LandMunit3 PDF - Learning & Memory

10/11/2024 LTP infers that learning is taking place - LTP experiments are somewhat artificial o LTP induced by a low intensity, high frequency electrical event that bypasses sensory input o Hippocampal slice is a model system that is 1 step removed fr...

10/11/2024 LTP infers that learning is taking place - LTP experiments are somewhat artificial o LTP induced by a low intensity, high frequency electrical event that bypasses sensory input o Hippocampal slice is a model system that is 1 step removed from the actual brain - Long-term changes at individual synapses that are associated with LTP do not constitute the full memory o Memories are the product of behaving organisms interacting with their environment o Memory trac (engram) = the physical basis of memory - Observing behavior to infer learning and memory o Experimental investigations of memory require: ▪ Training phase – learning takes place (thru experience) and establishes a memory ▪ Testing phase – attempting to detect the memory (thru behavioral change) - Behavioral change is influenced by more than just learning and memory o Many different processes interact to produce a behavior o Experimental methods must address alternative explanations for a behavior change o - Mouse model: behaviorally train mice to navigate through a difficult maze o Experiment ▪ Test/quantify the time it takes for the mouse to navigate thru a maze, post- training (test memory) ▪ Lesion brain area, then test/quantify the time it takes the mouse to navigate it o Ensure brain area is involved in spatial memory & not another variable/process ▪ Sensory impairments (loss of cues)  ▪ Motor impairments ▪ Motivation - Experimenters must demonstrate that the brain manipulation influenced behvaior by selectively influencing the memory component - Dimensions of a memory trace o A memory trace can be characterized by two dimensions ▪ Decay rate ▪ Vulnerability to disruption o There are at least two memory traces ▪ Short-term memory (STM) trace Decays rapidly Active state ▪ Long-term memory (LTM) trace Decays much more slowly In inactive state - A short-term memory can become a long-term memory when the memory trace progresses from an active to an inactive state - Memory traces that are inactive are less susceptible to disruption o IF brain trauma occurs when a memory (STM) is still active, when that memory is more prone to disruption - Memory Consolidation = memory trace becomes more stable and resistant to disruption with time o Originally proposed ~ 1900 psychologist; different from LTP consolidation - Is memory disruption due to a storage failure? o Amnesia due to disruptions in processes that store the memory - Is memory disruption due to a retrieval failure? o Memory is stored but can’t be accessed processes that enable us to retrieve a memory are disrupted o Can not be proven that a memory will not recover with time ▪ Can question: how much time to allow before concluding storage failure/no retrieval - Behavioral Tests for Studying Memory in Animal Models o Overview: ▪ Training phase ▪ Testing phase ▪ Can vary: # of training phases Duration of training phases Duration between each training phase Time between training & testing o Inhibitory avoidance conditioning o Fear conditioning o Water-escape task o Recognition memory task  - Inhibitory Avoidance conditioning o Procedure: ▪ Training phase: Rodent placed in the bright side of a box When it moves to the dark side, it will receive a shock Rodent removed from the box ▪ Test phase Place rodent on the bright side of a box Measure how long it takes for the rodent to cross to the dark side o Expected results: ▪ If learning occurred rodent will remember it received a shock on the dark side of the box ▪ Behavior: increased response latency(amt of time to cross to the dark side) if learning has occurred ▪ Behavior depends on Strength of electrical shock Number of trials ▪ It is assumed that memory trace is reflectd in the response latency (longer latency = stronger memory trace o Ceiling effect ▪ When a response is at its maximum Performance measure cannot go any higher If a manipulation is thought to strengthen a memory trace, a low intensity shock should be used(to avoid