lecture 18 - Using animal models to investigate genetic effects on behaviour Flashcards

Question

new genetic approaches

Answer 1

‘Optogenetics’ (Gero Miesenbock, University of Oxford, Karl Deisseroth, Stanford University) its a technique where you can selectively activate a bit of the brain that you're interested in and look at how that impacts upon behaviour. to do this you take a virus and stick a gene in it that encodes a receptor that responds to light - a gene from a particular algae. you then insert that virus and that gene into the brain regions and the neurons that you are particularly interested in. you then implant a cannula into the brain of your animal switch on a light which is then detected by the receptors in the particular brain region of interest and that then activates that specific brain circuity. you could then turn the light off and the activity on that Brains circitury goes back to normal. by using this technique by switching the light on and off you can activate and deactivate particular circuits within the brain and you can look at what activation of those particular circuits does to the animals behaviour

Answer 2

these mice tended to groom themselves compulsively. stroked their faces till they wiped all the fur of their faces and the researchers identified that this behavioural phenotype was due to hypo activity under activity of cortical striatal and circuitry using ontogenetic techniques they inserted this virus and algae gene into the skull of straital circuity of these mice, they implanted the light they switched the lights on activated the cortical striatal circuitry and show that by activating the circuity they restored function to normal levels and these mice could reduce levels of compulsive grooming optogenetics might be a therapeutic strategy for use in humans potentially and people with OCD who have hypoactive cortical striatal regions but theres issues with it in humans in terms of the human brain being much bigger than that of a mouse so its difficult for light to permeate through it so there lots of work now being done to try to develop a technique which gets around these problems.

Answer 3

* DNA encoding designer G-protein coupled receptors that do not respond to endogenous chemicals inserted into specific neurons/circuits * Drugs binding to these receptors introduced into the animal to modulate (turn up, turn down) the activity of the circuit → assess resultant effects on behaviour Ferguson et al. (2013) Direct-pathway striatal neurons regulate the retention of decision-making strategies J Neurosci 33(28): 11668-76

Answer 4

* Animal models offer greater experimental control and amenability than human studies for examining genetic effects on behaviour * There are a number of commonly used genetic animal models, each with its own set of advantages and disadvantages * Alterations to the genomic DNA sequence can lead to absence of gene expression (‘knockout’), altered gene function (‘knockin’) or increased gene expression (‘transgenesis’); these changes can be made to occur at selected timepoints/in selected tissues (‘conditional’ approaches) * Brain gene expression can also be altered in a specific manner by introducing small genetic sequences that impair transcription and/or translation * New genetic techniques (e.g. CRISPR, optogenetics and DREADDs) are revolutionising our knowledge about the genetic substrates and neural circuitry underlying particular behaviours

Answer 5

‘Discuss how research in animals might be used to develop new genetic therapies’

Answer 6

1. Introduction * Stress is a major risk factor for mental health disorders. * Chronic stress affects brain function, particularly in areas linked to emotion and cognition. * Understanding stress mechanisms can help develop better treatments. 2. Stress and the Brain * Chronic stress impacts key brain regions: ○ Hippocampus: Involved in memory; stress reduces its size and function. ○ Amygdala: Controls emotions; stress increases its activity, leading to heightened fear and anxiety. ○ Prefrontal Cortex: Regulates emotions and decision-making; stress impairs its function. * Stress alters neurotransmitters like serotonin, dopamine, and glutamate, which are critical for mood regulation. 3. The Role of Cortisol and the HPA Axis * The Hypothalamic-Pituitary-Adrenal (HPA) Axis controls stress responses by releasing cortisol. * Chronic stress leads to dysregulation of cortisol, which can contribute to anxiety and depression. * High cortisol levels damage brain structures and disrupt neural communication. 4. Early-Life Stress and Long-Term Effects * Childhood trauma and stress increase the risk of mental disorders in adulthood. * Early stress exposure alters brain development, making individuals more vulnerable to stress later in life. * Epigenetics plays a role in how stress effects can be passed down through generations. 5. Genetic and Environmental Interactions * Individual genetic differences affect stress responses. * Environmental factors like social support can buffer stress effects. * Gene-environment interactions determine resilience or susceptibility to stress-related disorders. 6. Stress and Mental Health Disorders * Chronic stress is a major contributor to: ○ Depression: Linked to reduced hippocampal volume and serotonin dysfunction. ○ Anxiety Disorders: Increased amygdala activation and heightened fear responses. ○ Post-Traumatic Stress Disorder (PTSD): Impaired memory processing and exaggerated fear responses. 7. Coping Strategies and Treatments * Pharmacological Approaches: ○ Antidepressants (SSRIs) help regulate serotonin and reduce stress effects. ○ Cortisol blockers are being researched as potential treatments. * Behavioral and Psychological Interventions: ○ Cognitive Behavioral Therapy (CBT) helps reframe negative thought patterns. ○ Mindfulness and meditation reduce stress and improve emotional regulation. ○ Exercise promotes neuroplasticity and reduces stress hormones. * Social Support and Lifestyle Changes: ○ Strong social connections help buffer stress. ○ Proper sleep, nutrition, and relaxation techniques are key for stress management. 8. Conclusion * Stress plays a central role in mental health disorders through its effects on the brain and body. * Understanding these mechanisms can improve prevention and treatment strategies. Future research should focus on personalized approaches based on genetics, environment, and lifestyle factors.

