SYNAPTIC ALTERATIONS IN A CELLULAR MODEL OF HUNTINGTON’S DISEASE 

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CHAPTER TWO:    METHODS

Neuronal cultures

Dissociated Hippocampal Cultures

Postnatal day zero (P0) Wistar rat pups of both sexes were used to prepare dissociated hippocampal primary neuronal cultures. The use of animals for this study was approved by the University of Auckland Ethics Committee. Culture preparations were performed under sterile conditions, using a Class II Biological Safety Cabinet (Heraeus). Dissection tools were sterilised by autoclaving prior to use. Depending on whether the neuronal cultures were intended for immunocytochemistry or electrophysiology studies, 22 millimeter square coverslips (Menzel-Glaser) or 13 millimeter round coverslips (Hurst Scientific) were used respectively during cell plating. All coverslips were prepared beforehand by overnight sterilisation in 69% nitric acid (Merck, #1017992500) followed by thorough individual washing in distilled water and storage in 100% ethanol (Merck, #1.00983). On the day preceding culture preparation, coverslips were flamed (to remove the ethanol and ensure sterility) and placed in 6-well culture plates (BD Bioscience, FAL353046). Then, 1.5 mL of 10 μg/mL poly-D-lysine (PDL; Sigma, #P1149) dissolved in sterile phosphate buffered saline (PBS: 136.89 mM NaCl [Sigma, #S7653], 2.68 mM KCl [Sigma, #60128], 10.15 mM NaH2PO4 [Fluka Chemika, #71504], 1.76 mM KH2PO4 [Sigma, #P5379], pH=7.4) was added to each well of the 6-well plate and incubated overnight at 37°C. The purpose of this step was to coat the coverslips with poly-D-lysine in order to facilitate the attachment and growth of neuronal cells.
On the day of culture preparation, P0 Wistar pups were collected from the Vernon Jansen Unit, the University of Auckland animal breeding facility, and culled by decapitation in a Class I dissection hood. The skin and skull were cut medially with a scalpel blade, then the skull was opened to expose the brain which was then removed using a spatula and placed in a plastic 35 millimeter Petri dish (Falcon, #353001) containing pre-chilled Hank’s Balanced Salt Solution (HBSS; 9.5 g HBSS [Sigma, #H2387], 2.38 g 4-[2-hydroxyethyl]-1-piperazineethanesulfonic acid [HEPES; Sigma, #H3375], pH=7.2 with 5 M NaOH [Sigma-Aldrich, #S5881] in 1 L of MilliQ water). Subsequently, the dish containing the brain was placed under a dissection microscope (Leica), and using two pairs of small curved forceps, the brain was first anchored at the level of cerebellum and the two hemispheres were teased apart to expose the hippocampi. The hippocampi were then gently removed by cutting the tissue around them and were transferred using a thin paintbrush into a new dish containing cold HBSS. The dish with hippocampi was then transferred to a Class II biological hood and the tissue was placed in a pre-warmed HBSS solution containing papain (≥20 U/mL; Worthington Biochem, #LK003178) and incubated for 15 minutes at 37°C.
During the enzymatic digestion in papain, the tube was gently inverted twice (at 5 min intervals) and then the enzymatic reaction was halted by exchanging the papain solution with pre-warmed high-glucose Minimum Essential Medium (MEM; Gibco, #A14518-01) containing 10% Fetal Bovine Serum (FBS; Moregate Biotech) and incubated for 2 min at 37°C. Subsequently, the MEM+FBS solution was removed and 1 mL of the culturing medium (NBM+B27; Neuro Basal Medium [NBM; Gibco, #21103-049] with B27 supplement [Gibco, #17504-044] and 1x GlutaMAX [Invitrogen #35050-061]) was added. The neurons were dissociated by gentle trituration of the digested hippocampal tissue using a fire-polished glass Pasteur pipette (Thermo Fisher, #KIM63B93), approximately 15-20 times. The undissociated pieces of tissue were allowed to settle at the bottom of a tube and the solution containing dissociated cells was transferred into a tube containing pre-warmed NBM+B27 culturing medium. The 6-well plate was then washed twice with 2 mL of sterile PBS to remove the excess PDL, and the neurons were plated at the density of ~105 cells/mL (2 mL/well). Primary neuronal cultures were incubated at 37°C and 5% CO2. At day in vitro 1 (DIV1), 50% of the medium was removed and replaced with fresh one. The cultures were maintained up to DIV14.

