date of hypothalamic neurons in embryos that lack leptin
Rationale and significance:
The birth of neurons, especially leptin-activated neurons, in the hypothalamus is
important in determining the feeding behavior for mice throughout their lifetime. For a healthy
weight throughout life, previous research has shown that leptin is needed to develop the
hypothalamus. Our goal in this experiment is to determine if leptin effects when neurons born in
the ARH, DMH, LHA, and PVH of the hypothalamus as well as the number of cells born.
Why is neurogenesis important?
The birth of neurons allows for development of the hypothalamus to continue correctly. Without
neurogenesis cells cannot get to their target or form circuits in the hypothalamus. This
experiment is important because leptin levels control the actions of some neurons in the
hypothalamus and we are determine the birth date of these neurons.
When does neurogenesis in WT mice occur?
From our most previous research on WT mice we have learned that neurons known to control
energy balance in the hypothalamus are born between E12-E16, with most born on E12. Neurons
in the DMH, PVH, and LHA are born between E12-E14. In the ARH, lots of neurons were born
on E12 but also as late as E16. The majority of leptin-activated cells in the adult hypothalamus
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were born on E12. Our experiments are designed to use the same protocol as this previous study
to determine the birth date of neurons in Lepob/Lepob mice.
What is the fate of hypothalamic neurons?
The neurons we will focus on are those in the ARH. The ARH contains neurons that coexpress
NPY and AgRP. It also contains another group of neurons which coexpresses α-melanocytestimulating hormone, which is derived from POMC and CART. These are the neurons controlled
by leptin and will be affected by the lack of leptin in the intrauterine environment of the mice in
our experiments.
Approach
How will we track the birth date of neurons, especially in mice that lack leptin? This will
have to be approached in a two ways using a similar procedure as the birth dating of WT mice
(Ishii and Bouret, 2012). The first method determines the birth date of the non-leptin-activated
neurons. The Lepob/Lepob pregnant mothers, the negative control, will be injected with BrdU, a
biomarker of dividing cells. By labeling the cells while still in the uterus, we can determine
where these cells end up in the hypothalamus and when they were born. The positive control, the
WT mice, was previously tested in our most recent work and will be repeated here for
consistency (Ishii and Bouret, 2012). The ip BrdU injections will take place in mice on E10, E12,
E14, E16, or E18 (Fig. 2). Injections need to take place at different times to determine when the
neurons are born. The male offspring will live until 10 days after they are born (P10) when they
will be anesthetized in order to perform immunhistochemistry. The mice brains will be removed
quickly and placed in a solution to store overnight. Then we will cut coronal sections and mount
them on slides for BrdU and HuC/D staining. A special specific procedure will be used to
visualize BrdU, which includes being incubated with either rat anti-BrdU and mouse anti-human
neuronal protein HuC/HuD (HuC/D) antibodies. A goat antirat IgG conjugated to a dye will be
used to visualize the anti-BrdU. We will use a lab microscope to image the results. The images
should indicate that where more staining occurs on a specific day, more neurons are being born
on that day. The results will be analyzed and quantified to gain statistical significance.
Figure 2. Timeline of BrdU, cFos and Leptin injections. There are two groups of mice in order to test neurons in the
hypothalamus (group 1) and leptin-activated neurons in the hypothalamus (group 2). There are differences in BrdU
injections and staining types. The timing of injections varies because it is known during E12-E16 most neurons are born.
The double staining is needed in group 2 in order to determine the birth date of leptin-activated neurons.
Julia Zalewski
Similar to the experiment above, the second part will look at leptin-activated cells in the
adult mice using a combination of BrdU and cFos staining. There are differences in the
procedure. The same strain of Lepob/Lepob pregnant mice will be used and ip BrdU injections
will take place on E12, E14 or E16 three times a day. Previous results from WT mice have
indicated that the majority of neurons are born between E12 and E16, which is why the timing of
injections differs from group 1. These mice will be allowed to live until 60 days after birth (P60)
when they will be injected with either leptin (positive control) or vehicle (negative control) and
then anesthetized 2 hours later. The tissues samples will be prepared in the a similar way. We
will stain the brains with HuC/D to visual BrdU and additionally cFos because changes in cFos
staining generally represent an increase in neuronal activity that can be expressed either by leptin
or transynaptic activation. Therefore the darker cFos stain means more neurons are present. The
immunohistochemical techniques, imaging, and analytical tests we will use are the same. Using
double staining, with both HuC/D and cFos, is important as it allows us to determine when
leptin-activated cells are born, by injecting them with BrdU using HuC/D to visualize and later in
life staining with cFos (Fig 3). The overlap of the two stains illustrates what happens when the
cells labeled at E12 with BrdU are present in the hypothalamus at P60 (Fig. 3).
