Department of Physics, Chemistry and Biology (IFM)
Final Thesis
Validation of the limited transgene mGluR5KD-D1
expression to D1R-expressed neurons in the striatum
Ali Nasr Esfahani
Supervisor: David Engblom, Linköpings universitet
Examiner: Matthias Laska , Linköpings universitet
Content
1. Abstract…………………………….………………............................................................... 1
2. List of abbreviation...................……………………………………..........................................1
3. Introduction………………………………................................………….....……………..... 1
4. Material and Methods………………….……….....................................…….…..………….....3
4.1. Animal Housing………………………….........................................………...……...........3
4.2. Tissue Processing and Preparation....……….……........................................…………3
4.3. Tissue Sectioning.......…………..........................................................................................3
4.4. Immunohistochemistry (IHC)...............................................................................3
4.5. Immunofluorescence (IFC) …………………………...………………..…..…….4
4.6. Light Microscopy ………………………………………...……………....………4
5. Results…………………………………………………………………..…......………4
5.1. MSNs markers are detectable by DAB Staining...................................................4
5.2. The mGluR5KD-D1 is only expressed in D1-R expressing neurons.......................5
6. Discussion....................................................................................................................6
7. Acknowledgment.........................................................................................................7
8. References....................................................................................................................8
1 Abstract
One of the main difficulties of addiction treatment is the high risk of relapse even after a long
abstinence. Therefore, discovering the underlying molecular principles of relapse is essential. The
metabotropic glutamate receptor, mGluR5, is considered to play an important role in this aspect.
One of the well distributed regions of mGluR5 in the brain is striatum, an area which is basically
compromised from medium-sized spiny neurons (MSNs). These neurons are divided into two
major subpopulations characterized based on their projections and protein properties. In our
constellation, we have generated a mouse line designed to have a selective mGluR5 knock-down
in dopamine D1 receptor (D1R) expressing neurons. It has however been unclear if the
expression of transgene is indeed limited to only D1R-expressed neurons. By
immunofluorescence, we here show that in the striatum the construct is expressed only in MSNs
and is restricted to the D1R-expressing cell population. Thus the transgenic mouse line is a good
tool for the study of mGluR5 selectively in D1R expressing neurons.
Keywords: Addiction, Reward, Cocaine, Drug Abuse, Relpase, Striatum, Dopamine, Medium
Spiny Neurons, mGluR5, Glutamate, immunofluorescence, DARPP-32, Enkephalin, GFP,
Mouse,
2 List of abbreviations
D1R – Dopamine Receptor D1
IFC- Immunofluorescence
D2R – Dopamine Receptor D2
IHC- Immunohistocehmistry
DA – Dopamine
mGluR5 - Metabotropic Glutamate Receptor
5
DARPP-32 - Dopamine And cAMP
Regulated Phosphoprotein of 32 kDa
GFP- Green Fluorescent Protein
MSNs- Medium Spiny Neurons
ppENK - pre-pro Enkephalin
3 Introduction:
Addiction is a major burden on every society. It is a complicated phenomenon caused by various
psychological and social consequences, and several biological processes underlie it. Drug
addiction is classically defined as a chronically relapsing disorder characterized by repeating of
compulsive drug seeking and taking, despite potential harms (1). Moreover, addiction is
recognized as a neuroadaptation disorder characterized by dys-regulation of the
mesocorticolimbic dopamine (DA) reward system (2).
Cocaine addiction, one of the most destructive forms drugs abuse has a high risk of relapse (3)
that can appear even after a long period of abstinence from drug administration and full
detoxification (4-7). Numerous experimental studies on both animals and humans reveal that
relapse to cocaine abuse occurs from extreme craving that may start from exposure to different
environmental stimuli including a drug-associated cues (8-12). There are several indications
showing the role of neuroadaptations related to behavioural sensitization which is participating in
addiction relapse (13-15). These lasting effects of cocaine stimuli are caused by the ability of the
drug to dysregulate reward-related associative learning and memory processes.