Dissociated cortico-striatal co-cultures

For the preparation of cortico-striatal co-cultures, P0 Wistar rat pups of both sexes were used. Equipment used for the preparation of these cultures was the same as for the hippocampal primary cultures, i.e. made under sterile conditions, using a Class I/Class II Biological Safety Cabinet and using autoclaved dissection tools. As these cultures were intended for immunocytochemistry experiments only, nitric acid-treated, PDL-coated 22 millimeter square coverslips (Menzel-Glaser) were used for cell plating.
On the day of culture preparation, the brain was removed from the skull, using the same method as described in Chapter 2.1.1, and placed in a plastic 35 millimeter Petri dish containing pre-chilled HBSS. To remove the striatum, the brain was first anchored using small curved forceps at the cerebellum and the olfactory bulbs were dissected away with a scalpel blade. Next, the rostral part of the cerebrum was removed by performing a coronal cut with the scalpel blade at approximately 1/3 of the brain’s length. The rostral fragment of the brain, containing the striatal and cortical tissue was transferred to a new dish with cold HBSS and dissected further, using two pairs of small forceps, in order to separate the striatum from the surrounding cortex. The striatal and cortical regions were transferred to the Class II biological hood and dissociated separately with papain, as described in Chapter 2.1.1. After the dissociation, both types of neurons were suspended in DMEM+ (DMEM [Gibco, #11965-092] + 10% FBS [Gibco, #16000-036]) and cell density was determined using hemocytometer.
In contrast to the hippocampal neurons, striatal neurons were transfected via nucleofection prior to plating (Kaufman et al., 2012). Nucleofection is an electroporation-based transfection method where voltage is used to deliver DNA into the nucleus of suspended cells. In order to nucleofect the striatal neurons, the dissociated cells were first centrifuged at 1,200 rpm for 3 min, to settle the cells at the bottom of the tube, the DMEM+ supernatant was removed and the cells were resuspended in 100 µL of pre-warmed electroporation buffer (Mirus Bio, #MIR50115) containing ~15 µg of the huntingtin DNA construct (described in Chapter 2.2). The suspension was then transferred to a nucleofection cuvette (Mirus Bio), keeping the cell number per cuvette no more than 2×106. The cuvette was inserted into the Nucleofector II device (Amaxa) and the neurons were electroporated using the programme #O-003 (intended for rat neurons). Immediately after the nucleofection, 500 µL of pre-warmed DMEM+ was added to the cuvette and the solution was transferred to a new tube.
Nucleofected striatal cells were mixed at a 1:1 ratio with previously dissociated cortical neurons, diluted to reach the final density of approximately 1-2×106 with DMEM+, and plated on a 6-well plate (2mL/well). After 2-4 hours, after the cells have attached to the coverslip surface, DMEM+ was removed and replaced with NBM+B27 culturing medium (see Chapter 2.1.1). Half of the medium was exchanged with fresh medium every 3-5 days until the neurons were immunolabelled on DIV13.

Expression vectors

In order to study the synaptic changes in HD, a cellular model of the disease was established where the dissociated neurons were transfected with pBWN-97Q Htt plasmid containing the 1st exon of the HTT gene followed by 97 CAA/CAG alternating repeats and a reporter enhanced green fluorescent protein (eGFP) gene at the 3’ end (Figure 2.1). The alternating CAA/CAG sequence ensures greater stability in transformed bacterial cells compared to CAG repeats alone (Kazantsev et al. 1999), thus avoiding the expansion or shortening of the repeat tract. The same plasmid type carrying 25 CAA/CAG repeats attached to the eGFP tag (pBWN-25Q Htt) as well as the plasmid with eGFP tag alone (pBWN-eGFP) served as controls. All three plasmids contain the CMV-β-actin promoter which directs the expression of the nuclear hormone receptor for ecdysone (EcR), a steroidal insect moulting hormone. The non-steroidal version of ecdysone, tebufenozide (TFZ; used at the concentration of 0.5 µM), was used to activate the heterodimeric receptor complex composed of EcR and endogenous retinoid X receptors (Suhr et al., 1998). This receptor complex binds to the ecdysone receptor-responsive promoter that controls the expression of the huntingtin/eGFP genes (Figure 2.2). pBWN plasmids also contain the ampicillin resistance gene (AmpR) used for bacterial selection. These constructs were originally developed by Dr Erik Schweitzer, UCLA (Aiken et al., 2004).
In order to study the localisation of surface NMDA and AMPA receptors, GluN1 and GluA1 subunits were overexpressed using plasmids where their genes are attached to the N-terminal Flag epitope (DYKDDDDK) (Cheyne & Montgomery, 2008; Lissin et al., 1998). These plasmids came from the laboratory of Professor Craig Garner, Charité University (Li et al., 2011; Waites et al., 2009). Other DNA constructs used for the transfection of primary neuronal cultures were subcloned for the purpose of this thesis and are described in Chapter 2.2.1. All the plasmids used in this thesis are listed in Table 2.1.

