Novel Tech Flashcards
- Define a viral vector
What are the advantages of AAV vectors?
How does a viral vector enters a cell?
Explain the function of different vectors
A viral vector is a tool commonly used in biological research and therapy, such as gene therapy, to deliver genetic material into cells. This process of transferring foreign DNA into host cells is called transduction. Viruses are an effective delivery system because they have evolved to specialize in infiltrating cells and incorporating their genetic material into the host’s genome. Scientists exploit this trait by removing the disease-causing aspects of a virus and inserting the desired genetic material, creating a viral vector.
- What are the advantages of AAV vectors?
Small, non-pathogenic, ssDNA packaging viruses
* Episomal (<90%) and integrated (>10%)
* Low immunogenicity, low inflammation profile
* Can infect a wide range of vertebrate hosts, including humans
* Require co-infection with adeno- or herpesviruses as helpers for replication lowers risk for random infection
* 150 naturally occurring AAV variants have been identified
* Is the main choice of vector used for basic neuroscience
* Also popular for clinical applications
They’re good for chronic conditions and work in non-dividing cells
They don’t typically integrate into the host genome –
AAV vectors have several advantages that make them attractive for gene therapy:
Safety: AAVs are considered non-pathogenic, meaning they do not cause disease. They also don’t trigger an inflammatory response as aggressively as some other viral vectors.
Stable Expression: AAVs can lead to long-term expression of the target gene, especially in non-dividing cells, making them suitable for chronic conditions.
Broad Tissue Tropism: Different serotypes of AAV can target different cell types (tissue tropism), providing a broad range of potential applications.
No Integration into Host Genome: AAV vectors primarily exist as episomes (extrachromosomal genetic material) in the host cell and do not frequently integrate into the host genome, reducing the risk of insertional mutagenesis.
How does a viral vector enters a cell?
It binds to receptors on the surface leading to endocytosis
Viral vectors enter a cell by first binding to specific receptor proteins on the cell surface. This binding triggers the cell to endocytose the virus, essentially engulfing and bringing the virus into the cell within a vesicle. The virus then escapes from the vesicle into the cytoplasm, often by causing the vesicle to break down or by piercing its membrane.
Once in the cytoplasm, viruses like adenoviruses and AAVs are transported to the nucleus, where the viral genome is released. If the vector is a retrovirus, it first reverse-transcribes its RNA genome into DNA in the cytoplasm, then this DNA is transported into the nucleus. Finally, the viral vector’s DNA is expressed by the cell’s machinery, producing the desired protein.
- Explain the function of different vectors
Different viral vectors have different characteristics that make them more suited to some applications than others:
Adenoviruses: Adenovirus vectors can infect both dividing and non-dividing cells, and they have a large cloning capacity. However, they induce a strong immune response and don’t integrate into the host genome, which can limit the duration of gene expression.
Retroviruses and Lentiviruses: These vectors integrate their genome into the host’s DNA, leading to long-term gene expression. Retroviruses can only infect dividing cells, but lentiviruses can infect both dividing and non-dividing cells.
Adeno-associated viruses (AAVs): As mentioned above, AAVs are non-pathogenic and cause a less intense immune response. They can infect both dividing and non-dividing cells and can lead to long-term gene expression in non-dividing cells.
Herpes Simplex Viruses (HSVs): HSV vectors have a large capacity for foreign DNA and are especially good at infecting neurons, making them useful for neurological applications.
In summary, the choice of viral vector depends on the requirements of the specific application, such as the type of cell to be targeted, whether long-term expression is needed, the size of the gene to be delivered, and the acceptable level of immune response.
- Define viral tropism
* How is cell-specificity obtained when using viral vectors?
* What is the function of the viral capsid?
* Explain retrograde and anterograde transport
- How is cell-specificity obtained when using viral vectors?
Cell specificity when using viral vectors is often obtained by leveraging the natural biology of the virus from which the vector is derived. Many viruses naturally infect certain types of cells (referred to as “tropism”), so researchers can select or engineer a viral vector based on its tropism.
Cell specificity can also be enhanced or altered by modifying the vector to include certain promoters or specific surface proteins (on the capsid) that interact with receptors present only on the target cell type. Additionally, cell specificity can be achieved by physically delivering the vectors directly to the target tissues.
- What is the function of the viral capsid?
The viral capsid is the protein shell that surrounds the genetic material of a virus. It serves several functions:
Protection: The capsid shields the viral genome from the environment, helping to ensure the genome’s stability until it can enter a host cell.
Delivery: The capsid facilitates the delivery of the viral genome into host cells. This involves recognizing and binding to host cell receptors, aiding in the virus’s entry into the cell, and releasing the viral genome within the cell.
Immunogenicity: The capsid is the primary viral structure recognized by the host’s immune system. Its shape and structure can stimulate an immune response, which can be beneficial for vaccine development but a challenge for gene therapy.
Designing capside for systemic administration in specific mouse strains only guarantees good transfection in said strain but not in other strains or species
- Explain retrograde and anterograde transport
Retrograde and anterograde transport refer to the movement of substances, such as viral vectors, within cells. The movement is facilitated by the cell’s cytoskeleton and associated motor proteins. Here’s a more detailed explanation of both:
Retrograde Transport: This refers to the movement of substances from the periphery of the cell towards the cell body or nucleus. In the context of viral vectors, this process is particularly important. Many viruses, including those used as vectors in gene therapy, take advantage of this cellular transport mechanism to reach the nucleus where they can integrate their genetic material into the host’s genome. The viruses bind to specific receptors on the cell surface and are endocytosed, or taken up into the cell, in vesicles. These vesicles then move along microtubules, a component of the cell’s cytoskeleton, towards the nucleus. This movement is facilitated by dynein, a motor protein that moves towards the minus-end of microtubules, which is generally oriented towards the cell center.
Anterograde Transport: This is the opposite of retrograde transport, meaning it refers to the movement of substances from the cell body towards the periphery. In the context of viral vectors, this could refer to the movement of newly synthesized viral particles out of the cell, although this is often accomplished by a different process called exocytosis. Anterograde transport also occurs along microtubules, but is facilitated by a different motor protein called kinesin, which moves towards the plus-end of microtubules, oriented towards the cell periphery. Anterograde Transport with AAVs also enables transsynaptic transport of the virus.
Examples would be the AAV1 and AAV9 serotypes
Viral vectors are a key tool in gene therapy, as they can be used to deliver therapeutic genes to target cells. Understanding and manipulating their use of the cellular transport machinery could lead to more effective therapies.
- Describe promoters used in viral vectors
* Define a promoter
* What is the function of the promoter?
* How can cell tropisms be controlled by the promoter?
* What is a pan-neuronal promoter?
* What is a constitutive promoter?
- Define a promoter
A promoter is a region of DNA located upstream (5’ end) of a gene that initiates transcription, the process of converting DNA into messenger RNA (mRNA). It’s essentially the “on-switch” for gene expression and is critical in determining when and where the gene is expressed.
A promoter is a sequence of DNA to which proteins bind that initiate transcription of a single mRNA stand from the DNA downstream of it
The promoter controls the binding of RNA polymerase to DNA
The promoter region is vital as it controls when and where in the organism the gene of interest (GOI) is expressed
AAV promoters have to be small, preferably <1 kb
Please note that a promoter is not an enhancer
They confer cell type specificity
- What is the function of the promoter?