ceiling effect) o Floor effect ▪ When a response is at its minimum ▪ Performance measured is too low to be further reduced ▪ If a manipulation is thought to impair memory processes, a high-intensity shock should be used 10/14/2024 - Behavioral tests for studying memory in animal models o Overview ▪ Training phase ▪ Testing phase ▪ Can vary: # of training phases Duration of training phases Duration between each training phase Time between training and testing o Inhibitory avoidance conditioning o Fear conditioning o Water-escape task o Recognition memory tasks - Fear Conditioning(pvalovian conditioning) o CS = auditory cue or context o US = electrical shock o CR = freezing behavior - Two types: o Auditory-cue fear conditioning ▪ Tone predicts a shock o Contextual fear conditioning ▪ Context predicts a shock - Procedure: o Training phase ▪ Rodent placed in a box ▪ Auditory cue administered ▪ Electrical foot shock administered ▪ Rodent removed from box o Testing phase ▪ Rodent placed in a box ▪ auditory cue administered ▪ Freezing behavior measured - Expected results o Increased freezing behvaior with shock o It is assumed that the memory trace is reflected in the freezing response (greater freezing behavior = stronger memory trace) - Water-escape task: testing spatial learning o AKA Morris Water Maze o Procedure: ▪ Training phase: Hide a platform underneath water Place a rodent in the water until it can find the hidden platform Rodent must use cues from around the room ▪ Testing phase See how long it takes for the rodent to find the platform o Expected results: ▪ If learning has occurred, the rodent would take less time (decreased escape latency) to find the hidden platform  ▪ - Recognition Memory Tasks o Tests Recognition memory o Rodents will spend more time exploring a novel object o Procedure: ▪ Training phase: Explore a context with unique and distinct objects in specific locations ▪ Testing phase: Move the objects and measure how much time an animal explores the novel object ▪ ▪ ▪ Three different types: Object-recognition task Object-place location task  Object-in-context task - Transgenic mice o o DNA with altered genes injected into pronucleus of the fertilized egg ▪ Transfer new genese into the genome ▪ Modify or delete a specific gene ▪ Utilize CRISPR for targeting accuracy o Transgenic mice can be: ▪ Knock-ins ▪ A full knockout ▪ A conditional knockout o o Conditional knockout – aspecific gene is knocked out in specific cells (allows precision)  o ▪ Uses the Cre-loxP system ▪ Two genes are genetically engineered: Gene specific to your cell of interest(e.g. albumin is only expressed in liver cells) has the gene for Cre recombinase attached to its promoter region Gene of interest is surrounded by loxP sites ▪ When Cre recombinase is translated, it interacts with loxP sites and cuts out the gene between the loxP sites - Lesion – damages the brain region, resulting in taking away function o Stereotaxic surgery is used to tareget specific brain areas ▪ Uses coordinate system to target a specific brain region ▪ Can implant an electrode to damage a brain region with electrical stimulation ▪ Can implant a cannula to inject chemicals/drugs into a particular brain region ▪ - Viral vector methodology o Use of viruses to deliver new genetic material into specific cells o Genetic construct is made that contains ▪ Gene of interest (GOI) ▪ Promoter region to initiate expression of GOI o Genetic construct inserted into virus o Virus is injected into targeted neurons via stereotaxic surgery  - Optogenetics o Using light (“opto”) to excite or inhibit neurons with extreme temporal precision (ms) ▪ Target specific cells o Opsins = proteins that respond to light ▪ Channelrhodopsin = blue light opens a Na+ channel -> neurons are activated ▪ Halorhodopsin = green light activates Cl- pump -> neurons are inhibited - Genetic engineering with DREADD o DREADD = designer receptor exclusively activated by designer drugs ▪ Viral vectors delivers delivers a novel receptor to specific neurons of interest ▪ Novel receptor only respond to synthetic drugs (e.g. clozapine N-oxide (CNO)) Novel receptors can be inhibitory or excitatory ▪ Takes ~1 hour for the synthetic ddrug to have effect 10/16/2024 - Recall: