PSYC3051 Tutorial Notes.docx

**Methods in Behavioural Neuroscience** ***Systemic Injections*** Involves the injection of a drug into the bloodstream, either directly or indirectly, that will affect the entire organism. - Quick and easy relative to other methods - Usually used for broader research questions - Not appr...

**Methods in Behavioural Neuroscience** ***Systemic Injections*** Involves the injection of a drug into the bloodstream, either directly or indirectly, that will affect the entire organism. - Quick and easy relative to other methods - Usually used for broader research questions - Not appropriate for observing a specific neurological structure Injection method: - Selecting dose - Adjusted to animal wight (mg/kg) - Dose dependent on the drug's dose-response curve and selecting the appropriate dosage to induce the behaviours that need to be tested - Selecting route of administration - Dosages may differ based on how the drug is administered - Some drugs work best when administered through a particular route - Subcutaneous (skin) - Intraperitoneal (abdomen) - Intravenous (vein) -- although more difficult and much better done with other methods of administration ***Lesions*** Involves permanent damage to the chosen brain region. - Injection of a drug (NMDA) into the target which over-excites and kills the neuron. Lesions are done to inactivate a structure for a long period but not when temporal specificity as the lesion is permanent. This is followed by histological analysis post-mortem to confirm the lesioned area. Lesions are done through stereotaxic surgery 1. Animal is anesthetised 2. Placed on stereotax 3. Landmarks on the skull and known coordinates are used to locate brain regions 4. Small hole is drilled, and the drug is injected at a particular depth 5. Rati is stitched and given time to recover (roughly 7 days). 6. After the experiment, post-mortem histological analysis to confirm placement. Consider the number and location of lesions ***Infusions*** Involves stereotaxic surgery to implant a guide cannula above a specific neural target. This allows direct infusion of a drug into the target structure by using a microinfusing syringe pump. Must consider: - Drug dose - Use post-mortem histology to confirm cannula placement Infusions are done to: - Inactivate the structure with a GABA agonist (e.g., muscimol) or infuse receptor agonists/antagonists - Appropriate when want to give a drug and know where it is having its effect (not systemic) - Appropriate when some temporal specificity is important as the drug effect is temporary (i.e., within-subjects design) - Repeated infusion of the drug will cause permanent damage ***Designer receptors activated by designer drugs (DREADDs)*** Allows chemogenetically turn on and off cells in target region. A viral vector contains the DREADD and a fluorescent tag. Using stereotaxic surgery, virus is injected into target structure where designer receptors are then expressed. Injection of CNO then activates these receptors, allowing for excitation or inhibition. - Good temporal specificity - Allows for targeting of specific cell types through genetic promotor sequences or cre-recombinase - More feasible for extended testing than infusions -- much easier to give a systemic injection of CNO than to use the infusion pump. Histological analysis postmortem to check: 1. Expression/injection location; using fluorescence microscopy for Green Fluorescent Protein (GFP) 2. Target cell type: e.g., GABAergic cells ***Optogenetics*** Turning cells on and off using a light. 1. Virus is injected into target structure where opsins are then expressed 2. Guide cannula is implanted during stereotaxic surgery. 3. Optic fibre is then fed through guide cannula which delivers light to target brain region allowing for excitation or inhibition of target cells. Important things to consider: - Choice of virus -- most often AAV serotypes as these do not replicate on their own to affect adjacent cell populations. - Choice of genetic promotor sequence or use cre-recombinase approach. - Rats have better behavioural repertoire compared to mice - Choice of opsin (e.g., Channel rhodopsin to excite cells, Halo rhodopsin to silence cells) - Wait time for virus expression -- most often 3-6 weeks depending on how far the virus needs to propagate - Not all cell types can be specifically targeted using a promotor driven viral vector (e.g., HABA, TH+/DA). In these cases it may be necessary to use a Cre-recombinase approach. - Transgenic animals that express Cre protein in certain cell types or neuronal populations. - The virus is then modified so that the virus is only expressed in these cell types containing Cre. Like DREADDS, histological analysis post mortem to check: 1. Expression/Injection location; using fluorescence microscopy 2. Target cell type: depending on genetic promotor. a. Researchers also do electrophysiological confirmation of the effects of light on electrical signalling. - Optogenetics is similar to DREADSS and can target specific cell types (through promotor sequences) in target brain regions. - Fantastic temporal specificity: can turn on and off within a testing session. ***Methodological controversies*** Some of these manipulations have unintended effects of their own. - E.g., CNO can have unknown consequences from the use of DREADDs - E.g., blue light increases neuronal activity-regulated gene expression in the absence of optogenetic proteins. **Papers** ***Dissociated Role of Thalamic and Cortical Input to the Lateral Amygdala for Consolidation of Long-Term Fear Memory*** Little is known about the neural circuit pathways during post-learning periods for consolidation of memories. As such the authors optogenetically inhibited the thalamic and cortical input into the LA. Found that inhibition of