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Subcloning

Primer design and gene amplification

For the experiments requiring simultaneous co-expression of huntingtin DNA constructs with SAP97 isoforms, DNA sequences coding for αSAP97 and βSAP97 had to be subcloned from the original eGFP gene-containing vectors (pFU-rSAP97I3-EG) into a vector with a different reporter gene type. For this purpose, the pmCherry-N2 vector was chosen, carrying the red fluorescent protein mCherry gene (Figure 2.3). The polymerase Chain Reaction (PCR) was used to amplify the αSAP97 and βSAP97-coding DNA fragments. Phusion High-Fidelity DNA Polymerase (Thermo Fisher, #F530S) was used in PCR reactions. All the primers used for gene amplification and sequencing were designed using the Primer3 online tool (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). The primers used are listed in Table 2.2 and the summary of subcloning procedure is presented in Figure 2.4.

Insert DNA preparation

Primers used for amplification contained EcoRI restriction sites in order to facilitate their ligation with the target vector backbone. The final volume of the PCR reaction was 50 µL and the reaction mix contained: H2O (27.5 µL), 5X Phusion HF Buffer (10 µL), 10 mM dNTPs (1 µL), 5 µM forward primer (5 µL), 5 µM reverse primer (5 µL), 1 U Phusion DNA Polymerase (0.5 µL). The PCR conditions used are displayed in Table 2.3.
After the reaction, 5 µL of the product was run on a 0.5% agarose gel (Invitrogen, #16500) to verify the size of amplified DNA fragments. Then, 18 µL of the product was mixed with 2µL of 10XEcoRI-HF RE-Mix restriction enzyme (New England Biolabs, #R3101) and incubated in a heat-block at 37°C for 30 min. After the restriction digestion, the entire solution was loaded on an agarose gel in order to separate the digested PCR products from other components of the original PCR reaction. The bands corresponding to either of the SAP97 isoforms (Figure 2.5) were excised from the gel with a razor blade and their DNA was purified with GeneJET Gel Extraction Kit (ThermoFisher, #K0691). First, the DNA containing gel fragments were placed in a 1.5 mL tube and 1:1 volume of the Binding Buffer was added. The gel mixture was then incubated at 60°C for 10 min to melt the agarose and vortexed. Melted gel mixture
was transferred into the GeneJET purification column and centrifuged for 1 min at 14,000 rpm. The flow-through was discarded and 100 µL of Binding Buffer was added to the column and centrifuged again for 1 min at 14,000 rpm. The DNA bound to the column was washed by adding 700 µL of the Wash Buffer and the column was centrifuged for 1 min, 14,000 rpm. After the washing step, the column was centrifuged for another minute (14,000 rpm) to ensure that all the liquids were removed from the column. Then, the column was placed in a new 1.5 mL tube and the DNA was eluted with 20 µL endotoxin-free H2O and centrifuged for 1 min at 14,000 rpm. The concentration of the prepared insert DNAs was determined using NanoDrop ND-1000 spectrophotometer.

Vector DNA preparation

In parallel to the insert DNA preparation, the target pmCherry-N2 vector was digested using the EcoRI restriction enzyme: 2 µg of maxiprepped pmCherry vector was diluted to 18 µL with H2O and mixed with 2 µL of 10X EcoRI-HF RE-Mix, then incubated for 1 hour at 37°C. For the last 15 min of the digestion reaction, 1µL of Heat Inactivated Alkaline Phosphatase (Invitrogen, #A14322) was added in order to hydrolyse the phosphate groups at the vector ends and prevent self-ligation. After the incubation, the alkaline phosphatase was inactivated at 65°C for 5 min. The digested and phosphorylated vector was purified using a GeneJET PCR Purification Kit. The mixture containing the digested vector was mixed at a 1:1 volume ratio with the Binding Buffer, vortexed and transferred onto a GeneJET column. The column was centrifuged for 1 min at 14,000 rpm, the flow-through was discarded and 700 µL of the Wash Buffer was added. After 1 min of centrifugation at 14,000 rpm the flow-through was discarded and centrifugation was repeated to dry the column. Vector DNA was then eluted with 20 µL of endotoxin-free H2O by centrifugation for 1 min at 14,000 rpm. The size of the resulting linear vector was verified with gel electrophoresis (Figure 2.6) and its concentration was determined using NanoDrop ND-1000 spectrophotometer.