what are memories? o Sensory inputs into a distributed set of weakly connected (dashed lines) collection of cell assemblies change the strength of connections among the neurons (solid lines) in the assemblies - CELLS THAT FIRE TOGETHER WIRE TOGETHER o Memories are the changes in the connectivity among a collection of neurons responding to a particular experience o Thus, memories are distributed throughout the neural systems engaged in a particular experience that produces a memory o Content of memories is determined by specific sets of cell assemblies activated by the memory-producing experience - What is important for LTP is important for memory formation o Glutamate binds to NMDA and AMPA receptors o Post-translational processes o Transcription and translation of new protein products - What molecules contribute to the early formation of memories? o NDMARs o AMPARS o CaMKII o Actin dynamics - NMDARs are necessary for LTP generation – are they necessary for memory formation? o Pharmacological approach  o o APV (NMDAR antagonist) administered daily into the ventricles, allowing it to enter the cerebrospinal fluid (CSF) for several days prior to training sessions o When APV is chronically administered into CSF, in vivo could be induced, but not expressed/sustained o o - The formation of a memory trace depend on the activation of NMDA receptors - Functional NMDA receptor channel o Comprised of 4 different subunits o All functional NMDARs have GluN1 o GluN1, GluN2A, and GluN2B - Genetic approach  o Selectively delete the GluN1 subunit in pyramidal cells in the CA1 yields the “CA1 knockout mouse” (CA1KO) o o Stimulate the perforant path, record fEPSP in dentate gyrus of CA1KO o o Other findings: ▪ Test the CA1KO in a modified water escape task where the platform is visible ▪ CA1KO mice are able to learn 10/21/2024 - NMDARs o NMDA receptors are comprised of 4 different subunits o All functional NMDARs have GluN1 ▪ Other subunits are GluN2A, GluN2B - Genetic approach: anatomy of the NMDA receptor o GluN2B subunits open for longer, letting more Ca2+ into the dendritic spine o So if there are more GluN2B subunits, then it should be easier to induce LTP o Genetic Approach: overexpression of GluN2B NMDAR complexes enhances LTP & memory  o - Recall: fear conditioning - - - - Enhanced contextual and auditory-cue fear conditioning - Also improved object recognition o Increasing GluN2B-contaiining NMDAR complexes enhances learning and memory - Are NMDARs necessary for memory formation? – Caveats and conflicting results o Some studies have reported learning despite NMDAR blockade o Some have suggested sensory or motor deficits from NMDAR blockade o Previous experience can modify the dependency of learning on NMDARs o There may be redundant mechanisms that support memory in the absence of the NMDAR ▪ Other Ca2+ sources o Mossy fibers -> CA3 pyramidal neurons does not need NMDARs to induce LTP - AMPAR involvement in in early formation of neurons – insertion of AMPAR into the PSD - Pharmacological Approach: AMPARs in Memory Formation & Retrieval o Perirhinal cortex = necessary for object-recognition memory task o CNQX = AMPAR antagonist ▪ Injected into the perirhinal cortex o  o o AMPARs are involved in both the formation & retrieval of memories - Role of AMPARs in fear conditioning o Fear conditioning drives GluA1-containing AMPARs into spines within the basolateral amygdala (BLA), a brain region important in fear conditioning - - - A behavioral experience that produces fear conditioning drives AMPARs into the synapse - What would happen if you prevent the trafficking of AMPARs into spines? - Genetic approach: Preventing AMPARs from being trafficked will weaken fear memory o Create & inject a nonfunctional version of GluA1 subunit(“dummy receptor”) to compete with functional receptors o Rats are injected with a viral vector of “dummy receptors” or control into their basolateral amygdala o Rats undergo fear conditioning  o o - Summary of the role of AMPARs in fear conditioning o Fear-conditioning drives GluA1-containing AMPARs into spines within BLA o Blocking functional AMPArs from being trafficked into the spine decrease fear memory and LTP - AMPAkines and cognitive enhancement o AMPAkines keep AMPA channels open for longer -> enhanced depolarization o AMPAkines enhance rate of fear conditioning o Are allosteric modulators that allow for more sodium into the cell - - NMDARs are critical for acquisition of memory, but not for its retrieval - AMPARs are important for both acquisition and retrieval of memories. 