Mesocorticolimbic DA reward pathways contribute to various drug-induced conditions including
physical dependence, craving and relapse (16-19). Striatum, including the nucleus accumbens, is
one of the main anatomical parts of this circuit that receives a large number of DA projections
participating in reward and relapse (20-26). This region also receives glutamatergic projections,
to where triggering of addiction relapse has been referred because of enhanced glutamatergic
neurotransmission (27-30). It is demonstrated that 95% of the striatal neurons consist of medium
spiny neurons (MSNs) (31) which are mainly divided into two subclasses base on their
projections and peptide content (32-34). One population expresses D1Rs (D1-MSNs), projects to
the substantia nigra and co-express the neuropeptide dynorphin (35). The other population
expresses D2Rs (D2-MSNs) (35) projects to the globus pallidus and co-express the neuropeptide
enkephalin.
Although the role of the striatum/nucleus accumbens in addiction is well established, the
proportional contribution of each D1 and D2 MSNs, and the specific underlying neurobiological
mechanisms of plasticity changes in striatal neurons after cocaine administrations that underlie
addiction relapse are poorly understood. In this perspective, the role of the metabotropic
glutamate receptor, mGluR5, which is a glutamatergice receptor expressed on both MSN
populations is potentially extremely interesting (36-45).
We aimed to point out more specific and restricted targets of the addiction relapse mechanisms
within the reward pathway. For this purpose, a novel mouse line has been generated in our
constellation in which mGluR5 is selectively knocked-down in dopamine D1 receptor (D1R)
expressing neurons. These mice (called mGluR5KD-D1 mice) show reduced levels of mGluR5 in
the striatum and a strongly reduced reinstatement of cocaine seeking, a model for relapse. In
addition they show deficiencies in specific aspects of reward-related learning that may explain
this reduced relapse tendency.
To link the function of mGluR5 in D1R neurons to these behaviour changes, it is important to
test if our construct is expressed in the accurate location which is D1-R expressing neurons.
Consequently, the aim of this study was to show if the construct is expressed only in D1R- and
not in D2R-expressed neurons in the striatum.
4 Materials and Methods:
4.1 Animal Housing:
Transgenic mice and control mice on C57BL6 background were used for this study. Animals
were all adult males aged 6-15 weeks old. They were housed in standard cages at a steady room
temperature (20ºC) on a regulated 12 hrs light/dark cycle (light was available at 7 a.m.) and
provided freely with food and water. All experiment procedures were performed in compliance
with the Swedish national guidelines and confirmed by the local Animal Care and Use
Committee.
4.2 Tissue Processing and Preparation:
Mice were deeply anesthetized with CO2 respiration and transcardially perfused with 20 ml of
0.9% saline followed by 50ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (PBS)
(pH 7.4). The whole brain was excised from the skull and post-fixed in 4% paraformaldehyde in
PBS for 3 h and then incubated in 30% sucrose in 0.1 M PBS overnight at 4°C.
4.3 Tissue Sectioning:
Brains were coronally cut into 30µm sections using a freezing microtome (1320 Leitz) and stored
for later usage in sterile bins containing cold cryoprotectant (0.1 M phosphate buffer, 30%
ethylene glycol, 20% glycerol) in -20ºC.
4.4 Immunohistochemistry (IHC):
Free floating immunohistochemistry was performed to achieve good specificity.
Sections were rinsed in PBS for 10 minutes and then bathed in 600µl block solution (PBS (1X) +
1% bovine albumin + 0.3% Triton) for 45 minutes. Sections were then incubated in primary
antibody including mouse polyclonal anti-DARPP-32 (1:500; BD Biosciences cat:611520,
lot:58902) or rabbit anti-enkephaline (1:500; Neuromics, cat: RA14124-50) overnight at room
temperature.
After three times rinsing with PBS for 10 minutes, sections were incubated in 600µl of 0.3%
H2O2 for 30 minutes. After rinsing 3X10 minutes with 1000 µl PBS, sections were incubated for
two hours in biotinylated anti mouse or anti rabbit (1:1000; Vector laboratories) secondary
antibodies. After rinsing, sections were put in ABC solution (1XPBS concentration for both A
and B 1:1000) for two hours and then washed with PBS for 20 minutes. Color was developed by
keeping sections in DAB solution (1 tablet of DAB (3,3 diaminobenzidine) in 15ml of Trisbuffered saline, PH 7.6, added to 12µl of fresh 30% hydrogen peroxide; 1:1000) for 2-5 minutes
in ventilation bench and then rinsed 2X10 minutes in PBS and finally mounted on slides and
dried over night. The glasses were put in xylene for about 4 hours and then mounted with DPX
and left in a flow bench to be dried. All the processes were performed at room temperature.