Figure 3. Staining with BrdU and cFos. These four cells represent what we will be seeing at different stages of the
experiment. When injecting BrdU at E12 we would see the stain one color. In the second part we would inject leptin at
P60 and stain with cFos in order to visualize the increase in neural activity. The overlap of the two stains seen in group 2
indicates that most of the cells that are present there were born at E12.
Potential Outcomes and Interpretation
If previous studies are any indication, we should find few neurons being born in the
hypothalamus (McNay et al., 2012) due to the loss of hypothalamic neural stem cells. The birth
date of cells could also be affected as our study on WT mice concluded that changes in the
intrauterine environment may affect hypothalamic neurogenesis. A Lepob/Lepob mother does not
have a normal intrauterine environment. Therefore, we believe this change could cause neurons
in the hypothalamus to be born later or not at all, which would have abnormal developmental
effects.
The results should not turn out like WT, as in figure 3. In other words, we do not expect
to see the two stains overlapped (Fig. 3d) which indicates that there are no BrdU labeled E12
cells at P60 because leptin is not present. Basically, neurons that are leptin-activated will not be
born because they cannot be born without leptin. A negative result in group 1 would show very
little staining indicating that no cells are born and that a few cells that may be born later (after
E12). We predict this because of previous studies that have shown there are fewer cells present
in Lepob/Lepob mice (McNay et al., 2012) and if new cells were being born we would assume that
there would be some alteration in the birth dating of neurons. This would affect development of
the hypothalamus, and therefore the necessary circuitry for neurons to travel through would not
Julia Zalewski
be set up. In the hypothalamus this could affect the feeding behavior of the mouse for the rest of
its life. The postnatal surge of leptin would not occur and mice would be more inclined to
become overweight as they don’t have the POMC neurons to tell the hypothalamus to decrease
our appetite. A positive result will indicate that most neurogenesis occurs at E12, as it does in
WT mice (Fig 3).
Potential Pitfalls
Due to little information on the birth date of leptin-activated cells in the hypothalamus,
we do not have much data to compare our results too. We will acknowledge this in our
discussion section. Additionally, when working with animals, subjects could die, and therefore
not survive until the date we need them to (either P10 or P60). This would cause our data to be
incomplete and would need to be started over. We are also only looking at male mice offspring,
and the data may be different if we were to include female offspring. Also when using leptin
injections we need to be careful where they are going. If leptin is injected in the wrong spot our
results could be altered. Therefore we need to be precise in where these injections occur.
Additionally, due to the nature of our experiment we need to make sure that if we get a positive
result that it is due to the lack of leptin and not experimental error. Therefore, multiple mice will
need to be tested, and this procedure will need to be followed by another lab in order to obtain
consistent results.
Aim 2: Determine if embryonic leptin has the ability to correct neurodevelopmental and
metabolic problems in Lepob/Lepob
Rationale and significance:
Leptin plays a key role in the development of the hypothalamus. The maternal
environment can be responsible for changes in the neurodevelopmental and metabolic effects
because there is a pre-natal critical period. The most recent birthdating study has found that ARH
neurons are born between E12-E16, with a large peak at E12 (Ishii and Bouret, 2012). The
neurons in the ARH are the first to express NPY and AgRP mRNA cell bodies at E14. These are
the important group of neurons (NPY and AgRP) that increase appetite. Previous studies have
shown that the development of these cell bodies is decreased in Lepob/lepob mice while POMC
and CART decreased (Duan et al., 2007), which indicates that leptin plays a role in their
development. The development of neurons in the ARH is important because they have been
found to be born in WT during this critical period, where changes in the intrauterine environment
can cause developmental and nutritional consequences for the embryo later in life. WT mice
normally contain leptin and have normal development while Lepob/lepob mice do not have leptin
and have neurodevelopmental and metabolic changes that occur later in life. Therefore, we will
use Lepob/Lepob pregnant mothers and inject leptin into the embryos to see if leptin can correct
these defects.