Answer 7

Gene editing tools are crucial for understanding brain function and neuro disorders. Recent advancements, particularly CRISPR/Cas9 and anti-sense nucleotide-based techniques, allow for efficient genome modifications in the adult nervous system. CRISPR/Cas9 is highly versatile, enabling gene knockouts, mutations, expression control, and epigenetic modifications, even targeting multiple genes simultaneously. Though its advanced applications in neuroscience are still developing, the technology is widely accessible. Anti-sense nucleotide-based methods offer a simpler, rapid way to knock down genes with minimal expertise. This article explores these tools and their potential impact on neuroscience research Genes play a crucial role in human behavior, and dysfunctions in gene expression contribute to various neurological and neuropsychiatric disorders. Understanding how genes influence behavior is essential for both basic neuroscience and developing new treatments. Since gene regulation varies across brain regions, gene editing must be precise in both timing and location. To address these challenges, advanced gene editing tools have been developed. This article highlights CRISPR/Cas9 for its flexibility and ease of use, as well as anti-sense oligonucleotides for their accessibility and simplicity in gene knockdown applications in neuroscience research THE EVOLUTION OF GENE EDITING TOOLS The evolution of gene editing tools has progressed from pharmacological approaches to advanced genetic manipulation techniques. Early methods used drugs to modulate protein activity, but they lacked specificity. The development of transgenic knockout mice allowed for targeted gene manipulation, though early methods lacked temporal control. The Cre-LoxP system improved tissue specificity, and later inducible Cre systems allowed for precise timing of gene deletion. Viral delivery methods further enhanced control but required additional technical steps. RNA interference (RNAi) techniques, such as shRNA, enabled gene knockdown in wild-type rodents without transgenic models, though they required careful design to avoid off-target effects. Despite these advancements, gene editing tools remained time-intensive and complex. Newer technologies like CRISPR/Cas9 offer a highly flexible, user-friendly approach, allowing multiple genes to be studied simultaneously. Additionally, anti-sense nucleotides (ASOs and Morpholinos) provide a simpler alternative by directly modulating gene expression in the brain without the need for viral delivery or transgenic mice. These innovations represent the next generation of gene editing tools in neuroscience. USING CRISPR/Cas9 FOR GENE MODULATION IN THE BRAIN CRISPR/Cas9 has revolutionized gene editing due to its simplicity, flexibility, and efficiency. Unlike RNA interference (RNAi), which is time-consuming and often requires multiple iterations, CRISPR/Cas9 reliably produces gene knockouts and allows for rapid switching between targets, even enabling multiple genes to be edited simultaneously. It is user-friendly, requiring minimal specialized knowledge, and free resources are available for designing targeting sequences. Originally discovered as a bacterial immune defense, CRISPR/Cas9 uses an RNA-guided Cas9 protein to cut DNA at specific sites. Scientists have adapted this system to target and modify genes, introducing double-stranded DNA breaks that facilitate gene knockouts or knock-ins. This makes CRISPR/Cas9 a powerful tool for gene modulation in neuroscience and other fields. Using CRISPR/Cas9 for Gene Knockout CRISPR/Cas9 enables efficient and straightforward gene knockout by creating targeted double-stranded DNA breaks using a single guide RNA (sgRNA) and the Cas9 enzyme. These breaks are repaired through non-homologous end joining (NHEJ), leading to mutations that disrupt gene function. This method is simpler and more efficient than previous techniques, allowing for easy gene targeting and the simultaneous knockout of multiple genes. Studies have demonstrated CRISPR/Cas9’s effectiveness in the brain. For example, Swiech et al. (2015) successfully knocked out the Mecp2 gene in mice, resulting in impaired memory, highlighting the system's potential for behavioral research. They also used CRISPR/Cas9 to target three DNA methyltransferase genes (Dnmt1, Dnmt3a, and Dnmt3b) simultaneously, showing that multi-gene knockouts can reveal complex genetic interactions affecting brain function. However, multi-gene editing efficiency decreased with additional sgRNAs, suggesting potential challenges in targeting multiple genes at once. Despite this, CRISPR/Cas9 remains a powerful tool for neuroscience, offering high flexibility and the