4.5 Immunofluorescence (IFC):
To detect colocalization of the proteins of interest, free floating triple staining was performed.
After rinsing with PBS for 10 minutes the brain sections were incubated with a mixture of mouse
polyclonal anti-DARPP-32 (1:500; BD Biosciences cat:611520, lot:58902), rabbit antienkephaline (1:500; Neuromics, cat: RA14124-50) and chicken polyclonal anti-GFP ( 1:1000;
abcam, cat:ab13970-100 , lot: 660556) overnight at room temperature. Next day sections were
rinsed in PBS 6X10 minutes and then incubated in the presence of anti-mouse Cy5 (1:500;
Jackson Immunoresearch) and anti-rabbit Alexa568(1:1000; Invitrogen) and anti-chicken
Alexa488(1:1000; Invitrogen) secondary antibodies for 2h at room temperature. Following three
washes in PBS, the sections were immediately moved to slides and mounted with Flouromount.
Then the slides were kept in 4ºC and protected from direct light.
4.6 Microscopy:
Slides obtained from IHC were observed with Nikon MD105 light microscope. Brain areas were
identified using a mouse brain atlas (George Paxinos and Keith B.J Franklin, 2001, The Mouse
Brain in Sterotaxic Coordinates, 2nd edition, Academic Press 46).
Then with confocal microscope images from IFC were acquired with a Nikon Eclipse E600
confocal microscope, using objective of 100+oil numerical aperture. Images were captured and
auto-averaged to reduce the noise. The images were taken from different locations which had the
best appearance and most cellular density.
5 Results:
5.1 MSNs markers are detectable by DAB Staining:
This step was done to confirm that the expression of markers for different MSN populations in
the striatum is detectable. The first antibody was used against dopamine and cAMP regulated
phosphoprotein of 32 kDa (DARPP-32) (46), which is a regulatory protein enriched in cytoplasm
of all MSNs (47-50) and works as a marker of both D1-R and D2-R expressing neurons, while
the second antibody was against pro-pre enkephaline (ppENK) that is present only in D2-R
expressing neurons (51).
Images collected from the sections after DAB staining are shown in the Figure 1. As seen in
Figure 1, both antibodies work and both markers are expressed in many striatal neurons whereas
they are not expressed in cortical neurons.
5.2 The mGluR5KD-D1 is only expressed in D1-R expressing neurons:
As the main aim of this study was to localize the expression of the transegene to be able to
interpret the behavioural findings in a correct way, we focused on the expression of the construct
in the striatum. Anti DARPP-32 antibody was used for DARPP32, which is abundant in both D1R and D2-R MSNs (48) to mark out all the cell bodies of MSNs. This should label all MSNs, as
shown by Matamales et al (52). In the confocal microscope, we observed DARPP-32-positive
cells in all sections (Fig.2 A; blue cells). These cells consist of two MSNs subpopulations, D1-R
and D-2 expressing neurons that compromise 95% of striatal neurons (31). The next step is to see
whether two subpopulations of MSN can be separately marked in our generated mice.
Figure 1. Expression of the MSNs markers in mGluR5KD-D1 generated mouse in medium
spiny neurons of the striatum. A) DARPP-32, a protein presented in all MSNs is
detected. B) Pre-pro Enkephalin (ppENK), the specific protein existing in only D2espressing medium spiny neurons was characterized. Examples of DARPP-32 and
ppENk are shown (►).
In the transgenic mice, the coding sequence for green fluorescent protein (GFP) was introduced to
facilitate tracking down the interested construct expression. Since GFP sequence was under D1-R
promoters, we anticipated that GFP will be only expressed on D1-R expressed neurons. Further,
we identified GFP–positive cell by immunohistochemistry. Subsequently, we, merged the
confocal images and saw that all GFP cells coexpressed DARPP-32 (Fig.2 B). However, around
half of the DARPP-32-positive cells did not co-express GFP. This shows that in the striatum the
construct is exclusively expressed in MSNs, but only in a sub-population of them(Matamales, M.,
Bertran-Gonzalez, J., Salomon, L., Degos, B., Deniau, J.-M., Valjent, E., Hervé, D., Girault, J.-A.