Ligation and colony PCR

After the SAP97 DNA inserts and the vector had been prepared, the ligation reaction was performed. Vector DNA was mixed with each of the insert DNAs at 3:1 molar ratio. 30 fmol of the vector DNA was added to 90 fmol of each of the inserts, then 4 µL of 5X Ligase Reaction buffer was added together with 1 µL (1 U) of the T4 DNA Ligase (Invitrogen, #15224-017). The reaction mixture was increased to 20 µL final volume, vortexed gently and incubated at room temperature for 45 min. The ligation reaction was performed for 45 min and then the mixture was used to transform the competent bacteria (Chapter 2.2.2). After transformation, the bacteria were plated onto 10 cm Petri dishes containing LB Agar (Invitrogen, #22700-025) and 50 μg/mL kanamycin (Roche, #10106801001) for clone selection. Agar plates were incubated at 37°C overnight and selected colonies were tested for the DNA construct with with inserts in correct orientation by colony PCR.
The colony PCR final volume was 15 µL, containing H2O (10.4 µL), 5X Phusion HF Buffer (4 µL), 10 mM dNTPs (0.4 µL), 5 µM forward primer (2 µL), 5 µM reverse primer (2 µL), Phusion DNA Polymerase 0.4 U (0.5 µL). SAP97seq_FC and mCh_Fout primers were used for this reaction. The reaction mixture was distributed into 0.2 mL PCR tubes, and a sample of the bacterial colony was transferred into the tube using a pipette tip and mixed. During the initial denaturation step of the PCR programme, bacterial cells are disrupted to release their contents, including plasmid DNA, and amplification reaction begins. The PCR programme was the same as shown in Table 2.3, with the annealing temperature of 58°C. Using the SAP97seq_FC and mCh_Fout primers, only fragments based on the plasmids containing inserts in the correct orientation could be amplified. Clones carrying the correct DNA constructs were then used to establish a 5 mL culture for the purpose of DNA minipreparation (Chapter 2.2.3). Prepared DNA, together with the sequencing primers (Table 2.2), was then sent to the University of Auckland DNA Sequencing Centre for Sanger sequencing in order to confirm that they carry desired insert DNAs that are in-frame with reporter gene (mCherry) and free of point mutations. The maps of resulting SAP97 isoform plasmids are presented in Figure 2.7.

Transformation

Two types of competent E. coli strains were used for transformation. DH5α E. coli (Invitrogen, #18265-017) was a general purpose strain used to amplify most of the plasmids. Due to low-efficiency of transformation of αSAP97-mCherry and βSAP97-mCherry plasmids with DH5α competent cells, XL10-Gold Ultracompetent cells (Agilent, #200315) were used for these two constructs. In order to transform the bacteria, an aliquot of 50 µL (DH5α) or 15 µL (XL10-Gold) was thawed on ice and 0.5 µg of plasmid DNA was added to it. The bacteria and DNA were mixed gently by tapping on the tube and then the tube was incubated on ice for 30 min. The transformation was induced using heat-shock at 42°C for 20 (DH5α) or 30 (XL10-Gold) seconds. The tubes were returned to ice for a brief (1 min) incubation and µL of pre-warmed Super Optimal Broth (DH5α) (SOC; Invitrogen, #15544-034) or LB Broth (XL10-Gold) (Invitrogen, #A1374301) was added. Bacteria were then allowed to proliferate for 1 hour at 37°C and 230 rpm agitation. After the incubation, 100 µL of each of the transformed E. coli strains was streaked at two different concentrations (undiluted and diluted at a 1:4 ratio in corresponding bacterial medium) on agar plates containing antibiotics (ampicillin [Invitrogen, #11593-027], 100 µg/mL, or kanamycin [Roche, #10106801001], 50 µg/mL). The plates were kept overnight at 37°C.