The primary function of a promoter is to guide the cell’s transcription machinery, including RNA polymerase and various transcription factors, to the correct location on the DNA strand to start transcription. Promoters contain specific sequences that are recognized by these proteins, allowing the initiation of transcription at the right place and the right time. The specific sequence and structure of a promoter can influence how frequently a gene is transcribed, making some promoters “stronger” (leading to more transcription) and others “weaker” (less transcription).
They confer cell type specificity - How can cell tropisms be controlled by the promoter?
Cell tropism, or the types of cells a virus can infect, is primarily determined by the interactions between viral proteins and cell surface receptors. However, promoters can also play a role in defining which cells express the transgene once the viral vector is inside the cell.
By choosing a promoter that is active only in certain cell types (a tissue-specific promoter), researchers can ensure that the transgene is expressed primarily in those cells. This doesn’t change which cells the viral vector can enter, but it does control where the gene of interest is expressed, adding a level of specificity and safety to gene therapy applications.
- What is a pan-neuronal promoter?
A pan-neuronal promoter is a type of promoter that initiates transcription specifically in neuronal cells, i.e., nerve cells. When this type of promoter is used in a viral vector, the gene of interest will be expressed primarily in neurons. This is beneficial when the target of gene therapy is a neurological disease. - What is a constitutive promoter?
A constitutive promoter is a type of promoter that is active in all cell types and at all times. When a gene is under the control of a constitutive promoter, it is continuously transcribed into mRNA, and therefore its protein product is continually produced. Examples of constitutive promoters include the cytomegalovirus (CMV) promoter and the chicken beta-actin (CAG) promoter. These are often used in experiments where high, consistent levels of gene expression are needed.
- Describe vectors used to map neural circuits
* Explain the basic principles of using rabies virus to label neural circuits
* Which other viral vectors are often used in combination with rabies virus. Why?
- Explain the basic principles of using rabies virus to label neural circuits
Rabies virus is highly infectious, particularly neuro-invasive, and quickly propagates through the nervous system through transcellular transfer
G-protein-coated RABV infects cells via the G receptor.
In neurons, the G receptor is restricted to presynaptic nerve terminals. Therefore, once RABV is released from an infected cell, it is only taken up by the presynaptic nerve terminals of adjacent neurons
For RABV to crawl through the whole brain, it first infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses to retrogradely infect neurons that project to the starter cell.
1. It is highly toxic!
2. It will eventually infect the whole brain!
Rabies virus is commonly used to label and study neural circuits because of its ability to jump synapses in a retrograde manner (from the synaptic terminal back to the neuron’s cell body) and infect upstream neurons. This ability provides a unique tool for researchers to trace and map the connections between neurons, thereby understanding the structure and function of neural circuits.
A genetically modified rabies virus is often used in these studies. This virus is engineered to express a fluorescent protein, which allows infected neurons to be easily identified under a microscope. Additionally, to control the infection to specific neurons and to limit the infection to only one synaptic jump, a version of the rabies virus with a deleted glycoprotein gene (ΔG) is often used.
However, the rabies virus needs the glycoprotein to spread across synapses. Researchers cleverly solve this problem by using another virus to deliver the glycoprotein specifically to the target neurons. The ΔG rabies virus can then infect these neurons, produce the fluorescent protein, and spread to directly connected neurons, but no further.
- Which other viral vectors are often used in combination with rabies virus. Why?
To enable the ΔG rabies virus to infect neurons and jump synapses, another viral vector is used to ‘complement’ the missing glycoprotein in the target neurons. This is typically an adeno-associated virus (AAV) or a lentivirus.
The helper virus is engineered to express the rabies glycoprotein (Rgp) under a cell-type specific promoter, ensuring that only the desired target neurons will produce the glycoprotein. The helper virus also often includes a fluorescent protein under a different promoter, allowing the target neurons to be distinguished from the neurons infected by the rabies virus.
Once the helper virus has been used to deliver the glycoprotein to the target neurons, the ΔG rabies virus can be applied. The rabies virus will infect the target neurons (since they express the necessary glycoprotein), then spread retrogradely to neurons connected to the target neurons.
By this method, a whole neural circuit connected to the target neurons can be labeled and visualized, providing valuable insights into the structure and function of the nervous system. This technique, known as trans-synaptic tracing, has revolutionized our understanding of neural circuitry.
- AAV vector design
* What are the basic DNA elements found within an AAV vector
* What is the function of each of these elements?
* How can expression of the gene-of- interest (GOI) be directed towards a desired cell-type population? Explain different solutions.
- What are the basic DNA elements found within an AAV vector
AAV vectors typically contain a few key elements in their DNA payload:
Inverted Terminal Repeats (ITRs): These are short (approximately 145 base pair) sequences located at each end of the genome. They form hairpin loops that are crucial for packaging the AAV genome into the capsid and for integrating the vector genome into the host cell genome.
Promoter: This is a DNA sequence that controls when and where the gene-of-interest is expressed. It guides the transcription machinery to the start of the gene.
Gene-of-Interest (GOI): This is the gene that the researchers want to introduce into the host cells. It could be a therapeutic gene, a reporter gene (like GFP, which encodes a green fluorescent protein), or any other gene that serves the purpose of the experiment or treatment.
localization signal: used to ‘tag’ the GOI at the N or C terminus to create a signal for transport, localization and recognition. the tagging consists of small sequences which can be inserted in multiple repeats to boost the desired property
Polyadenylation Signal (PolyA Signal): This is a sequence that signals the end of transcription and triggers the addition of a poly(A) tail to the mRNA. This poly(A) tail is crucial for the stability and translation efficiency of the mRNA.
- What is the function of each of these elements?
ITRs: The ITRs are necessary for replication and packaging of the AAV genome. They are the only parts of the wild-type AAV genome that are needed in the AAV vector, as they contain the recognition sequences needed for replication and packaging.
Promoter: The promoter controls the expression of the gene-of-interest. It can be selected to be active in all cells (a constitutive promoter) or to be active only in certain cell types or under certain conditions (an inducible or cell-specific promoter).
GOI: This is the gene that is being delivered by the AAV vector. The choice of gene depends on the goal of the experiment or therapy. other possibilities are to cut out a target gene or to silence its transcription if its endogenously expressed in the species
PolyA Signal: The polyA signal is necessary for proper mRNA processing and stability. Without it, the mRNA would degrade rapidly and not be efficiently translated into protein.
Localization signals: Desired properties (for wanted transport, localization, recognition) can be added by tagging the GOI. The tagging consists of small sequences (sometimes inserted in multiple repeats for boosting the desired property) - happens at C or C terminal
WPRE (Woodshuck post-transcriptional enhancer): enhances expression of the GOI
- How can expression of the gene-of- interest (GOI) be directed towards a desired cell-type population? Explain different solutions.
There are a few strategies to direct the expression of the GOI to a specific cell type:
Use of Cell-Type Specific Promoters: One way is to use a promoter that is only active in the desired cell type. For example, if you wanted to express the GOI in neurons, you might use a promoter like Synapsin or CaMKII. This can be combined with cre-loxP or cre-FLEx.
Use of Specific AAV Serotypes: Different AAV serotypes have different tropisms, or preferences for certain cell types. By selecting the appropriate serotype (capside), you can target specific cells or tissues.
Physical Delivery: The AAV vector can be physically delivered to the specific location where the target cells reside, such as by direct injection into a particular tissue or organ.
Transductional Targeting: The AAV capsid can be engineered to alter its tropism and enhance its specificity for certain cell types. This could be done by inserting specific peptides into the capsid proteins that have high affinity for receptors on the desired cell type. This method is still experimental and is an active area of research.