(2009) Striatal medium-sized spiny neurons: Identification by nuclear staining and study of
neuronal subpopulations in BAC transgenic mice ,PLoS ONE 4 (3), art. no. e4770 ).
To distinguish D1-R from D2-R expressed neurons, we looked for pre-pro Enkephalin (ppENK),
a protein which is only expressed in D2-R expression neurons of striatum (53,58). ppENK
expression (red in Fig.2) was seen in around half of the MSNs. Importantly we saw no
colocalization between ppENK and GFP, showing that the construct is not expressed in D2Rexpressing MSNs. Since all MSNs showed either GFP or ppENK labelling (Fig.2D), we can
conclude that the construct is expressed selectively in D1R-expressing cells.
Figure 2. Immunoflourescent labeling showing that the expression of the transgene is
selective to D1-MSNs. (A) Immunofluorescent labeling for DARPP-32 (blue) identifying
medium spiny neurons (MSNs). (DARPP-32; blue). (B) The expression is restricted to
MSNs and involves around half of them (GFP; green). (C) GFP is not expressed in D2MSNs (ppENK; red). (D) All the MSNs (blue) express either GFP or ppENK. Examples of
GFP-expressing () and non-GFP-expressing (►) MSNs.
6 Discussion:
One of the major obstacles in addiction treatment is the high tendency of relapse, particularly
after exposure to environmental stimuli associated with previous drug-use. This project is a part
of an ongoing study observing the role of mGluR5 receptors on dopamine D1 receptorexpressing neurons in incentive learning underlying cocaine relapse. However, in order to
interpret the behavioral experiments we had to be confident that our mGluR5KD-D1 construct was
properly located and expressed in the desired site. In the present study we show that the
transgene, tracked by immunofluorescence, is indeed expressed in D1R-expressed neurons of
straitum.
It is generally accepted that relapse to drug addiction involves the dopamine system in the
striatum (59), however, the deficiency of dopaminergic therapies on relapse highlights the
probable existence of other neuroteransmitters in the relapse process (61,62). One of the
potentially important neurotransmitters is glutamate (63,64). There are various studies
emphasizing the possible roles of glutamatergic circuit, particularly mGluRs in drug addiction
relapse, and different forms of plasticity in the striatum (65-69). The impairment of cocaine selfadministration seen in in mGluR5 -/- mice and the fact that pharmacological blockade of mGluR5
inhibits aspects of cocaine’s stimulant and rewarding effects suggest that mGluR5 signalling may
be critical for drug-seeking behavior(70-73).
According to our results, all the blue MSNs are expressing either ppEnkephalin or GFP (Fig2. D),
and there is no coexpression of them. This reveales that the expression of the mGluR5KD-D1
construct is limited to D1-R expressing neurons as we desired.
Considering the significances of both dopaminergic and glutamatergic systems, we focused on
the role of mGluR5 in striatum expressed on D1-R subpopulation of MSNs which dissociated
from D2-R expressing neuron subclass. However, there is some evidence indicating that a small
population of MSNs expresses both D1 and D2 types of receptor. This third subpopulation of
MSNs is also expressing ppENK together with both D1 and D2 receptors (74,75). Looking at our
captured images, there was no cell body containing both GFP and ppEnkephalin. This could be
due to that this subpopulation is either very small, that we did not have enough images from
different regions of striatum or that the construct is not expressed in this population. However,
this contrast does not question our results since the majority of MSNs are D1R and D2R neurons
and the impact of the third group may not be relatively significantly effective.
To conclude, our experiment shows that the construct in the mGluR5KD-D1 mice is expressed
accurately. Thus we can draw firm conclusions from the behavioral studies and say that mGluR5
on D1R-expressing cells is important for incentive learning processes underlying relapse. Further
our results show that the mGluR5KD-D1 mouse line is a good tool for further studies of mGluR5
on D1R-expressing cells. (To David: is it good to say that we could compare the contribution to
WT)
7 Acknowledgements:
I would like to express my deepest gratitude to my supervisor David Engblom whose helps,
guidance and encouragement was always motivating and supportive to me. I am also indebted to
my colleagues, Milen Kirlov, Anna..., Nina..., Daniel... and Anna..., for their friendly and helpful
(supports) provided in our lab.
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