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Plasmid DNA Minipreparation

A Plasmid Miniprep kit (Invitrogen, #K2100-10) was used to isolate plasmid DNA from a small E. coli culture in order to confirm the identity of the DNA before plasmid maxipreparation. 5 mL of bacterial culture was incubated overnight in LB Broth (Invitrogen, #12780-052) containing antibiotics. 4 mL was taken and centrifuged at 14,500 rpm for 5 min to pellet the cells. The supernatant was discarded and the pellet was resuspended in 150 µL of RNAse A-containing resuspension buffer. Then, 250 µL of lysis buffer was added and the tube was inverted up to 10 times and incubated at room temperature for 2 min. The cell lysis reaction was terminated by the addition of 350 µL of neutralisation buffer. The tube was inverted again up to 10 times and then centrifuged for 3 min at 14,500 rpm to spin down the cell debris. Plasmid DNA-containing supernatant was then collected and transferred onto a NucleoSpin Plasmid Easy Pure Column. The column was centrifuged for 30 seconds at low speed (2,000-3,000 rpm) for plasmid DNA binding to the column. The flow-through was discarded and 450 µL of wash buffer was added, in order to purify the plasmid DNA, and the tube was centrifuged for 1 min at 14,500 rpm. The flow-through was discarded again and the tube centrifuged to dry the column. Finally, 25 µL of molecular biology-grade endotoxin-free water was added to the column to elute the DNA, which was collected in a new 1.5 mL tube during 1-minute centrifugation at 14,500 rpm. The concentration of the DNA was measured using a NanoDrop ND-1000 spectrophotometer.
DNA isolated during minipreparation was used to confirm the identity of the construct either via restriction digestion, followed by agarose gel electrophoresis, or via Sanger sequencing performed at the University of Auckland DNA Sequencing Centre.
Gel electrophoresis
After the PCR or restriction digestion, DNA fragments were separated using gel electrophoresis in order to confirm their identity based on their size. Agarose gels (0.5-1%) were prepared by dissolving UltraPure Agarose (Invitrogen, #16500-100) in SYBR Safe DNA gel stain in 0.5x TBE buffer (Invitrogen, #S33101) in a microwave oven. The agarose solution was then poured onto the gel tray located in the gel electrophoresis tank with a comb insert. When the gel was set, the comb was removed and the gel immersed in 0.5x TBE buffer (40 mM Tris-acetate, 20 mM EDTA, pH=8.0). To determine the size of the DNA products, 5 µL of 1 kb Plus DNA Ladder (Invitrogen, #10787-018) mixed with 10x BlueJuice loading buffer (Invitrogen, #10816-015) was loaded into one of the gel wells. 10x BlueJuice loading buffer was added to the samples of post-PCR mixtures, stirred by pipetting up and down, and carefully applied to separate gel lanes. In the case of DNA digested with 10x EcoRI-HF RE-Mix, the loading buffer was already present in the enzyme mixture and thus was applied directly onto a gel. Electrophoresis was performed at 100 mV, using an Electrophoresis Power Supply (Thermo, #EC250-90), for at least 30 min or until the loading dye had approached the end of the gel. The gel was imaged under ultraviolet light using a Gel Doc XR+ imaging system (BioRad) to visualise the DNA bands.

Plasmid DNA Maxipreparation

DNA Maxiprep kits (Qiagen, #12362) were used to prepare large quantities of high-quality endotoxin-free plasmid DNA that was later used for the transfection of neuronal cultures. An Erlenmeyer flask with 150 mL of LB medium was inoculated with leftover mini-culture using a sterile plastic loop. In the case of αSAP97-mCherry and βSAP97-mCherry, Terrific Broth (ThermoFisher, #A1374301) was used instead of LB for higher plasmid yield. Antibiotics (100 µg/mL ampicillin or 50 µg/mL kanamycin) were added to the medium and the culture was incubated overnight at 37°C and 230 rpm agitation. After the incubation, the bacterial culture was decanted into 50 mL Falcon tubes and centrifuged at 6,000 x g for 15 min at 4°C to pellet the cells. The supernatant was discarded and the bacterial cells were resuspended in 10 mL of resuspension buffer. Then, 10 mL of lysis buffer was added and the tube was inverted 5-7 times and left for 5 min at room temperature to allow the lysis of bacteria. The reaction was neutralized by the addition of 10 mL of neutralisation buffer and the tube was mixed by inverting 5-7 times. The solution was subsequently poured into the QIAfilter cartridge, incubated for 10 min at room temperature and then pushed with a plunger in order to filter the liquid fraction through a layer of filtering resin, separating it from the bacterial debris. 2.5 mL of endotoxin removal buffer was added and the solution was incubated on ice for 30 min. During that incubation period, the QIAGEN-tip 500 was equilibrated with 10 mL of equilibration/QBT buffer. After the incubation had finished, the filtered solution was transferred into the QIAGEN-tip 500 and the DNA was allowed to bind to the column while the solution was passing through by gravity flow. Then, the column was washed twice with 30 mL of QC buffer and the DNA was eluted with 15 mL of elution/QN buffer into a new 50 mL Falcon tube.
Eluted DNA was then purified further by precipitation. First, 10.5 mL of isopropanol was added and the tube was centrifuged at 15,000 x g for 30 min at 4°C. The supernatant was removed and DNA pellet was rinsed with 5 mL of 70% ethanol and the tube was centrifuged again for 30 min at 4°C, 15,000 x g. The supernatant was discarded, the DNA pellet was allowed to air-dry for approximately 10 min and then the DNA was resuspended in 50-200 µL (depending on the pellet size) of endotoxin-free molecular biology-grade H2O. The DNA concentration, measured using a NanoDrop ND-1000 spectrophotometer, was determined by averaging the results of 2-3 measurements and DNA stock solutions were stored at – 20°C.