By using one or more of these strategies, researchers can increase the specificity of gene delivery and limit off-target effects, making AAV vectors a powerful tool for gene therapy and basic research.
- Describe the Cre-lox system
* How does the Cre-lox system work?
* How is Cre-lox system useful in neuroscience?
* Explain how Cre-driver mouse lines can be used in conjunction with AAV vectors
- How does the Cre-lox system work?
The Cre-lox system is a method of genetic recombination that allows researchers to precisely manipulate genes within an organism at specific (developmental) times or in specific tissues.
This system consists of two main elements: the Cre recombinase enzyme and loxP sites. Cre is an enzyme that recognizes loxP sites, which are specific sequences of DNA. When a DNA molecule contains two loxP sites, the Cre enzyme can cause recombination between these sites, leading to the excision, inversion, or translocation of the DNA sequence between them.
If the loxP sites are oriented in the same direction, the DNA segment between them will be excised when Cre is present. If the loxP sites are in opposite directions, the DNA segment between them will be inverted.
- Derived from P1 bacteriophage
- A potent and specific system for controlling gene expression
- The protein Cre recombinase recognizes 34 bp loxP sites
- The orientation and location of the loxP sites determines how the genetic material will be rearranged
- How is Cre-lox system useful in neuroscience?
The Cre-lox system has been incredibly useful in neuroscience for creating conditional knockout animals and for controlling gene expression in specific cell types or at specific developmental stages.
For instance, researchers can insert loxP sites on either side of a specific gene in mouse embryonic stem cells, and then use these cells to generate transgenic mice. In the absence of Cre, the gene functions normally. However, if these mice are bred with mice that express Cre recombinase under a specific promoter (such as a promoter active only in a certain subset of neurons), the gene will be excised only in the cells where Cre is expressed, creating a tissue-specific or cell-type specific knockout.
Cre-dependent Gene Expression: Placing a stop codon with loxP sites on either side (often called a “loxstop- lox” or “LSL” cassette) upstream of a gene of interest will prevent gene expression in the absence of Cre. In the presence of Cre, the stop codon is excised, and gene expression proceeds.
Cre-dependent Gene Knockout: Conversely, putting the loxP sites on either side of a gene (called “floxing”, for “flanked by loxP”), will permit gene expression until Cre is present, at which time the gene will be disrupted or deleted.
Cre-dependent shRNA Expression: Cre-lox can be used to turn shRNA constructs on or off. In floxed-shRNA constructs, Cre can excise the shRNA to return gene expression to physiological levels. In lox-STOP-lox shRNA constructs, Cre expression promotes shRNA expression.
Gene Switch: These constructs contain two genes of interest, Genes A and B. When Cre is absent, only Gene A is translated correctly. Cre expression excises Gene A and alters the reading frame to allow in-frame translation of Gene B.
FLEx Switch: This system allows scientists to utilize recombination elements such as Cre to turn off the expression of one gene while simultaneously turning on another. The FLEx switch system takes advantage of the orientation specificity of Cre and the different types of target sites available, both mutant and wild type. The ability to manipulate the number, orientation, and type of target sites that flox your genes of interest makes FLEx switch a powerful experimental tool.
- Explain how Cre-driver mouse lines can be used in conjunction with AAV vectors
Cre-driver mouse lines are strains of mice that express Cre recombinase under control of a specific promoter, thus restricting its expression to a certain cell type or tissue.
This system can be used in conjunction with AAV vectors in a method called “Cre-dependent expression”. In this approach, an AAV vector is designed to contain a gene-of-interest (GOI) that is flanked by loxP sites. However, a ‘stop’ sequence is also included between the loxP sites and before the GOI. This stop sequence prevents the expression of the GOI until it is removed.
When this AAV vector is injected into a Cre-driver mouse line, the AAV infects cells indiscriminately, but the GOI is only expressed in cells where Cre is present. In these cells, Cre recombinase excises the stop sequence, allowing the GOI to be expressed.
This provides a powerful tool for researchers, as it allows for gene expression to be controlled with high spatial specificity (by injecting the AAV into specific tissues) and cell-type specificity (by choosing a Cre-driver line that expresses Cre in the desired cell type). As such, it is a commonly used technique in neuroscience for manipulating gene expression in specific neural circuits.
- Genome editing using CRISPR technology
* Explain the function of the Cas9 enzyme
* Explain the function of the guide RNA
* How is CRISPR technology useful in neuroscience?
* What is ex vivo gene therapy?
* What is in vivo gene therapy?
- Explain the function of the Cas9 enzyme
Cas9 is an enzyme that acts as a pair of “molecular scissors” in the CRISPR-Cas9 genome editing system. It originates from a type of adaptive immune system found in certain bacteria. Cas9 can cut DNA at specific locations, allowing researchers to add, delete, or modify certain genetic elements.
Its activity is guided by a molecule called guide RNA (gRNA) which leads Cas9 to the appropriate part of the genome based on sequence complementarity. Once Cas9 is guided to the right place, it can make a double-strand break in the DNA, which the cell then repairs. If the researchers provide a DNA template, the cell can repair the break in a way that incorporates the new DNA sequence, allowing specific changes to be made to the genome.
- Explain the function of the guide RNA
The guide RNA (gRNA) in the CRISPR-Cas9 system is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-binding and a user-defined ~20 nucleotide “spacer” or “guide” sequence that defines the genomic target to be modified. The gRNA is complementary to the target DNA sequence, and its primary function is to guide the Cas9 enzyme to the correct location in the genome for cutting. It is able to find this sequence based on the PAM sequence, which is where the gRNA can recognize a potential binding site. - How is CRISPR technology useful in neuroscience?
CRISPR technology provides a powerful tool for neuroscientists to study the function of specific genes and genetic changes in the brain. For example, researchers can use CRISPR to create model organisms with specific mutations in genes of interest, or to knock out specific genes in specific types of neurons to study their function.
In addition to basic research, CRISPR also holds promise for the treatment of neurological disorders. For example, it could potentially be used to correct genetic mutations that cause diseases like Huntington’s or Parkinson’s. While this application is still largely in the experimental stage, the potential of CRISPR for treating neurological diseases is a major area of research.
- What is ex vivo gene therapy?
Ex vivo gene therapy involves removing cells from the patient, genetically modifying them in the laboratory, and then returning them to the patient. This approach is useful for diseases that involve a specific type of cell that can be easily removed and returned to the body. For example, this approach has been used successfully to treat certain types of immune deficiencies and blood disorders. - What is in vivo gene therapy?
In vivo gene therapy involves directly delivering the gene of interest into the patient’s body. This can be achieved by using viral vectors (like AAV) or non-viral methods to deliver the therapeutic gene directly to the cells in the body. This approach can be used to treat diseases that involve cells or tissues that are difficult to remove from the body, such as neurons in the brain for the treatment of neurological disorders.
Both ex vivo and in vivo gene therapies have their advantages and challenges, and the choice between them depends on the specific disease and cell type being targeted.
- Calcium-biosensors
* How does a genetically-encoded Ca2+ biosensor work?
* What protein(s) is used to sense Ca2+?
* How is a genetically encoded Ca2+ biosensor useful in neuroscience or cell biology?
* How is it expressed?
- How does a genetically-encoded Ca2+ biosensor work?
A genetically-encoded calcium biosensor is a protein designed to emit a fluorescent signal when calcium ion (Ca2+) levels in the cell change. Calcium is an important second messenger in cells and changes in intracellular calcium levels can indicate cell activity, including neuronal firing.
