Bishop Lab employs a wide range of laboratory techniques and tests. These allow us to reliably paint a picture of the changes occurring in the hemiparkinsonian rat brain which often lead to undesirable side effects such as L-DOPA-induced Dyskinesia (LID) and Parkinson's Disease Associated Psychosis (PDAP).
You may navigate our techniques using the following Table of Contents:
In Vivo Assays
Our lab employs a wide variety of rat behavioral tests in order to monitor the development and severity of Parkinson's Disease (PD), L-DOPA-Induced Dyskinesia (LID), and/or Parkinson's Disease-Associated Psychosis (PDAP). Undergraduate students in Bishop Lab can play a large role in running behavior as it is not uncommon for a study to call for up to 2-5 behavioral assays in a single test day.
Stereotaxic Surgery
A vast majority of our studies begin with a unilateral 6-hydroxydopamine (6-OHDA) lesion to the medial forebrain bundle to simulate a hemiparkinsonian model with a within-subjects control, however we also implement bilateral lesions when necessary. In addition to lesions, we frequently implement cannula for either microinjection or microdialysis studies during this procedure. Certain studies will also call for the infusion of viral vectors to modify the expression of receptors, proteins, or cell signaling molecules.
Chronic L-DOPA Priming & Common Behavioral Assays
As a vast majority of our studies focus on reducing the severity and duration of L-DOPA-induced Dyskinesia (LID), the bread and butter for Bishop lab commonly is the 6-OHDA lesion paired with L-DOPA priming. While the 6-OHDA lesion elicits bradykinesia and a parkinsonian phenotype, chronic L-DOPA priming (typically once daily for 14 days; 6mg/kg) is necessary for the development of Abnormal Involuntary Movements (AIMs) known as dyskinesia.
In order to approximate lesion severity, the Forepaw Adjustment Stepping (FAS) Test (video) is employed following a lesion stabilization period. Rats displaying significant impairments are considered hemiparkinsonian and may then begin L-DOPA priming. On occasion, the Amphetamine-Induced Rotation Test will also be utilized to further approximate lesion severity.
The AIMs Test (video) is employed throughout L-DOPA priming to measure the development and severity of LID. Three subcategories of dyskinetic behavior are rated: axial - contortional twisting of the body, limb - shaking/flailing movements of the lesioned forepaw, and oralingual - side-to-side jaw movements and tongue protrusions.
Transgenic Rat Models
In the Bishop Lab colony room, you will often find 2-3 transgenic rat models employed throughout various studies. Often times we will breed a strain we plan to use across numerous studies.
Cre-lines including D1-Cre, ChAT-Cre, and TPH2-Cre are often employed to allow for cell-specific uptake of infused adeno-associated viral (AAV) vectors via the FLEX approach, allowing for precise targeting of specific sub-populations of neurons.
A c-Fos-driven LacZ model is also employed to allow for the post-mortem identification, quantification of active neurons. This model also allows for behavioral testing following temporary inactivation of active neurons in conscious animals via Daun02 micro-infusions.
Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)
DREADDs are man-made G-protein-coupled receptors that are exclusively activated by synthetic ligands like clozapine-N-oxide (CNO). Following delivery via viral vector during a stereotaxic surgery, DREADDs allow for selective activation, inhibition, or modulation of neuron activity. When paired with a Cre-dependent AAV, specific neuron-types within a selected region can be targeted for study with DREADDs. When embedded in a cellular membrane, DREADDs allow an investigator to uncover the entire range of influence a single neuron-type may exert on a system.
Locomotor Activity Chambers
Locomotor Activity Chambers are surrounded by infrared photocell arrays, which record vertical and horizontal infrared beam breaks. This tool is connected to a computer for analysis of locomotor measures including, but not limited to: Center Time, Stereotypy Count, Rearing Activity, Total Distance, and Movement Number.
Catwalk
The Catwalk can be described as a narrow tunnel of about 1 meter in length. As the rat scurries across the platform, a camera records its movements. The glass floor is illuminated with green light, while the ceiling is illuminated red, allowing for later software to determine the amount of pressure being put on each paw, the time spent on each paw, gait patterns, and many more variables.
Prepulse Inhibition
Prepulse Inhibition (PPI) chambers (video) allow for the measurement of sensory gating behaviors. A weight sensor beneath the rat measures startle amplitude resulting from loud, unexpected sound stimuli. Presentation of a quieter sound (prepulse) preceeding the louder stimulus (pulse) reduces the startle amplitude in healthy animals. PPI chambers allow us to investigate the impacts of Parkinson's Disease, LID, Parkinson's Disease-Associated Psychosis (PDAP), and pharmacotherapies on sensory gating capabilities.
Anxiety Behavioral Tests
Head Twitch Response Test
The Head Twitch Response (HTR) Test (video) is used to measure unconditioned hallucinogenic behavior. The HTR is a paroxysmal rotational shaking of the head. The response is a mixture of head shakes and whole-body shakes.
Exploratory Rearing Test
The Exploratory Rearing Test (right) is also used to measure unconditioned hallucinogenic behavior. This response is characterized by the rat standing on its hind paws to sniff beyond the rat's container and is based on the animal's motivation to explore its surroundings.
Microdialysis
Our lab uses in vivo microdialysis in awake and freely-moving rats to study the involvement of specific regions in dyskinesia. After a surgery implanting a guide cannula, a membranous probe is lowered into the cannula to sample cerebrospinalfluid (CSF) containing neurotransmitters and their metabolites. As artificial CSF (aCSF) is perfused into the probe, positive pressure forces the CSF through through flexible tubing into a collection tube. Following completion of a study, High Performance Liquid Chromatography is used to quantify Norepinepherine, Dopamine, DOPAC, 5-HIAA, and Serotonin.