Glycerol stocks

Glycerol stocks were prepared for bacteria carrying each of the plasmid used. To do that, 1 mL of the maxiprep culture was transferred into a cryotube (Raylab, #GR122278) containing 1 mL of 100% glycerol (Sigma, #G5516) and mixed by pipetting up and down. Tubes were then stored at -80°C. Glycerol stocks were used to inoculate antibiotic containing bacterial medium for each maxipreparation.

TABLE OF CONTENTS
ABSTRACT 
ACKNOWLEDGEMENTS 
LIST OF FIGURES 
LIST OF TABLES 
LIST OF ABBREVIATIONS 
CHAPTER ONE: GENERAL INTRODUCTION 
1.1. HUNTINGTON’S DISEASE – HISTORICAL OUTLINE AND GENETICS
1.2. HD NEUROPATHOLOGY AND ASSOCIATED SYMPTOMS
1.3. THE STRUCTURE AND ROLE OF HUNTINGTIN
1.4. CHEMICAL SYNAPSES IN THE CNS
1.5. GLUTAMATE EXCITOTOXICITY
1.6. SAP97 AS A SYNAPSE REGULATOR
1.7. THESIS AIMS
CHAPTER TWO: METHODS 
2.1. NEURONAL CULTURES
2.2. EXPRESSION VECTORS
2.3. HIPPOCAMPAL CULTURE TRANSFECTION
2.4. IMMUNOCYTOCHEMISTRY
2.5. EPIFLUORESCENT IMAGING
2.6. SUPER-RESOLUTION IMAGING
2.7. WHOLE-CELL PATCH CLAMP RECORDINGS
2.8. WESTERN BLOTTING
CHAPTER THREE: SYNAPTIC ALTERATIONS IN A CELLULAR MODEL OF HUNTINGTON’S DISEASE 
3.1. INTRODUCTION
3.2. AIMS
3.3. RESULTS
3.4. DISCUSSION
3.5. CONCLUSIONS
CHAPTER FOUR: THE ROLE OF SAP97 IN HD PATHOGENESIS 
4.1. INTRODUCTION
4.2. AIMS
4.3. RESULTS
4.4. DISCUSSION
4.5. CONCLUSION
CHAPTER FIVE: SUPER-RESOLUTION IMAGING OF GLUTAMATE RECEPTORS IN A CELLULAR HD MODEL 
5.1. INTRODUCTION
5.2. AIMS
5.3. RESULTS
5.4. DISCUSSION
5.5. CONCLUSION
CHAPTER SIX: ELECTROPHYSIOLOGICAL VALIDATION OF FUNCTIONAL GLUTAMATE RECEPTOR CHANGES IN A CELLULAR MODEL OF HD 
6.1. INTRODUCTION
6.2. AIMS
6.3. RESULTS
6.4. DISCUSSION
6.5. CONCLUSION
CHAPTER SEVEN: GENERAL DISCUSSION 
7.1. GLUTAMATE RECEPTORS IN HD – WHAT HAVE WE LEARNT FROM THE LABORATORY MODELS?
7.2. SAP97 AND RECEPTOR LOCALISATION IN HD
7.3. OUTLOOK: FUTURE DIRECTIONS FOR HD RESEARCH
7.4. SUMMARY
REFERENCES
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Regulation of NMDA receptor surface distribution in a neuronal model of Huntington’s disease

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