Overall Hypothesis
Our hypothesis is that leptin injections into Lepob/Lepob mice embryos will be able to
correct the development of neurons in the ARH, which in turn should be able to enable normal
development to take place.
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(i) Hypothalamic connections
The embryonic leptin injections into the Lepob/Lepob embryos will cause the Lepob/Lepob
offspring to have increased ARH projections to their targets.
(ii) mRNA expression patterns
The embryonic leptin injections into Lepob/Lepob embryos will rescue the development of mRNA
expression of leptin activated neurons such as NPY/AgRP and POMC/CART in their offspring.
(iii) Hypothalamic Neurogenesis
Embryonic leptin injections into the Lepob/Lepob embryos will cause an increase in hypothalamic
neurogenesis.
Approach
The aim is broken up into three experiments, but all experiments follow the same
procedure for embryonic leptin injections. In order to determine if leptin can correct
neurodevelopment and metabolic problems in mice, we obtained pregnant mice that lack leptin
(Lepob/Lepob). We also have WT mice and we will compare our results to this as well. We will
be following a similar procedure used to see the development of the cortical neurons in the
cerebral cortex (Udagawa et al., 2006). The Lepob/Lepob pregnant mothers will be injected with
BrdU intraperitoneally in order to mark the cells that could be present later in life. The biomarker
is needed to obtain results for phase iii. Two hours later exo utero surgery will be performed as
previously described (Hatta et al., 2002). The next step is important to do quickly because we do
not want to cause too much stress on the mother and developing embryos. The pregnant mothers
will be anesthetized and the abdominal wall and uterine wall will be cut. At this point 200 ng of
leptin will be injected into the lateral ventricle of positive control E12 and E16 ob/ob embryos in
the right uterine horn (Figure 4). The injections need to take place during this time because it is
the pre-natal critical period. A vehicle will be injected in negative control ob/ob embryos in the
left uterine horn of the same mother. The vehicle is needed in order to determine whether or not
leptin has an effect. The uterus will then be placed back into the abdominal wall and sutured, the
mother will recover. Recovery is important for development of the embryos. The next steps take
place after the embryos develop and are born.
Figure 4. Leptin injections into Lepob/Lepob embryos. 200ng of leptin will be injected into the lateral ventricle of positive
control E12 and E16 ob/ob embryos in the right uterine horn. A vehicle will be injected into the left uterine horn.
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(i) Hypothalamic connections
This experiment has previously been done on WT mice, and we will run the same
experiments on WT mice in order to compare our results. The Lepob/lepob mothers will give birth
to two different groups of pups. One group will have received leptin injections (positive control)
and the other received the vehicle (negative control). Using a previously similar procedure
(Bouret et al., 2004) the offspring will be anesthetized on P4, P8, P10, P12, P14, P16, P21, or
P60. The mice need to be anesthetized at different days in order to determine how ARH
projections develop. The brains will then be removed and numerically coded to prevent biases
during analysis. DiI crystals will be implanted in the ARH of each brain using an insect pin
(Figure 5). DiI is used because it is a fluorescent tracer that labels axonal projections in tissues.
After, we will incubate them in the dark for six weeks, we will take sections through the
hypothalamus and evaluate with conventional fluorescence and confocal microscopy.
Figure 5. Implantation of DiI crystals into the ARH (coronal view). The DiI crystal will be inserted into the ARH of the
hypothalamus using a insect pin. The DiI is a tracer that we will use to image the projections. (Horvath and Bruning,
2006) Note: Arc=ARH
To test the activity of leptin on ARH projections in adult Lepob/Lepob mice, we used
immunohistochemical labeling of AgRP. Due to the efficiency of DiI labeling decreasing in
adults, we had to use this technique. It is known that in adult rodents AgRP expression is
confined to NPY neurons in the ARH, AgRP immunoreactive fibers will represent these
projections. The anesthetized mice will have their brains frozen, sectioned and then we will
perform immunohistochemistry. The sections will be incubated for 48 hours in either a rabbit
anti-AgRP or a sheep anti-α-MSH. For the α-MSH staining, sections will be incubated in donkey
anti-sheep IgG for 1 hour. For the AgRP staining, the antibodies will be localized with a goat
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anti-rabbit IgG. It is necessary to use different stains depending on which cell body we are
looking for. Sections will then be prepared for microscopic identification. Images will be
analyzed using imaging software to obtain the results.