Most genetically-encoded calcium biosensors are based on a protein called Green Fluorescent Protein (GFP), or one of its color variants, fused to another protein that can bind calcium.
In a common design, the calcium-binding protein is calmodulin, and the GFP is linked to a calmodulin-binding peptide. In the absence of calcium, the calmodulin and its binding peptide interact in a way that causes the GFP to fluoresce at a low level. When calcium is present, it binds to calmodulin, causing a conformational change that alters the interaction with the binding peptide. This conformational change causes the GFP to fluoresce more brightly. Therefore, by monitoring the brightness of the GFP, one can monitor changes in calcium levels.
- What protein(s) is used to sense Ca2+?
A genetically-encoded calcium biosensor is a protein designed to emit a fluorescent signal when calcium ion (Ca2+) levels in the cell change. Calcium is an important second messenger in cells and changes in intracellular calcium levels can indicate cell activity, including neuronal firing.
Most genetically-encoded calcium biosensors are based on a protein called Green Fluorescent Protein (GFP), or one of its color variants, fused to another protein that can bind calcium.
In a common design, the calcium-binding protein is calmodulin, and the GFP is linked to a calmodulin-binding peptide. In the absence of calcium, the calmodulin and its binding peptide interact in a way that causes the GFP to fluoresce at a low level. When calcium is present, it binds to calmodulin, causing a conformational change that alters the interaction with the binding peptide. This conformational change causes the GFP to fluoresce more brightly. Therefore, by monitoring the brightness of the GFP, one can monitor changes in calcium levels.
- How is a genetically encoded Ca2+ biosensor useful in neuroscience or cell biology?
Genetically-encoded calcium biosensors have numerous uses in neuroscience and cell biology. Because calcium is an important second messenger in neurons and is involved in processes like neurotransmitter release and synaptic plasticity, these sensors allow researchers to monitor neuronal activity in real-time.
By using genetically-encoded sensors, researchers can specifically express the sensor in certain cell types or compartments within the cell, allowing them to monitor calcium dynamics with high spatial resolution. This can provide insights into the functioning of neural circuits or the roles of calcium in various cellular processes.
Moreover, unlike chemical calcium indicators, genetically-encoded biosensors can be targeted to specific cell types using genetic methods, offering much greater specificity.
- How is it expressed?
Genetically-encoded calcium biosensors are typically delivered into cells using a plasmid or viral vector. This vector contains the DNA sequence encoding the biosensor under the control of a specific promoter to drive its expression.
In many cases, researchers use a viral vector (such as an adeno-associated virus, or AAV) to deliver the biosensor to cells in vivo. Alternatively, the biosensor can be introduced into cells in culture via transfection or transduction.
Once the DNA encoding the biosensor is inside the cell, the cell’s own machinery transcribes and translates it, resulting in the production of the biosensor protein. The sensor protein can then fluoresce in response to changes in calcium levels, and this fluorescence can be detected using various imaging techniques.
- Optogenetics
* Explain the basic principles behind optogenetics
* Define the “opto” part
* Define the “genetics” part
* Why is optogenetics useful in neuroscience?
- Explain the basic principles behind optogenetics
Optogenetics is a technique that involves the use of light to control cells in living tissue, typically neurons, that have been genetically modified to express light-sensitive ion channels or other proteins. This allows researchers to precisely control the activity of specific cells in response to light, enabling the study of the contributions of individual cells or networks of cells to overall tissue or organism function. - Define the “opto” part
The “opto” part of optogenetics refers to the use of light in this technique. The light-sensitive proteins that are used in optogenetics, known as opsins, respond to light of specific wavelengths. Different types of opsins respond to different wavelengths of light, allowing for different responses to be triggered by different colors of light. This enables researchers to use light to control cell activity with a high degree of spatial and temporal precision. - Define the “genetics” part
The “genetics” part of optogenetics refers to the genetic manipulation used to introduce the light-sensitive proteins into specific cells. This is typically achieved by creating a genetically modified organism that expresses these proteins in certain cell types, or by using viral vectors to deliver the genes encoding these proteins into specific cells in a living organism. - Why is optogenetics useful in neuroscience?
Optogenetics has revolutionized neuroscience by providing a tool for controlling and studying the behavior of individual neurons and neural circuits with unprecedented precision.
Traditional techniques for studying the brain, like electrical stimulation or pharmacological methods, do not provide the same level of control and specificity. Electrical stimulation affects all neurons in the vicinity of the electrode, making it difficult to target specific cell types or pathways. Pharmacological methods are often not fast enough to keep up with the rapid dynamics of neural activity.
In contrast, optogenetics allows researchers to target specific types of neurons and to turn them on or off on the millisecond timescale, matching the speed of actual neural processes. This can help researchers understand how different types of neurons and neural circuits contribute to behavior and cognition, and it can also potentially provide new therapeutic strategies for neurological disorders.
- Optogenetics – excitation
* How does ChR2 work?
* What are the main advantages of using ChR2
* How is ChR2 expressed?
* Explain potential limitations of ChR2
- How does ChR2 work?
Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel derived from a type of single-celled algae. It is one of the most commonly used tools in optogenetics.
When ChR2 is exposed to blue light (around 470 nm wavelength), it undergoes a conformational change that opens the channel, allowing positively charged ions (like sodium and potassium) to flow across the cell membrane. This influx of positive charge can depolarize the neuron, leading to an action potential if the depolarization is strong enough. Thus, by delivering a flash of blue light, researchers can cause a neuron expressing ChR2 to fire an action potential.
- What are the main advantages of using ChR2
ChR2 has several key advantages that have made it a popular tool in optogenetics:
Speed: ChR2 responds to light almost instantaneously and can follow high frequency stimulation, allowing researchers to manipulate neural activity with millisecond precision.
Specificity: By using specific promoters to drive ChR2 expression, researchers can target specific types of neurons, allowing for cell-type specific manipulations.
Bidirectionality: While ChR2 itself only allows for the activation of neurons, it can be used in combination with other opsins that inhibit neuronal activity, allowing researchers to both turn neurons on and off.
Non Invasiveness: Because it is activated by light, ChR2 can be used to control neural activity without the need for electrical wires or other invasive equipment.
- How is ChR2 expressed?
ChR2 is typically introduced into neurons using genetic techniques. The gene encoding ChR2 is inserted into a plasmid or a viral vector, often along with a fluorescent marker to help identify the cells that express it. This vector can then be delivered into the brain via injection. Once inside a neuron, the cell’s own machinery will express the ChR2 protein, embedding it in the cell membrane where it can respond to light.
The ChR2 gene can be placed under the control of specific promoters to ensure it is only expressed in certain types of neurons. Additionally, Cre-Lox recombination can be used to further restrict ChR2 expression to specific cell types.
- Explain potential limitations of ChR2
While ChR2 is a powerful tool, there are some limitations to its use:
Light Delivery: In order to activate ChR2, light must be delivered to the neurons, which can be challenging in deep brain structures. This usually requires implantation of an optical fiber, which can be invasive.
Heat Generation: High-intensity light can generate heat, which can potentially cause damage to brain tissue or spur artifacts in experimental observations.
Non-Physiological Stimulation: The activation of neurons with ChR2, especially if it is expressed at high levels, might not fully mimic natural neuronal activity. High levels of activation could lead to unusual firing patterns or excessive calcium influx.