10/23/2024 - Testing the role of NMDARs & AMPARs in memory acquisition and memory retrieval - One-trial memory task o o Acquisition Phase: ▪ Uncovered sand well 1 contains a distinct flavored food pellet(flavor 1) underneath the sand ▪ Uncovered sand well 2 contains another distinct flavored food pellet (flavor2) underneath the sand o Retrieval Phase:  ▪ ▪ Rat is given flavor 1 in the “start box” ▪ Rat will only get a reward if it digs in sand well 1 (corresponding to flavor 1) o Experiment 1: Administer APV (NMDAR antagonist) v. vehicle o Experiment 2: Administer CNQX (AMPAR antagonist)v. vehicle o o AMPA and NMDA receptors are necessary for memory acquisition o AMPAR are required for memory recall - CaMKII o CaMKII is necessary for LTP generation and stabilization o CaMKII is different from PKM-zeta; it has an autoinhibitory-regulatory domain ▪ Ca2+ calmodulin can bind to this regulatory domain, changing its conformation o CaMKII can autophosphorylate itself ▪ This happens at the Thr286 residue ▪ This makes the kinase “autonomously active” (i.e the enzyme is in an always active state, even in the absence of Ca2+-calmodulin) o Genetic Approach: ▪ CaMKII knockout mice (CaMKII KO) LTP deficient  Never learn the location of the hidden platform in the place-learning water-escape task Don’t spend more time in target quadrant on probe trials Eventually learn to swim to the visible platform water-escape task ▪ Critique: behavioral results don’t rule out the possibility that there may be sensory/motor deficits associated with the knockout o - CaMKII: role in memory formation o Genetic approach: Engineer mice with a CaMKII protein that cannot be autophosphorylated (T286A mutation mice) ▪ LTP deficient ▪ o Behaviorally test them on: ▪ Inhibitory avoidance learning task ▪ Auditory-cue fear conditioning task ▪ Contextual fear conditioning task  o o With 1-3 trials, T286A mice have learning deficits in fear conditioning o But with more trials, T286A mice learn to associate the auditory cue & the context with shock o Suggests the autophosphorylation of CaMKII is important for rapid memory formation o Suggests that compensatory mechanisms exist when multiple training trials occur - Rats were exposed to fear conditioning, in which a tone was paired with a shock o Fear conditioning resulted in increased phosphorylated CaMKII in the dendritic spines of the BLA o o Pharmacological blockade of CaMKII phosphorylation also blocks the acquisition of both contextual & auditory-cue fear conditioning o - Actin dynamics o Recall: inhibiting actin polymerization causes LTP deficits  o Behavioral experience that results in learning and memory would activate LIMK and increase the phosphorylation of cofilin -> increase in actin polymerization - Object-place recognition task - - Training phase increases ratio of phosphorylated cofilin:total cofilin In the hippocampus - Blocking actin polymerization prevents formation of memory needed to perform object- place location task 10/25/2024 - Which molecules contribute to the early formation of memories? o NMDARs o AMPARs o CaMKII o Actin dynamics - Similarities of LTP generation and early memory formation o Generation – NMDARs activated, AMPARs eventually inserted into the PSD: requires post-translational mods (CaMKII activation and phosphorylation events) o Actin cytoskeleton is reorganized and enlarged: Also requires post-translational mods (cofilin phosphorylation; actin polymerization) - Paper 2: sleep deprivation o Background sleep deprivation ▪ Sleep contributes to memory consolidation ▪ Sleep deprivation impairs memory consolidation ▪ What is the mechanism behind this? o - Consolidation but for long term memory consolidation during sleep o Recall of mTOR in LTP consolidation for local protein synthesis o BDNF binds to TrkB receptor o mTOR is activated  o mTOR inhibits 4E-BP to remove 4E-BP’s inhibitory influence on elongation/initiation factors o