potential for large-scale genetic studies in the brain. Use of Cas9 for Knock-in Mutations CRISPR/Cas9 can also introduce knock-in mutations, which are useful for studying disease-related genetic changes. This is typically achieved through the homology-dependent repair (HDR) pathway, which repairs double-stranded DNA (dsDNA) breaks by using a homologous DNA template. However, HDR is inefficient in neurons, making its application in neuroscience challenging. Alternative methods are being explored, such as using the non-homologous end joining (NHEJ) pathway, which is active in neurons, and Cpf1, a Cas9-like enzyme that produces “sticky ends” to facilitate NHEJ-based knock-ins. These methods could allow for the introduction of disease-specific mutations or regulatory sequences like loxP sites directly into the brain. Beyond direct gene editing, CRISPR/Cas9 can also modulate gene expression without cutting DNA. This is achieved using a modified, nuclease-inactive dCas9, which acts as a scaffold to recruit other enzymes that regulate gene activity. Two strategies exist: one modifies dCas9 itself, while the other modifies the single guide RNA (sgRNA), both enabling precise control over gene expression without altering the genome. Using sgRNA/Cas9 as a Scaffold to Inhibit or Activate Genes CRISPR/Cas9 can be used as a scaffold to modify gene expression without altering the genome. By modifying the single guide RNA (sgRNA), transcriptional activators or repressors can be recruited to specific gene regions, such as promoters or enhancers, to either activate or inhibit gene activity. This can be done by fusing transcriptional regulators like HSF1 to the MS2 coat protein (MCP) or by using other RNA-binding proteins. An advantage of this method is the ability to multiplex and regulate multiple genes simultaneously, turning some genes on and others off within the same model. This is done by engineering sgRNAs with different stem loops that recruit specific proteins for activation or repression. This strategy is especially useful for studying neural behaviors by modulating gene expression in the brain. Additionally, this approach allows for position-specific epigenetic modifications, such as histone acetylation, at targeted gene regions, providing new insights into gene regulation and its relationship to behavior. While the use of CRISPRi (repression) and CRISPRa (activation) in the brain has not been fully demonstrated, there is potential for these methods to be applied in the CNS, especially when combined with light-activation techniques for temporal control. However, the main challenge lies in delivering the necessary constructs into the brain using viral vectors. OFF-TARGET EFFECTS OF CRISPR/Cas9 CRISPR/Cas9 can sometimes cause off-target effects, where the Cas9–sgRNA complex binds to sites in the genome that are similar but not identical to the intended target. The frequency of off-target binding varies depending on the guide sequence. To reduce off-target effects, strategies include: 1. Optimizing sgRNAs to minimize homology with other genomic sites. 2. Shortening sgRNA sequences (e.g., from 20 to 17–19 base pairs) to reduce off-target activity. 3. Modifying Cas9 (e.g., using eCas9) to decrease off-target activity. 4. Verifying results by using multiple sgRNAs targeting the same gene to confirm the results and minimize errors. 5. Paired nickases or FokI: This method requires two sgRNAs targeting different DNA strands to induce a double-stranded break (DSB), reducing off-target effects. FokI fuses to dCas9 (a mutated form of Cas9 without nuclease activity), and only when both sgRNAs bind close together will FokI dimerize to cut the DNA. While these methods reduce off-target effects, they can complicate experiments, especially for in vivo brain editing, as they may require additional vectors or viral delivery systems. DELIVERING CRISPR–Cas9 INTO THE BRAIN The application of CRISPR/Cas9 in neuroscience has been slow, partly due to the challenge of delivering the large Cas9 protein into the brain. Cas9's size often exceeds the packaging limit of AAV vectors, which typically range from 4 to 5 kb. However, there are several strategies to overcome this: 1. Cas9 Transgenic Mice: A mouse model with Cas9 knocked into the ROSA26 locus allows viral vectors to deliver sgRNAs and other proteins. This method eliminates packaging issues but requires a dedicated mouse line, limiting flexibility. 2. Multiple AAVs: One AAV delivers Cas9, while another delivers sgRNA and other proteins. This approach has been effective, with high co-transduction rates, and allows for multiplexing gene knockouts. Larger packaging viruses like HSV can be used to overcome size limitations. 