Off-Target Effects: Although the use of specific promoters can restrict ChR2 expression to specific cell types, there is always a risk of off-target expression, which could lead to the inadvertent activation of other cells.
- Optogenetics – inhibition
* How does inhibitory opsins work?
* What are the main advantages of using inhibitory opsins
* How is inhibitory opsins expressed?
* Explain potential limitations of inhibitory opsins
- Optogenetics – inhibition
* How does inhibitory opsins work?
Inhibitory opsins are used in optogenetics, a biological technique that involves the use of light to control cells in living tissue, often neurons, that have been genetically modified to express light-sensitive ion channels.
Inhibitory opsins like halorhodopsin and archaerhodopsin work by pumping negative chloride ions or protons into the neuron when activated by light, typically of a certain wavelength (for example, yellow light for halorhodopsin and green light for archaerhodopsin). This leads to hyperpolarization of the neuron, or an increase in the cell’s resting membrane potential.
This hyperpolarization makes it more difficult for the neuron to reach the action potential threshold, thus inhibiting the neuron from firing. In this way, inhibitory opsins are used to quiet or turn off specific neurons, allowin
Chloride pumps: One of the most efficient and widely used inhibitory opsins, NpHR, is a halorhodopsin from the archaeon Natronomonas pharaonis - Proton pumps can also be used to inhibit neurons through hyperpolarization, by pumping protons out of the cell, and have some features that make them desirable alternatives to chloride pumps, which include fast recovery from inactivation and high light-driven currents
- What are the main advantages of using inhibitory opsins
The use of inhibitory opsins, especially within the context of optogenetics, has several key advantages:
Precision: Inhibitory opsins provide a high degree of precision both spatially and temporally. Spatially, they allow researchers to target specific neurons or groups of neurons. Temporally, they can control neuron activity at the millisecond timescale, which aligns with the timescale of neuronal communication.
Reversible Manipulation: The effects of optogenetic manipulation are reversible. Once the light is turned off, the targeted neurons return to their normal state. This allows for sophisticated experimental designs that manipulate and then restore neuronal activity.
Non-Invasive: Compared to other techniques like deep-brain stimulation, optogenetics can be relatively non-invasive. Light can be delivered to neurons via fiber optic cables or LED devices implanted in the brain.
Specificity: Opsins can be selectively introduced to specific neuron types through the use of targeted genetic techniques, allowing researchers to study the role of specific cell types in the overall functioning of neural circuits.
Better Understanding of Brain Function: By selectively inhibiting certain neurons or brain areas, researchers can study the specific roles those neurons or areas play in behavior, perception, cognition, and emotion. This could not only enhance our understanding of the brain but also pave the way for new therapeutic strategies for a variety of neurological and psychiatric conditions.
Therapeutic Potential: Experimental treatments using optogenetics are being developed for a variety of conditions such as Parkinson’s disease, depression, and blindness. Inhibitory opsins could be used in these therapies to quiet overactive neural circuits.
It’s important to note, however, that the use of optogenetics and inhibitory opsins also have certain limitations and ethical considerations, such as the need for genetic modification of target cells, potential tissue damage from light exposure, and challenges with delivering light to deeper brain regions.
- How is inhibitory opsins expressed?
Inhibitory opsins, like all opsins used in optogenetics, are typically expressed in neurons through the use of viral vectors or transgenic techniques.
Viral vectors: In this method, a virus is used as a delivery vehicle to transport the opsin gene into neurons. The virus is engineered to be safe and will not cause disease. It contains the opsin gene, which is inserted into the genome of the neurons it infects, causing them to produce the opsin protein. Specificity can be achieved by injecting the virus into a particular region of the brain or by using a virus that only infects certain types of neurons. It’s also possible to use a type of virus that only infects neurons if they express a certain other gene, allowing for very precise targeting.
Transgenic techniques: In this method, the opsin gene is inserted into the genome of an organism at an early stage of development (often before birth), causing certain neurons to produce the opsin throughout the organism’s life. Transgenic animals, such as mice, are often used in optogenetic research. It’s possible to control which neurons express the opsin by linking the opsin gene to a promoter sequence that is only active in certain types of neurons.
The expression of inhibitory opsins allows researchers to control the activity of specific neurons using light, with the goal of understanding how these neurons contribute to brain function and behavior. However, the use of these techniques requires careful ethical consideration, as they involve genetic modification and potentially invasive procedures.
- Explain potential limitations of inhibitory opsins
While inhibitory opsins and optogenetics overall provide many advantages for neuroscience research, there are also several potential limitations to be aware of:
Invasiveness: Despite being less invasive than some other methods, optogenetics does still typically require surgery to deliver the light to the neurons of interest, which can lead to potential complications or damage to surrounding tissues.
Difficulty in reaching deep brain structures: Light delivery can be challenging for neurons located deep within the brain, as light scattering by the overlying tissue can reduce the intensity and precision of the stimulation.
Genetic modification: Optogenetics requires genetic modification of the target cells, which can be a complex process, and might not be applicable or ethical in all settings, especially in humans. It might also cause unforeseen alterations in the function of the target cells or tissues.
Specificity: While opsins can be targeted to specific cell types, there can still be off-target effects, where opsins are expressed in unintended cells. Additionally, light used to activate the opsins might also affect other light-sensitive processes in the body.
Physiological side effects: Chronic or intense illumination can lead to tissue heating or damage, which can cause undesired physiological effects.
Temporal limitations: Despite having high temporal precision, the speed of opsin deactivation can limit the frequency at which neurons can be inhibited.
Long-term effects: The long-term effects of optogenetic manipulation on neural tissue health and function are not completely known, which could limit its use for chronic studies or therapeutic applications.
Limitation of behavioral studies: The behavioral changes observed in optogenetic experiments are often complex and difficult to interpret. The activation or inhibition of certain neurons might result in multiple simultaneous effects, making it challenging to associate them with a specific behavior.
These pumps move only one ion per absorbed photon, which makes them inefficient compared to some of the currently available excitatory channel opsins that allow flow of ions through an open pore.
These limitations do not diminish the power and potential of optogenetics and inhibitory opsins but underscore the need for careful experimental design, interpretation, and ongoing technological refinement.
- Chemogenetics
* Explain the basic principles of chemogenetics (DREADDs)
* Define the “chemo” part
* Define the “genetics” part
* What are the main advantages of using DREADDs?
* What kind of limitations should be considered when using DREADDs
* How is DREADDs expressed?
- Explain the basic principles of chemogenetics (DREADDs)
Chemogenetics refers to a technology in neuroscience research that uses engineered proteins to manipulate cell signaling and function in living organisms in response to administered small molecules. A common example of a chemogenetic technique involves the use of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs).
Here are the basic principles:
Designer Receptors: DREADDs are engineered receptors, most commonly modified from muscarinic G protein-coupled receptors (GPCRs). They are modified in such a way that they no longer respond to their natural ligand (acetylcholine in the case of muscarinic receptors), but instead respond to a synthetic drug.
Exclusively Activated by Designer Drugs: The most commonly used synthetic drug is Clozapine-N-Oxide (CNO). When CNO is administered, it binds to the DREADD and activates the receptor. Importantly, CNO is otherwise pharmacologically inert and does not bind to other receptors in the body.
Expression of DREADDs: Similar to opsins in optogenetics, DREADDs are expressed in specific neurons by genetic engineering techniques, usually involving viral vectors or transgenic animals. A promoter sequence is used to ensure that DREADDs are expressed only in the desired cell type.