Wet Lab Assays
Wet lab assays in Bishop lab are more complex and involved than our rat behavior assays, and as such are typically ran by senior undergraduate students, lab technicians, graduate students, and post-docs. These assays are commonly used for lesion verification, cannula placement verification, or quantification and/or localization of proteins, nucleic acids, neurotransmitters, and active cells.
High Performance Liquid Chromatography
Bishop lab routinely performs High Performance Liquid Chromatography on both tissue (HPLC - Early Detection) and dialysate (HPLC - Reverse Phase) samples. Our tried and true methodology and reputation for precision has gained industry attention, so we also analyze samples for other institutions from time to time.
We primarily use HPLC for lesion verification, as all 6-OHDA lesioned rats will show a reduction in Dopamine within the lesioned striatum by 95 to 99.9%. Dialysate is analyzed to determine region-specific neurochemistry, and subsequent alterations as a result of disease and treatment.
Immunohistochemistry
Immunohistochemistry (IHC) allows for the quantification and localization of proteins within intact cells. Tissue slices are incubated with primary antibodies that target and bind the protein of interest. Numerous biotinylated-secondary antibodies then target each primary antibody in order to amplify the signal. Next, Horseradish peroxidase-avidin complexes bind to the biotinylated-secondary antibodies. Lastly, reporters react with secondary antibodies to indicate the presence of the protein of interest in a tissue.
Similarly to HPLC, we often use Tyrosine Hydroxylase (TH) immunoreactivity to measure lesion efficacy in perfused hemiparkinsonian rats. The intact striatum and substantia nigra will stain robustly for TH whereas the lesioned hemisphere will show a 95-99.9% reduction in dopaminergic tone.
Like IHC, X-gal is a histochemical stain used on LacZ animal lines that allows for the identification of active neurons in tissue. This stain does not require antibodies, however, because active LacZ enzymes in cells active at the time of sacrifice will cleave the X-gal staining molecule.
RNAscope
RNAscope (link) is one of the newer techniques to the Bishop lab arsenal of techniques. RNAscope is a groundbreaking successor to in situ hybridization assays, allowing for quantification and localization of target RNA within intact cells. RNAscope requires the binding of 2 independent, Z-shaped probes to the target RNA before any amplification may occur, resulting in excellent target-specificity. In short, this technique allows for highly-specific in situ hybridization to be performed with ease using a methodology more closely related to immunohistochemistry.
The images to the left depict rat striatum imaged at 40x.
Top left: DRD1 in green, DRD2 in red, DAPI in blue.
Top right: DRD1 in green, DRD2 in red, DRD3 in red, DAPI in white.
When RNAscope image stacks are analyzed using supercomputing software, both stained cells and mRNA transcripts may be visualized in three-dimensions. This allows for highly precise transcript counting, localization, and analysis.
Brightfield & Fluorescent Microscopy
Bishop lab employs both Brightfield and Fluorescent microscopy up to 40x magnification to allow for identification of active cells, receptors, and other proteins and signaling molecules. Microscopy is frequently used to view cells, receptors, and proteins in their undisturbed natural setting.
Brightfield is commonly used for cannula, and lesion verification, as well as cell-counts, while Fluorescent microscopy is more valuable for its ability to co-stain numerous proteins, particularly those engaged in heteromer complexes. Common Brightfield stains employed in our lab include Cresyl Violet, Tyrosine Hydroxylase immunohistochemistry, and X-Gal.
Western Blots
Western blotting provides a useful tool for quantifying protein concentration in a tissue sample. Sodium dodecyl sulfate (SDS) is mixed with the sample to normalize mass-to-charge ratios across proteins within the sample, allowing for separation based on mass alone. When a molecular ruler is ran concurrently, proteins of interest may be identified within samples based on their molecular weight. After transferring proteins to a blotting membrane, protein quantities are then measured using antibodies that target the protein of interest. These antibodies are conjugated to a chemiluminescent reporter that is later activated by reporter enzymes like horseradish peroxidase (HRP) for fluorescent detection, allowing for protein quanitication.
Bishop lab has used western blots to quantify monoamine transporters including DAT, SERT, and NET, as well as cell signalling molecules like phospho-extracellular-signal-regulated kinase.
Co-Immunoprecipitation
Co-Immunoprecipitation (Co-IP) is a complex and effective technique involving the isolation of two (or more) proteins for analysis of protein-protein interactions. In Co-IP, primary antibodies bind to the protein of interest before secondary antibodies are introduced to bind to magnetic beads. Magnets are then used to pull the magnetic beads, and thus the protein complex of interest, out of solution. Proteins are then eluted for later SDS-PAGE or Western Blot analysis.
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) allows for the detection and quantification of even the scarcest mRNA or DNA transcripts within a sample by exponentially amplifying specific sequences. Transcripts are normalized to housekeeper genes like β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) that are constitutively expressed in stable quantities.
In Bishop Lab, PCR is has been used to quanitfy a variety of DNA and RNA sequences ranging from immediate-early genes (IEGs) including preprodynorphin (PPD), preproenkephalin (PPE), and c-Fos; monoamine receptors including the D1, D2, and D3 dopamine receptors, as well as the 5-HT-1a Serotonin receptor; interleukins including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, and IL-12p70; and chemokines and cytokines including TNF-α, Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF), Interferon-γ (IFN-γ), and Monocyte Chemoattractant Protein-1 (MCP-1). PCR is also a useful tool for our verifying genotype in the transgenic lines that are bred in our colony room.