thalamic input impaired consolidation of fear memory for both recent and remote fear memories. For cortical input, inhibition disrupted remote fear memory but not recent memory. Underlines a dissociated role of thalamic and cortical input to the LA during early post-learning periods for consolidation of long-term fear memory. ***Forced swim stress activates rat hippocampal serotonergic neurotransmission involving a corticotropin-releasing hormone receptor-dependent mechanism*** Serotonin is important for adequate coping with stress. Aberrant serotonin function is implicated in a lot of depression and anxiety disorders. Dysregulation of HPA is related to stress-related illnesses. This study looked at the role of serotonin in the hippocampus and the role of the corticotropin-releasing hormone. Rats subjected to a force swim test saw a spike in hippocampal serotonin which depended on activation of CRH receptors. Additionally, administration of CRH led to increases in hippocampal serotonin. CRH-induced serotonin were adrenally independent. This suggests that the hippocampal serotonin system is able to mount CRH receptor-dependent responses to specific stressful situations that surpass the usually observed maximal increases of about 300% of baseline during stress and enhanced vigilance. ***Animal models in psychiatric research: the RDoC system as a new framework for endophenotype-oriented translational neuroscience*** The Research Domain Criteria aims to define psychopathology as phenomena of multilevel neurobiological existence and assigns them to 5 behavioural domains. Authors found that RDoC system uses biological determinism to explain the pathogenesis of distinct psychiatric symptoms and emphasises exploration of endophenotypes but not complex diseases. This allows the evasion of strict disease-to-model correspondence.\ The units of analysis of RDoC serve as a novel matrix for model validation. The general regulation and arousal, positive valence, negative valence, and social interactions behavioural domains of RDoC are good. Cognitive behavioural domain of the RDoC system requires more clarification. - These homologies in the 4 domains justifies the validity, reliability, and translatability of animal models appearing as endophenotypes of the negative and positive affect, social interaction and general regulation and arousal systems' dysfunction. - The recently proposed RDoC diagnostic framework is a new strategy of symptom-based classification. It revives and upgrades the idea of characterizing the basic function of brain rather than the typical pathological traits - The reductionism of the RDoC system makes it an important methodological tool. The RDoC system offers a good platform for the integral study of clinical data and for the critical evaluation of existing animal models based on systematic examination of endophenotypes. To summarise our analysis of the RDoC system, we may conclude that: 1. The RDoC system implies the same mechanisms underlie normal and dysfunctional brain function. The RDoc system also suggests the existence of a psychopathological continuum. 2. The RDoC system does not consider dynamical and developmental aspects of a disease, psychosomatic co-morbidities, paradigms of therapeutic interventions nor interactions between domain and components of Units of Analysis. 3. Four of the 5 RDoC domains correspond to The Affective Neuroscience Scale. A homology of the affective systems in humans and animals supports experimental models of the emotion and defence response dysfunction. However, the high translational value of these models is limited by the dissimilarities between developmental programs and psycho-social contexts of human disorders. 4. Nonetheless, the RDoC system offers a valid and instructive framework for basic research allowing one to focus on domains and their constructs and to explore endophenotype-based models. This avoids a major challenge of translational studies, which assume a strict disease-to-model correspondence. - Reporting effect size estimation and Bayes factors instead of p-values - Gives more quantitative information than p-values which merely indicates significance - Convergent evidence from cross-species studies provide more solid ground for replication. - Instead of replicating word for word, identifying convergent evidence from different species can be just as if not more effective in demonstrating replication - Preregistration avoids P-hacking and HARKing - The ARRIVE guidelines lead to a comprehensible methodology - ARRIVE (Animal Research Reporting of In Vivo Experiments) guidelines is a well-established instrument for transparency in methodology - Systematic reviews and meta-analyses lead to generalised conclusions - Multi-site preclinical confirmatory trials as a new module for translating animal findings to a heterogenous human population - Use multi-site preclinical confirmatory trials for better prediction and translating of animal findings to contemporary clinical situations. - Publishing negative results to counteract the publication bias for positive results - Open science for scientific data and publications ***The use of chemogenetics in behavioural neuroscience: receptor variants, targeting approaches and caveats*** Chemogenetics is the reversible remote control of cell populations and neural circuitry via systematic injection or micro infusion of an activating ligand. Utilises engineered receptors and biologically inert ligands called designer drugs. - Compared with optogenetics - No need for optical fibre probes and tethers = non-invasive technique - Lower temporal resolution - Necessary conditions - The modified receptor must not be receptive to any endogenous ligand - The modified receptor needs to have minimal or no endogenous activity in the absence of