Activation of DREADDs: When activated by CNO, DREADDs can either stimulate or inhibit neuronal activity depending on the type of G protein they are coupled to. For example, DREADDs coupled to Gi proteins inhibit neuronal activity, whereas DREADDs coupled to Gq proteins stimulate neuronal activity.
Behavioral and Physiological Changes: By activating DREADDs in specific types of neurons, researchers can observe the behavioral and physiological changes that result, helping to understand the function of these neurons in the brain.
Chemogenetics and DREADDs provide a powerful method for manipulating neuronal activity in living organisms, and offer several advantages such as the ability to target deep brain structures and the ability to administer the drug systemically (e.g., by injection or oral administration) rather than needing to deliver light to the neurons as in optogenetics. However, like all experimental techniques, they also have limitations and potential side effects.
- Define the “chemo” part
When CNO is administered, it binds to the DREADD receptors that have been genetically engineered to respond to it, which then triggers the receptor and influences the activity of the cell. The DREADD receptors are designed to respond exclusively to these synthetic drugs and not to any naturally occurring compounds in the body.
The advantage of using chemical compounds is that they can be administered in a variety of ways, such as oral administration or injection, and can reach cells throughout the body, including those located deep within the brain. This gives chemogenetics a level of versatility and broad applicability that complements other techniques like optogenetics, which require light delivery to the specific site of the neurons.
- Define the “genetics” part
The “genetics” part of chemogenetics refers to the use of genetic engineering techniques to modify cells so that they produce certain proteins – in this case, the designer receptors like DREADDs (Designer Receptors Exclusively Activated by Designer Drugs).
To achieve this, the DNA sequence encoding the DREADD protein is inserted into the genome of the target cells. This can be done in a variety of ways, including using viral vectors, which are engineered viruses that deliver the DREADD gene into cells, or through the creation of transgenic organisms, in which the DREADD gene is inserted into the genome at an early stage of development.
These genetic techniques can often be targeted to specific cell types by including a promoter sequence with the DREADD gene that is only active in certain cells. For example, a promoter sequence that is only active in certain types of neurons could be used to ensure that only those neurons produce the DREADD protein.
Once the DREADD gene has been incorporated into the genome of the target cells, those cells will produce the DREADD protein and express it on their surface. This allows the cells to be influenced by the administration of the designer drug (the “chemo” part of chemogenetics), and allows researchers to manipulate cell activity in a highly specific and controlled way.
- What are the main advantages of using DREADDs?
DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) offer several advantages for neuroscience research, particularly in comparison to other techniques for manipulating cell activity:
Non-Invasiveness: DREADDs can be activated by drugs that are administered systemically (e.g., by injection or oral administration), making the method less invasive than techniques like optogenetics that require light delivery to specific locations in the brain.
Long Duration of Action: DREADDs can have a long duration of action. Once the designer drug is administered, it can continue to have effects for several hours, allowing for prolonged manipulation of neuronal activity.
Spatial Specificity: Similar to optogenetics, DREADDs can be expressed in specific cell types or in specific regions of the brain, allowing for spatially precise manipulation of cell activity.
Temporal Control: The activation of DREADDs can be controlled based on when the designer drug is administered, providing control over when the cells are manipulated.
Bidirectional Control: Depending on the type of DREADD used, neuronal activity can be either increased or decreased, allowing for bidirectional control over cell activity.
Deep Brain Structure Targeting: As the DREADDs’ activation does not rely on light, but on a systemic drug, it is useful for targeting deep brain structures that are otherwise difficult to reach.
Safe and Controlled Experimentation: As DREADDs are only activated by their specific designer drug, and not by naturally occurring compounds in the body, there is less risk of off-target effects.
Behavioural Studies: They allow for the study of more naturalistic behaviors over longer time scales, due to the less invasive nature and longer duration of action of DREADD activation.
However, it’s important to bear in mind that DREADDs also have their limitations, such as potential off-target effects of the designer drug and the slower onset and offset of action compared to optogenetics.
- What kind of limitations should be considered when using DREADDs
While DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) provide a useful tool for manipulating neuronal activity, there are several potential limitations and considerations to be aware of:
low temporal and spatial resolution
Slow Onset and Offset: The effects of DREADDs are not as immediate as other methods like optogenetics. It takes time for the designer drug to be administered, absorbed, and reach the target cells. Similarly, the effects of the drug can linger, meaning that there is less precise control over the timing of cell activity manipulation.
Lack of Immediate Temporal Precision: Unlike optogenetic methods, which allow for manipulation of neuronal activity on a millisecond timescale, DREADDs do not offer this level of temporal precision due to the pharmacokinetics of the designer drug.
Off-Target Effects: While DREADDs are designed to be activated exclusively by a specific designer drug, and the commonly used drug Clozapine N-Oxide (CNO) is typically inert in mammals, recent research has suggested that CNO may be metabolized back into clozapine, an atypical antipsychotic drug, which can have off-target effects.
Need for Genetic Modification: Like optogenetics, the use of DREADDs requires genetic modification of target cells, which can be a complex process and might not be applicable or ethical in all settings, especially in humans.
Dose-Response Curve: As DREADDs operate via a drug that needs to circulate systemically to reach its target, there’s a risk of over- or under-stimulation. Precise dosing can be difficult to determine and may need extensive calibration.
Inter-individual variability: The effects of DREADDs can vary from one individual to another due to differences in drug metabolism and absorption, complicating the interpretation of results.
Side Effects: Like any drug, the compounds used to activate DREADDs can have side effects, especially at high doses.
Despite these limitations, DREADDs continue to be a powerful tool in neuroscience research, offering unique capabilities for manipulating neuronal activity in living organisms. As always, careful experimental design and interpretation can help to mitigate these limitations.
- How is DREADDs expressed?
The expression of DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) involves genetic engineering techniques, similar to the expression of opsins in optogenetics. Here are the common steps involved:
Creation of the DREADD Gene: The first step is the creation of the DREADD gene. This involves modifying the DNA sequence of a G protein-coupled receptor (GPCR) so that it no longer responds to its natural ligand, but instead responds to a synthetic drug.
Packaging into a Vector: The DREADD gene is then packaged into a vector, usually a virus, that can deliver the gene into cells. The vector may also contain other elements, such as a promoter sequence that ensures the DREADD gene is only expressed in certain types of cells.
Delivery of the Vector: The vector is then delivered to the target cells. This could be done by injecting the vector into a specific region of the brain, or by creating a transgenic organism where the vector is incorporated into the genome at an early stage of development. The vector inserts the DREADD gene into the genome of the target cells.
Expression of the DREADD Protein: Once the DREADD gene is in the genome of the target cells, those cells will produce the DREADD protein. The protein is expressed on the surface of the cell, where it can be activated by the synthetic drug.
Activation of DREADDs: When the synthetic drug is administered, it binds to the DREADD protein, activating it and causing a change in cell activity. Depending on the type of DREADD, this could either increase or decrease cell activity.
Expressing DREADDs provides a powerful method for manipulating cell activity in living organisms, but also involves complex genetic engineering techniques and potential ethical considerations.
- Fluorescent proteins
What is fluorescence?
Describe FRET (Fönster/Fluorescence Resonance Energy Transfer)?
Describe application of fluorescent proteins in imaging
Describe limitations and potential issues of fluorescence protein imaging
What is fluorescence?