3. Smaller Cas9 Homologs: Cas9 from Streptococcus aureus is smaller than the commonly used Streptococcus pyogenes Cas9, allowing it to be packaged with sgRNA and GFP into a single AAV. These strategies, along with ongoing innovations, are making CRISPR/Cas9 more feasible for in vivo use in neuroscience. Although advanced methods like CRISPRi, CRISPRa, and epigenetic modulation have not been widely tested in vivo, they are poised to transform brain gene editing and neuroepigenetics research. Disrupting gene expression in the brain without using viral vectors or mouse models is possible through anti-sense oligonucleotides (ASOs), which are short DNA sequences that bind to target mRNA, leading to its degradation or translational inhibition. ASOs are advantageous because they can be easily administered in vivo without the need for viral vectors or transgenic animals. However, the use of ASOs in vivo faces challenges, primarily due to their susceptibility to degradation by nucleases. To address this, researchers have developed modified ASOs with more stable backbones, such as phosphorothioate bonds, which improve their stability but may require higher doses and can lead to cytotoxicity. Another modification involves using ribose in the sugar ring, improving binding affinity and protecting ASOs from degradation. Some ASOs also use exon skipping, which can produce functionally inert proteins or restore gene function, providing potential therapeutic benefits, such as in the treatment of spinal muscular atrophy (SMA) by restoring function to the Smn2 gene. While ASOs are primarily used for gene knockdown, their potential to restore gene activity in specific contexts is also being explored, offering new possibilities for gene therapy and neuroscience research. Using Locked Nucleotides for Gene Knockdown Locked nucleic acids (LNAs) are a modification of antisense oligonucleotides (ASOs) that enhance binding affinity and knockdown efficiency by "locking" the ribose backbone into a stable structure. This modification also protects the oligos from degradation and allows them to cross the blood-brain barrier. However, LNAs face limitations, including the inability to recruit RNase H for degradation and potential toxic effects and off-target actions, which hinder their widespread use in vivo. To address these issues, a new generation of ASOs, called "gapmers," was developed. Gapmers combine LNA-modified regions at the ends with unmodified DNA in the middle, allowing RNase H-mediated degradation of the mRNA while benefiting from the stability and improved affinity of the LNA modifications. Gapmers have shown promise in vivo, successfully reducing gene expression in diseases like Huntington's disease and showing long-lasting effects with minimal off-target impacts. Despite their potential, gapmers require caution due to the risk of toxic side effects, especially at high doses. Advances in ASO design are needed to minimize these risks and maximize their effectiveness in neuroscience research. Overall, LNA-based gapmers are a powerful tool for gene knockdown in the brain and offer great promise for future therapeutic applications. USE OF MORPHOLINOS TO REGULATE GENE EXPRESSION IN THE BRAIN Morpholinos (PMOs) are synthetic oligonucleotides with a unique backbone that makes them resistant to nuclease degradation, allowing them to remain active for extended periods. Unlike ASOs, PMOs do not recruit RNase H, but they can still achieve gene knockdown by interfering with splicing, translation, or miRNA maturation. PMOs are unable to cross cell membranes unassisted, limiting their general use. However, modifications like vivo-Morpholinos, which link PMOs to dendrimers or cell-penetrating peptides, improve their cell permeability and make them more effective in vivo, especially when directly injected into the brain. vivo-Morpholinos have shown significant promise in gene suppression in the brain with minimal cytotoxicity, making them a valuable tool for gene editing in brain research. These modifications, along with gapmer-based ASOs, offer direct application to target areas without the need for viral vectors or mouse models, providing a powerful approach to gene regulation. In conclusion, recent gene-editing approaches like CRISPR/Cas9 and anti-sense mRNA inhibition are transforming neuroscience research by offering flexible, effective, and accessible tools for studying gene function in the brain. These methods allow for targeted gene disruption and rapid knockdown, providing essential insights into gene-behavior relationships.

lecture 18 - Using animal models to investigate genetic effects on behaviour Flashcards

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