(ii) mRNA expression patterns
The Lepob/lepob mothers will give birth to two different groups of pups. One group will
have received leptin injections (positive control) and the other received the vehicle (negative
control). In order to determine if embryonic leptin injections can rescue the expression of
metabolic defects in the offspring we need look at the development of NPY/AgRP and
POMC/CART neurons in the ARH. This quantitative analysis has been performed previously
using Lepob/Lepob hypothalamic tissues(Duan et al., 2007), but not with leptin injections. We
will wait sixty days after birth (P60) of the mice to anesthetize them. The brains will be removed
and hypothalamic dissection will take place shortly after. Tissue homogenization and RNA
extraction will be performed. We need to obtain hypothalamic RNA in order to determine the
mRNA expression in the hypothalamus.
We will perform real time PCR because it is the most sensitive method and can
discriminate between closely related mRNAs. In this case we will use it to compare the
hypothalamic mRNA expression of NPY/AgRP and POMC/CART. We will take the RNA to
reverse transcribe and then incubate. Quantitative PCR assays will then be performed. The cycle
conditions will be: 94.5 °C for 15 minutes, followed by 40 cycles at 97 °C for 30 seconds, 59.7
°C for 1 minute. The expression levels of mRNA will be expressed as either the high numbers of
cell containing the mRNA or the same number of cells containing more mRNA The experiment
in part 3 will help us determine which is true. Statistical significance will be quantified by twoway ANOVA to determine the possible effects of genotype and leptin treatment. This model will
allow us to determine the earliest responders among these genes and which ones have the highest
concentration.
(iii) Hypothalamic neurogenesis
After the embryonic leptin injections, this experiment will follow the same procedure as
Aim 1. There are some differences in that we will be comparing the leptin injected offspring with
the vehicle offspring. The negative controls are the Lepob/Lepob mice that receive leptin
injections while the positive controls are those that receive the vehicle injection.
Potential Outcomes and Interpretation
(i) A negative control would show a difference in the volume of ARH projections to their target
areas. The ARH projections to different target areas should be increased in Lepob/Lepob mice that
received leptin injections compared to those mice that received the vehicle. Specific target areas
that would have increased projections to them include the PVH, DMH and LHA. The images
would show stained ARH fibers indicating more fibers as the mice got older. By this we mean
that the volume of fibers would be higher at P8 than P6, and this trend would continue as time
went on. This outcome would suggest that projections are developing during pre-natal critical
period and need leptin to develop correctly.
(ii) Previous experiments have shown that the expression of POMC and CART, which are the
neurons that inhibit appetite, are decreased in Lepob/Lepob mice. Therefore, we expect to observe
a negative result that the mRNA expression of POMC and CART cell bodies will be increased in
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Lepob/Lepob mice because they received pre-natal leptin injections. The standard error (SE) will
represent this on the graphs. We will make graphs representing the cell bodies vs. rate of
expression would indicate which are expressed the most. This outcome would suggest that leptin
levels are responsible for the development of POMC & CART as well as NPY & AgRP cell
bodies. Additionally, with increased POMC and CART neuron cell bodies, which inhibit
appetite, we would expect to see the mouse that received pre-natal leptin injections weigh less
than then the mouse that received vehicle.
(iii) As previously explained, we expect to see a difference in the hypothalamic neurogenesis of
Lepob/lepob mice (Aim 1) specifically that less neurogenesis occurs. We would not expect to see
the two stains (HuC/D and cFos) overlapped (Fig. 3d) which indicates that there are no BrdU
labeled E12 cells at P60 because leptin is not present. Therefore, in this experiment embryonic
leptin injections would increase neurogenesis. This would be shown by an increase in cells that
have the overlapped staining (fig 3d). Neurons that are leptin-activated can be born in this case
because we are injecting leptin into the embryos. The positive result would show very little
staining indicating that no cells are born and that a few cells that may be born later (after E12)
because some mice have not been injected with leptin. This experiment will help us determine if
the increase expression of mRNA is due to more cells or less cells containing more or similar
amounts of mRNA. In other words, if we see increased neurogenesis it would be that there is an
increase in the number of cells which would increase the mRNA expression. If there is not an
increase in neurogenesis but increased mRNA expression, it could be that there is the same
number of cells that have high concentration of mRNA. The results from this experiment could
also help explain the projection results. Increased neurogenesis would indicate that there are
more cells which would mean more ARH projections could reach their targets.