this allows for the translation of TOP proteins o ultimately results in local protein synthesis o 4E-BP2 is the most abundant repressor protein in the brain o 4E-BP is a TOP protein o 4E-BP2 binds elF4E (eukaryotic initiation factor 4E), which prevents elF4E from initiating translation - mTOR forms two different complexes of proteins: o mTORC1 (mTOR complex 1) ▪ sensitive to rapamycin ▪ activation of mTORC1 results in local protein synthesis o mTORC2 (mTOR complex 2) ▪ insensitive to rapamycin ▪ activation of mTORC2 results in regulation of actin polymerization - Paper 2: materials and methods o Overall experimental timeline: ▪ Stereotaxic ssurgery to deliver viral vector (for genetic manipulation) to hippocampus ▪ Training on the object-place task ▪ Sleep deprivation for 5 hours ▪ Testing-phase o Viral vector ▪ Promoter for CaMKII-alpha = restricts expression to excitatory neurons ▪ Gene inserted – elF4EBP2 gene o Sleep deprivation protocol ▪ Gentle handling for 5 hours o Object-place recognition task ▪ Hippocampal-dependent - Helpful hints o Nascent proteins = newly made proteins o AMPK signaling pathway ▪ Activation of AMPK blocks mTOR o Puromycin was used to tag nascent proteins ▪ Can measure puromycin levels on a western blot o Using GADPH as a reference protein to normalize protein amount in western blots ▪ GADPH = enzyme in glycolysis ▪ Expressed everywhere & used as reference protein to normalize protein levels across biological samples ▪ Such reference proteins can be called “housekeeping proteins/genes” ▪ By normalizing to a reference protein that is considered to be expressed at a constant level across samples, researchers can compare protein expression levels relative to a consistent baseline - Memory consolidation  o Memory traces o - Memory traces that are inactive (& older) are less susceptible to disruption - If brain trauma occurs when memory (STM) is still active, when the memory is more prone to disruption - - - LTP consolidation requires CREB phosphorylation, protein degradation, and mTOR activation - Memory Consolidation = a process that stabilizes the memory and makes it resistant to disruption - Three main principles o Enduring memories require that the bahevioral experience that initiated them also induce translation and transcription  o Consolidation processes occur in waves that can continue for at least 24 hours o These processes are sustained by autoregulatory positive feedback loops that ensure a continuous supply of synaptic mRNAs for local translation - The research paradigm o Variables that can be manipulated: ▪ Independent variable: Pharmacological or genetic manipulation to influence a molecule hypothesized as important for memory ▪ Retention interval = time between the training experience that establishes the memory and the test used to retrieve the memory o  - - A memory is consolidated when it is no longer vulnerable to pharmacological treatments that interfere with the translation and transcription events initiated by the memory- producing behavioral experience 10/28/2024 - Inhibiting Protein Synthesis: experimental protocol o Train rats on an inhibitory avoidance task ▪ Learning requires longer latency to cross into dark side o Infuse anisomycin (drug to block protein synthesis) into the hippocampus of these rats(anisomycin binds to ribosomal complex and inhibits protein synthesis of everything) o Vary: ▪ The time when anisomycin is infused ▪ the retention interval(time between last training session and first test session) o Blocking protein syntethesis broadly prior to training ▪ No effect on STM ▪ Prevents consolidation of lTM (long term memory) o STM does not depend on new protein synthesis, but new protein synthesis is required for memories to endure o When anisomycin is injected into the hippocampus immediately after training: ▪ “The memory trace endures for ~2 days, then it decays” (NO) ▪ Higher retention (relative to acquisition) at 2d – note that there is impairment o o Thus, a few min of protein synthesis following training allows some memory retention for ~2 days, then it decays further o When anisomycin is injected into the hippocampus 1d after training: ▪ The memory trace is present for up to 3 days after training ▪ Memory