Fluorescence is a physical phenomenon where a substance absorbs light (or other forms of electromagnetic radiation) of a specific wavelength and then re-emits light of a longer wavelength. This process happens in three stages:
Absorption: The fluorescent substance (fluorophore) absorbs a photon of light, which excites an electron from its ground state to a higher energy excited state.
Relaxation: The electron quickly loses some energy through non-radiative processes, leading to a relaxed excited state.
Emission: The electron then returns to its ground state, releasing the energy difference as a photon of light. Because some energy was lost during the relaxation phase, the emitted photon has less energy, and therefore a longer wavelength, than the absorbed photon.
Fluorescence is used in many scientific applications, including molecular biology, biochemistry, and medical diagnostics. Fluorescent proteins, in particular, have been a transformative tool in biology, allowing researchers to visualize and track the behavior of specific proteins, cells, and processes within living organisms.
Notably, the green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its many colorful derivatives, which were developed through mutagenesis, have found widespread use as tags in molecular and cellular biology to visualize a variety of biological phenomena.
Describe FRET (Fönster/Fluorescence Resonance Energy Transfer)?
Förster Resonance Energy Transfer (FRET) is a mechanism of energy transfer between two light-sensitive molecules or chromophores. A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through non-radiative dipole-dipole coupling. This process is also known as Fluorescence Resonance Energy Transfer, although the original name recognizes the scientist Theodor Förster, who introduced the theory.
The efficiency of FRET depends on several factors, including:
Spectral Overlap: The emission spectrum of the donor must overlap with the absorption spectrum of the acceptor.
Distance: The donor and acceptor must be very close, typically within a range of 1 to 10 nanometers. As a result, FRET is often used to measure distances or changes in distances within molecules, or to determine if two molecules are interacting.
Orientation: The orientation of the donor and acceptor dipoles relative to each other can also influence FRET efficiency.
When FRET occurs, it results in a decrease in the fluorescence intensity of the donor, reduced fluorescence lifetime of the donor, and an increase in the fluorescence of the acceptor. By measuring these changes, researchers can detect and quantify FRET, and hence the interactions or changes they are interested in.
FRET is an important technique in molecular and cellular biology. It can be used to study protein interactions, confirm protein conformational changes, measure intracellular distances, and validate if certain biological processes have occurred. It has been critical in many studies including investigations of signal transduction, apoptosis, and neuronal synapse activity.
Describe application of fluorescent proteins in imaging
Fluorescent proteins have revolutionized imaging in biological research. They have a wide array of applications that provide insights into a host of cellular processes. Here are a few ways fluorescent proteins are used:
Protein Localization: By fusing a fluorescent protein to the gene encoding the protein of interest, researchers can visualize where this protein is located within the cell or organism.
Protein Movement and Dynamics: Fluorescent proteins can be used to track the movement of proteins within a cell in real time. This is particularly useful in studying dynamic processes such as cell division, endocytosis, and exocytosis.
Interaction Studies: Techniques such as Fluorescence Resonance Energy Transfer (FRET) can be used to study the interaction between two proteins. If the two proteins are close enough for energy transfer to occur, the acceptor fluorescent protein will emit light.
Reporter Genes: Fluorescent proteins are often used as reporter genes to study the activity of a particular promoter. If the promoter is active, the fluorescent protein is produced and can be detected by fluorescence microscopy.
Cell Lineage Tracing: By using different fluorescent proteins, researchers can label different cells within an organism and trace their lineage as they divide and differentiate.
Calcium Imaging: Some fluorescent proteins can respond to the presence of certain ions or molecules. For instance, GCaMP is a green fluorescent protein that increases its fluorescence in the presence of calcium ions, allowing researchers to visualize changes in intracellular calcium levels.
Super-Resolution Microscopy: Certain techniques such as STED (Stimulated Emission Depletion) and PALM (Photo-Activated Localization Microscopy) use properties of fluorescent proteins to achieve resolution below the diffraction limit of light.
Optogenetics: Fluorescent proteins can be paired with light-sensitive proteins to control and monitor the activity of specific neurons with light.
These are just a few examples of how fluorescent proteins are used in imaging. The discovery and development of these proteins have provided scientists with a powerful toolset to visualize and understand many biological processes.
Describe limitations and potential issues of fluorescence protein imaging
While the use of fluorescent proteins in imaging has revolutionized many fields of biology, there are several potential limitations and issues that researchers must consider:
Photobleaching: Fluorescent proteins can lose their ability to fluoresce over time when exposed to light, a process known as photobleaching. This can limit the duration of imaging experiments.
Phototoxicity: The light used to excite the fluorescent proteins can potentially cause damage to the cells or tissues being imaged, a phenomenon known as phototoxicity.
Maturation Time: Some fluorescent proteins require time to mature and become fluorescent after they are produced inside cells. This can delay the ability to visualize these proteins and might not accurately represent real-time protein synthesis.
Spectral Overlap: If multiple fluorescent proteins are used in the same experiment, their emission spectra may overlap, making it difficult to distinguish signals from different proteins.
False-Positives/Negatives: Misfolding of fluorescent proteins could result in a non-fluorescent protein, leading to false negatives. Alternatively, free floating fluorescent proteins from dead cells or protein degradation might lead to false positives.
Perturbation of Function: The addition of a fluorescent protein to a protein of interest could potentially interfere with the normal function of the protein, including its localization, mobility, and ability to interact with other proteins.
Expression Levels: Overexpression of a protein due to the use of strong promoters in the plasmid carrying the fluorescent protein can lead to artifacts and may not represent the protein’s natural state in the cell.
Size of Fluorescent Proteins: Fluorescent proteins are quite large compared to many cellular proteins, and fusing a fluorescent protein to a protein of interest can potentially interfere with the protein’s function or localization.
Despite these challenges, the advantages of using fluorescent proteins often outweigh these potential issues, and many strategies can be used to minimize these limitations, such as using lower light intensities, optimizing the maturation conditions, and carefully validating the function of the fusion protein.
- Population genetics and transgenic animals in psychiatric disease
Explain how to evaluate the validity of an animal disease model.
In context of validity-discuss the challenges in developing animal models for mental
disorders
Give examples of behavioral tests to assess general health and phenotypes related to mental processes.
Why do genetic associations hold significant value in understanding causality compared to other association, considering the deterministic nature of genes?
Explain how to evaluate the validity of an animal disease model.
Internal validity: The results are true/relevant for the population from which it was sampled. It comprends:
Selection bias: Biased allocation to treatment groups. solution= randomization
Performance bias: Systematic difference in care or testing. solution= blinding
Detection bias: Systematic distortion in data collection when assessor is aware of the treatment assignment
External validity: the extent to which the results are true/relevant for other populations.
Face validity: How well are disease phenotype in human are replicated by the model
Construct validity: Does the model involve/ measure a relevant mechanism or process? How Well it explained the underlying mechanism
Predictive validity: How well does the model predict aspect of the disease (e.g treatment effect)
In context of validity-discuss the challenges in developing animal models for mental
disorders
Developing animal models for mental disorders presents several unique challenges. These challenges largely arise from the complex nature of these disorders, the high degree of variability in symptoms among patients, and the difficulties in translating human psychiatric symptoms to animal behaviors. Here are a few specific challenges:
Symptom Complexity and Replication: Mental disorders often involve complex emotional, cognitive, and behavioral symptoms, many of which cannot be replicated in animals. For example, it’s not possible to directly assess feelings of worthlessness or suicidal thoughts in an animal. We can only measure proxies of these symptoms, like changes in social interaction or learned helplessness, but these may not fully capture the human experience of the disorder.