Potential Pitfalls
The insertion of leptin into living embryos could be potentially fatal. Not only could it
stress the mother out, but the embryos could be damaged and therefore there could have
developmental defects or even die from the surgery itself. We will be extremely careful during
this phase following the protocol from previous experiments that used this technique. In order to
obtain enough data we will have multiple mothers. Additionally, the amount of pre-natal leptin
injected into the embryos may not be enough to have an effect on the metabolic and
neurodevelopmental defects. Therefore, the experiments would be repeated with leptin levels
increased to determine how much is needed. When dealing with animals death could also occur
at any point which would cause us to start over because we need to be able to compare mice that
received leptin injections and those that received vehicle from the same litter.
(i) As previously mentioned, the pre-natal leptin level injections may not be sufficient enough to
cause the ARH projections to reach their targets. Leptin levels would be increased to determine
the optimal level. Lepob/Lepob mice with pre-natal leptin injections may have fewer projections
than the WT mice, but they will have more than mice injected with vehicle. Therefore, leptin
levels may need to altered until the correct amount is found where projections in the Lepob/Lepob
are the same in WT mice. Also, because there is a second critical period that occurs postnatally,
additional leptin injections could be needed in neonates to reach the normal volume of
projections from the ARH. An experiment that uses both pre-natal and post-natal leptin
Julia Zalewski
injections will be needed to be performed in order to determine if this is true or which is most
beneficial to restoring normal levels.
(ii) PCR is extremely sensitive and even minute amounts of contamination by genomic DNA or
previously amplified PCR products can lead to abnormal results, so steps must be taken to avoid
this pitfall. We will take time and be precise when preparing samples for PCR to avoid mistakes.
Like any experiment using animals, death can occur before the mice reach P60, therefore we
need to have a large number of Lepob/Lepob mothers. Additionally, we will monitor pups after
birth to maintain their health. Also, in this case the pre-natal leptin level injections may not be
high enough to have effect on the development of POMC/CART and NPY/AgRP cell bodies.
Therefore, leptin levels would gradually be increased to determine the optimal level.
(iii) As we are following the same procedure as in Aim 1, many of the pitfalls that could occur
there apply here too, such as death and not having data to compare it to. In order to compare our
results with that in Aim 1 we need only male offspring. This could pose a challenge as it may
take multiple litters to obtain a large enough sample of males for sufficient data. Additionally,
the stress of receiving vehicle injection could be enough to cause insufficient results. By this we
mean that the rate of neurogenesis could be lower than in the Lepob/Lepob mice from Aim 1
because they were not opened up and injected.
Summary
Our research here will work to understand how leptin works pre-natally within the
hypothalamus to cause abnormal feeding regulation and neurodevelopmental problems. Our
study will use mice that lack leptin (Lepob/Lepob) in order to determine the effects leptin has on
neurogenesis and development in the hypothalamus. Additionally this work can have
implications for understanding on how to prevent obesity. Understanding how the hypothalamus
functions, when neurons are born and what neurons are controlled by leptin, can help lead to the
possible discovery to preventing obesity and type II diabetes. It could also have implications on
pregnancy care guidelines. There are suggestions of what you should and shouldn’t eat while
pregnant but if there is scientific evidence of maternal nutrition effecting the embryo, then there
may be rules or guidelines made that prevent a pregnant women from eating certain foods to
prevent from maternal nutritional deficiencies.
Julia Zalewski
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Real-time PCR has become one of the most widely used methods of gene quantitation because it has a large dynamic range,
boasts tremendous sensitivity, can be highly sequence-specific,
Julia Zalewski
PCR is the most sensitive method and can discriminate closely related mRNAs.
In contrast to regular reverse transcriptase-PCR and analysis by agarose gels, real-time PCR
gives quantitative results. An additional advantage of real-time PCR is the relative ease and
convenience of use compared to some older methods
-leptin injections into the mouse effects of neurogenesis. (same procedure compare mice that
have leptin injections vs vechicle)
Same number of cells? Diff? or expressing different levels of NPY/CART
Correlation between the number of cells and expression of mRNA , could it be that there are the
increased cells with increased mRNA expression in cell