significantly decayed by day 8 o o When anisomycin is injected into the hippocampus 2 d after training: ▪ Memory consolidation is unaffected  o - A short-term memory can be established without new protein synthesis - It takes 1-2 days for the memory to be fully consolidated o Consolidation is dependent on protein synthesis - Inhibiting protein synthesis: conclusions o At least two waves of protein synthesis are required for a emomory to be fully consolidated ▪ First wave occurs a few minutes following training Allows memories to persist for ~2 days ▪ Second wave occurs ~1 day following training Allows memories to persist for ~7 days - Remember the processes important in LTP consolidation: o First wave: local protein synthesis o Second wave: genomic signaling -> new protein synthesis (transcription and translation) - - First wave – local protein synthesis o BDNF activates TrkB receptor o Activates mTOR o Inactivates 4E-BP (repressor protein) o TOP mRNAs can be translated into translational factors - Hypothesis: BDNF-TrkB-mTOR-TOP pathway is responsible for memories that persist for ~2 days  - Slide 16 clicker Q answer is A – prior to training - - Block mTOR in hippocampus prior to inhibitory avoidance training - Block mTOR in hippocampus a few minutes after inhibitory avoidance training - Local protein synthsis( initiated by mTOR) is responsible for the initial wave or protein synthesis - Unless local protein synthesis occurs, there will be no second wave of protein synthesis 11/1/2024 - Recall: Molecular basis of memory Consolidation o Remember the processes important in LTP consolidation ▪ First wave: local protein synthesis ▪ Second wave: genomic signaling -> new protein synthesis(transcription & translation) o The first wave – Local protein synthesis ▪ BDNF activates TrkB receptor ▪ Activates mTOR ▪ Inactivates 4E-BP (repressor protein) ▪ TOP mRNAs can be translated into translational factors o Conclusions with the rapamycin experiments: ▪ Local protein synthesis (initiated by mTOR) is responsible for the initial wave of protein synthesis ▪ Unless local protein synthesis occurs, there will be no second wave of protein synthesis - If mTOR is critical for memory consolidation then, BDNF/TrkB receptor activation should be as well o Two peaks of BDNF protein expression in the BLA after fear conditioning:  o - What happens if we block the first peak of BDNF protein expression? o TrkB-IgG = nonfunctional TrkB receptors that compete wit endogenous TrkB receptors for BDNF; depletes availability of extracellular BDNF o K252a = TrkB receptor antagonist - - Interfering with BDNF function 30 min before training (block the first wave): o 1 day & 7 day retention impaired (both waves were prevented) o Blocking the first wave of protein synthesis prevents LTM formation - Blocking 9 hours after training - o Interfering with BDNF function at 9 hours after training(block the 2 nd wave): o Only 7 day retention was impaired o Blocking the second wave of protein synthesis prevents maintenance of the LTM - The second wave – genomic signaling o Activation of transcription factors to initiate the synthesis of new mRNA in the nucleus o CREB is phosphorylated by BDNF/TrkB/mTOR pathway o Phosphorylated CREB is a transcription factor o Target CREB in order to prevent the transcription of new mRNAs; this might stop the second wave of protein synthesis - Blocking CREB with antisense oligodeoxynucleotide 6 hours prior to training o - CREB is necessary for LTM - CREB activates C/EBP-beta, another transcription factor - C/EBP-beta increase ~9-12 hours after inhibitory avoidance training - o Is C/EBP-beta responsible for the second wave of BDNF - Inhibiting BDNF decreases phosphorylated CREB - Inhibitory avoidance training initiates BDNF-TrkB-mTOR pathway that results in increased pCREB - Inhibiting C-EBP-beta 5 hours after training using antisense oligodeoxynucleotide o Results in decreased learning - Infusing BDNF at 12 houEprs post-training rescues learning o BDNF infused 2 days post training has no effect o C/EBP-beta is responsible for the second peak of BDNF o BDNF is necessary for the second wave - 1. BDNF/TrkB/mTOR pathway is activated (dendritic spine) 2. CREB is phosphorylated(soma) 3. pCREB induces transcription