Heterogeneity of Disorders: Many mental disorders are highly heterogeneous, with symptoms and disease progression varying significantly between individuals. This variability makes it difficult to create a single animal model that accurately represents all cases of the disorder.
Unknown Etiology: For many mental disorders, the underlying causes are not well understood. These disorders likely involve complex interactions between genetics, brain chemistry, and environmental factors. Without a clear understanding of the cause, it’s difficult to create an animal model that has good construct validity.
Species Differences: There are substantial differences in brain structure and function between humans and other animals. Furthermore, animals don’t share the same social structures, self-awareness, or language capabilities as humans. These differences can limit the extent to which animal models can replicate human mental disorders.
Translational Challenges: Even when an animal model does show similar behaviors to a human with a mental disorder, it can be challenging to translate these findings to develop effective treatments for humans. Many drugs that were effective in animal models have failed in clinical trials, raising questions about the predictive validity of these models.
Despite these challenges, animal models remain an invaluable tool in psychiatric research, providing insights into the biology of these disorders and potential treatment strategies. They are continually being refined and improved, with newer models incorporating genetic information and environmental factors to better mimic the complexity of human mental disorders.
Give examples of behavioral tests to assess general health and phenotypes related to mental processes.
General health:
breeding
weight
fur/whiskers
homecage behavior: activity, fighting, nesting
reactions to handling
Neuropsychiatric disease:
anxiety tests - sensitive to anxiolytics: open field, elevated plus maze, O-maze
depressive-like behaviors - sensitive to antidepressants:
(an)hedonia: glucose preference, intracranial selfstimulation threshold
despair tests: forced swim test, tail suspension test
learning and memory tasks
spatial navigation and working memory: morris water maze, T-maze
operant tasks: skinner box, continuous performance tasks
social interaction tests: three-chamber sociability and social novelty, tube dominance test
Why do genetic associations hold significant value in understanding causality compared to other association, considering the deterministic nature of genes?
Genetic associations are indeed a powerful tool for understanding causality in biology and medicine for several reasons:
Stability of Genetic Material: Genetic material, unlike other biological variables, remains relatively constant throughout an individual’s lifetime. Environment, behavior, and other biological factors can change, making it difficult to establish their role in disease onset. In contrast, the genome of an individual is established at conception and remains relatively stable, allowing for clear causal inferences.
Mendelian Randomization: This is a method used in genetics to infer causal relationships between risk factors and outcomes. It uses the fact that genetic variants are randomly allocated at conception and therefore can serve as proxies for potential risk factors to establish causality. This method can overcome problems of confounding and reverse causality that often affect observational studies.
Population Genetics: Genetic associations can be studied across populations, which can help control for confounding factors. For example, by comparing disease rates in people with and without a particular genetic variant within the same population, researchers can reduce the influence of environmental factors that might be different between populations.
Functional Insights: Genetic studies can help to unravel the underlying biology of a disease. By identifying genetic variants associated with a disease, researchers can pinpoint the genes and biological pathways involved. This can lead to the development of new therapeutic targets.
Polygenic Risk Scores: These scores are a sum of risk factors across many genetic variants for an individual. These scores can be used to quantify an individual’s genetic risk for a disease and are increasingly being used in personalized medicine.
However, while genetics provides a powerful tool for understanding causality, it’s important to remember that most diseases are not solely determined by genetics. Environmental factors and lifestyle play a crucial role in many diseases, and their interactions with genetic factors (gene-environment interactions) are often key to understanding disease risk and progression. Furthermore, genetics itself is a complex field with phenomena such as epigenetics (changes in gene expression without changes in DNA sequence) adding another layer of complexity.
- Biosensors based on GPCRs
How does GPCR-based biosensor work?
What other techniques can measure neurochemical in vivo?
What are the main advantages of GPCR- based biosensors?
What are the main weakness of GPCR-based biosensors
- Biosensors based on GPCRs
How does GPCR-based biosensor work?
G protein-coupled receptors (GPCRs) are a large family of cell membrane proteins that play a crucial role in cellular signaling. GPCRs interact with a wide range of molecules outside the cell and trigger a specific response inside the cell. GPCR-based biosensors take advantage of this signaling mechanism to detect specific molecules or cellular events.
Here’s a basic outline of how a GPCR-based biosensor might work:
Sensing Element: The biosensor is designed so that the GPCR interacts with the target molecule or event. This could be a ligand that binds to the GPCR, or a change in the cellular environment that modifies the GPCR’s activity. For instance, in some biosensors, the GPCR might be engineered to respond to a specific drug or hormone.
Signal Transduction: Once the GPCR interacts with the target, it undergoes a conformational change, triggering the activation of G proteins inside the cell. The activated G proteins then trigger a cascade of intracellular events.
Signal Amplification: The G protein signaling cascade can lead to the activation of many downstream molecules, effectively amplifying the signal from the original GPCR-target interaction.
Signal Detection: The signaling cascade leads to a detectable change inside the cell. This could be a change in the concentration of a certain molecule, the activation of a specific gene, or a change in cellular behavior. This change can be detected and quantified, providing information about the presence or concentration of the target molecule, or the occurrence of the target event.
For example, some GPCR-based biosensors are designed to produce a fluorescent signal in response to the target. The GPCR might activate a signaling pathway that leads to the production of a fluorescent protein, or the GPCR itself might be tagged with a fluorescent protein that changes its fluorescence properties when the GPCR is activated.
Such GPCR-based biosensors have a wide range of potential applications, from drug discovery to environmental monitoring. They offer a highly sensitive and specific method for detecting target molecules or cellular events.
What other techniques can measure neurochemical in vivo?
There are several methods that can be used to measure neurochemicals in vivo (i.e., within a living organism). Here are a few key ones:
Microdialysis: This technique involves implanting a small probe into the brain. The probe has a semi-permeable membrane that allows small molecules, like neurochemicals, to diffuse into the probe. The probe is continually perfused with a solution, which collects the neurochemicals and carries them out of the brain for analysis.
Fast-Scan Cyclic Voltammetry (FSCV): This is an electrochemical technique that can be used to measure neurotransmitter levels in real time. A small electrode is inserted into the brain, and rapid voltage changes are applied to the electrode. The resulting currents are measured and used to identify and quantify neurotransmitters.
Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT): These imaging techniques can be used to measure the distribution and density of specific types of receptors or transporters in the brain, which can provide indirect information about neurochemical levels. They involve injecting a radioactive tracer into the body, which binds to the target of interest in the brain.
What are the main advantages of GPCR-based biosensors?
Many applications – all imaging approaches
–> Cell culture, ex vivo, in vivo
High temporal resolution (millisecond to second) (sensor dependent)
Different sensors can be used for different purposes
Possibilty of genetic specificity
–> Using different promoters
Molecular specificity (use of the endogenous receptor)
–> NB, some receptors can bind multiple ligands
Multisite longterm recordings are feasible
–> Fiber photometry
Population imaging with cellular or even subcellular resolution is possible
–> Microendoscopes and multi-photon microscopy
–> Not possible with microdialysis or FSCV!
What are the main weakness of GPCR-based biosensors
Only relative measurements – Changes, not concentrations
Photobleaching –> Difficult to find optimal light intensity: Good signal + minimal bleaching
Endogenous receptor – Also a target of pharmacological agonists/antagonists
Which sensor is the right one?
Genetically encoded – not suitable for human use
Potential buffering of the studied molecule by the sensor-receptor –> Depends on affinity of the sensor