ligand binding - The modified receptor must have a high affinity for the lignad that has no pharmacological activity at other endogenous receptrs. - Types of chemogenetic receptors - Muscarinic-based DREADDs - K-opioid-based receptor DREADD (KORD) - Other This review is focused on three key areas to explore in chemogenetics - Different strategies that have been used to restrict chemogenetic receptors to defined neuronal populations - The use of a dual virus approach to targeting projection neurons and the effectiveness of different routes of administration of designer drugs. - Potential caveats associated with chemogenetics. Viral methods for chemogenetic receptor expression Strategies to target defined neural populations - Cre System - Cre-dependent viral vectors permit restriction of DREADDs in neurons defined by the expression of specific genetic markers - Cre-recombinase expressing animals are injected with viral vectors but expression of the genetic material is restricted to the neuronal population that expresses the Cre-recombinase. - Many Cre lines now available = greater ability to specifically target DREADD expression to a selective cell type. - Limitation -- there is a risk of tumour causing off-target effects in vivo. - Transgenic mice expressing DREADD receptors Strategies to target neural circuits - Selective manipulation of DREADD-expressing terminals in the target region - Non-selective expression of DREADDs in the projection region using AAV - Intracranial cannulas are implanted above the projection target region and local infusion of CNO given. - A dual-viral vector approach to manipulate neurons defined by their anatomical projections. - Cre-vector is retrograde transport type and is injected into a brain region that has anatomical connectivity with the brain region that receives the Cre-dependent vector. - DREADD expression is limited to neurons that project to the brian region that receives the Cre-dependent vector. - Systemic administration of activating ligand then occurs Strategies to administer designer drugs Caveats of chemogenetics - Collateral projections in circuit-specific manipulation - Non-specific effects of ligands used to activate DREADDs - Effects of chronic administration ***A common limiter circuit for opioid choice and relapse identified in a rodent addiction model*** The experiment's rationale is that no circuits that limit heroin seeking and opioid choice have been identified. The study looked at how the IL to Nashell pathway drives heroin choice and relapse. Inactivation of the IL shows differential effects on opioid choice versus relapse but however the IL-Nashell pathway itself is a common limiter of opioid choice and relapse. - Subjects - 2 groups: behavioural economics assay and IL-Nashell pathway inhibition, IL inactivation - Chemogenetics and DREADDS was used to inhibit the neural pathways - Training - Two levers one for heroin and one for food and they are trained to self-administer each lever separately - Choice procedure - Both levers extended and the rats are given a choice. - Cued relapse tests - Relapse tests conducted during acute withdrawal. Levers presented simultaneously (IL inactivation study) or in an alternating fashion (IL-Nashell study) Results: - Rats will work harder for heroin than food and heroin is considered more essential at higher prices. As prices increase (increased number of lever presses), responding for food drops more than heroin. However, rats will consume more food than heroin when its free (no lever presses). - Motivation for heroin is significantly higher than motivation for food. - Relatively even choice preference for heroin vs food, relapse rates are also similar. Motivational indices correlate with intake and relapse, but not preferred choice. Motivation for heroin correlates with relapse, but motivation for food does not. - Choice and relapse are distinct behavioural constructs: relapse tied to motivational factors, while choice may be more cognitive - Inhibiting the IL-Nashell pathway increases heroin choice and relapse but not food relapse. Stimulating the pathway does not impact heroin choice or heroin or food relapse. - IL-Nashell pathway responsible in modulating relapse only for opioids. - Differences may be due to structural features of dendritic spines which differ for heroin and food features - Inactivating the IL cortex increases heroin choice but also decreases heroin relapse but not food relapse ***Harris et al. (2019) The Partial Reinforcement Extinction Effect Depends on Learning About Nonreinforced Trials Rather Than Reinforcement Rate*** Partial Reinforcement Extinction Effect (PREE) is related to how responding extinguishes more slowly following conditioning on partial reinforcement schedule compared to a continuous reinforcement schedule. This effect has been observed in both Pavlovian and Instrumental conditioning contexts. - Partial reinforcement leads to: - Slower extinction generally - Higher responding than continuous reinforcement for instrumental responses - Lower responding than continuous reinforcement for conditioning. - Sequential account -- idea that animals learn about a sequence of trials as a single reinforced unit. The preceding non-reinforced trials and the reinforced trial are viewed as one - Trial-based account: notion that rate of extinction relates to detecting a change in the per-trial probability of reinforcement - Time-Based account: notion that extinction relates to detecting a change in the rate of reinforcement (defined with respect to time, not trials) - Results are consistent with the sequential account of PREE, which states that slower extinction depends on learning about nonreinforced trials during partial reinforcement and is not because of any difference in per-trial probability of reinforcement.