of C/EBP-beta (soma) 4. C/EBP induces transcription of BDNF(soma) - > 2nd wave of BDNF  - 11/4/2024 - Arousing experiences are memorable - Memory modulation & Arousal framework - - Arousal stimulates the adrenal gland - - Memory Modulation :early experiments o A low dose of strychnin (glycine receptor antagonist) produces a state of arousal that can influence memory consolidation o Inject strychnin immediately after training on a complicated maze ▪ Improved retention performance o Inject strychnine before testing on the maze ▪ No effect on retention performance o There is a brief period of time after the memory-inducing behavioral experience when the strength of the memory trace can be modified - Basolateral amygdala (BLA) o “The great modulator”: where epinephrine acts to modulate the memory o BLA has extensive projections to brain areas that are likely to store memories o o In order for the BLA to be the “great modulator” it must not be a site for storage itself ▪ The memory must be able to be retrieved even when the amygdala is removed  o Place-learning and visible- platform versions of the water escape task (aka morris water maze) can be learned even when the BLA is significantly damaged o Amphetamine, a stimulant is injected into the BLA following training o - Inhibitory avoidance task o Lidocaine (suppresses neural activity) is injected immediately after inhibitory avoidance training o Rats are tested 48 hours later o  o o A o o A - The role of epinephrine in memory retention - o Epinephrine is released from your adrenal gland o Epinephrine cannot cross the blood-brain barrier o How is epinephrine exerting its effect on the BLA?  o Adrenal medulla releases Epi -> Epi activates vagus nerve o Vagus nerve release glutamate onto NTS -> activates NTS neurons o NTS release glutamate onto LC -> activates LC neurons o LC neurons release NE onto BLA -> activates BLA o BLA neurons releases glutamate onto target brain areas o Glutamate induces more protein synthesis o Memories are strengthened - - Using in vivo microdialysis to measure neurotransmitter levels in the brain - Insert microdialysis probe in your brain region of interest - Pump in artificial CSF, collect samples from the extracellular collect samples from the extracellular space - Run samples to measure neurotransmitter concentrations  - - B - Increasing NE in the BLA o Greater concentrations of NE are in the BLA with greater intensity of shock in the inhibitory avoidance task ▪ Greater shock associated o Exploring a novel environment AND shock are both necessary to induce NE release in the BLA - Test NE sufficiency by administering NE directly into the BLA after training - Test NE necessity by administering NE receptor antagonist directly into the BLA after training 11/6/2024 - Memory Modulation o There is a brief period of time after the memory inducing behvaioral experience when the strength of the memory trace can be modified o So why do we perform our experimental manipulations (e.g. applying agonists/antagonists) immediately following behavioral training and not before training o For behavioral testing, we need a baseline of behavioral training to ensure the memory is already being formed o The pharmacological manipulations of epinephrine are affecting “modulation” or possible enhancement/decrement of the memory - Administering NE in the BLA immediately following training enhances memory retention o Suggests NE is sufficient for memory retention -  o Answer is Arc and Phosphorylated CREB – E - Arc = immediate early gene (transcribed rapidly in response to strong synaptic activity) - Arc is necessary for L-LTP - BDNF/TrkB/mTOR pathway activates the transcription of Arc mRNA in the soma - Arc is quickly transported from the soma to the activated dendritic spine - o Rats undergo inhibitory avoidance training o Independent variable: vehicale vs. lidocaine vs. clenbuterol (adrenergic receptor agonist) into the BLA o Dependent variable: arc protein in the hippocampus - o Decreasing BLA activity -> decreased levels of Arc in the hippocampus o Increasing adrenergic receptor activity in the BLA -> increased levels of Arc in the hippocampus o Decreasing BLA activity -> impaired memory retention on the inhibitory avoidance task o Increasing adrenergic receptor activity in the BLA -> improved memory retention on the inhibitory avoidance task - In addition to the vagus nerve, epinpephrine can modulate memory through increased glucose release - Liver stuff - - - Hypothesis: epinephrine is able to modulate memory by increasing levels of glucose, providing energy stores for protein synthesis - Glucose modulates memory - - Memory declines with age o Aged rats can acquire new memories, but they forget more rapidly o Are there deficits in memory systems with age? - - Arousing events can increase epinephrine in aged animals o Increase shock intensity = increase in epinephrine release - Aged reats don’t have an associated increase in glucose blood levels with an arousing event - o Rapid forgetting by old rats may be due to the failure of the liver to respond to epinephrine by secreting glucose - Injecting aged rats systematically with glucose reverses the age-related rapid forgetting in an inhibitory avoidance task o Injection of glucose immediately after inhibitory avoidance training o Tested rats 7 days later  o - Aged rats have impaired CREB activation in response to memory-inducing behavioral training o o Phosphorylated CREB in the dentate gyrus o Avoidance training does not lead to ncreased pCREB in the dentate Gyrus(DG) of old rats o Increased pCREB in DG can be restored with injection of glucose following training - Memory maintenance o Long-lasting, multiple waves of protein synthesis are associated with longer durations of a memory o Recall from LTP: ▪ synaptic tag = enlarged actin cytoskeleton ▪ allows for that dendritic spine to capture new proteins o PKM-zeta mRNA found in dendritic spines o PKM-zeta available within 10 minutes of an LTP-inducing stimulus o Once activated, PKM-zeta remains active o Positive feedback loop to perpetuate increased translation of PKM-zeta - Inhibiting PKM-zeta erases Taste Aversion Memory  o Memory for taste aversion is mediated by the insular cortex o Inject ZIP into the insular cortex at: 3 days, 7 days, or 25 days after training o - Taste - o Injecting Zip into the insular cortex erases taste aversion memory ▪ There is no time-dependent effect for ZIP after 3d ▪ Injecting ZIP into the hippocampus has no effect - PKM-zeta: o Inject a viral vector to administer more PKM-zeta to insular cortex o Establish a weak taste aversion o Conditions ▪ Inject 5 days prior to establishing a weak taste aversion Enhanced memory taste aversion ▪ Inject 9 days after establishing a weak taste aversion Prevents forgetting - PKM-zeta prevents endocytosis of AMPARs  o o Infusing ZIP alone (black bars) into BLA erases fear memory o Infusing ZIP+GluR23y (blue bars) into the the BLA has no effect on fear memory o Suggests that inhibiting PKM-zeta results in endocytosis of AMPARs 11/8/2024 - Psychological view of forgetting o Forgetting = the inability to recall something that could be retrieved on an earlier occasion o Sources of forgetting (amnesia) ▪ Interference of memory A second learning even could produce amnesia for a prior experience if that learning even was similar to the first and occurred closely after it o The second learning event interferes with the consolidation of the first learning event ▪ Retrieval Failures Memory was formed but(usually temporarily) cannot be retrieved - Active cellular & molecular neural processes regulate forgetting o Therefore, blocking these cellular & molecular processes will improve memory retention o What do you predict contributes to the neurobiology of forgetting - AMPAR endocytosis regulates forgetting o AMPARs that contain GluA2 subunits are more stable and less prone to endocytosis o Is forgetting due to removal of GluA2-containing AMPAR over time  o o o GluR23Y blocks endocytosis of GluA2-containing AMPARs improves memory - Contextual fear conditioning o  o o Blocking GluA2 AMPAR endocytosis improves memory/decreases forgetting o Figure F is happening at 14 days later when vehicle animals typically generalize their contextual fear conditioning for new spaces, from their fear conditioning – the GluR23y remember the old context well enough they don’t confuse it with the new context - NMARs regulate forgetting(in vivo LTP effects) o Induce LTP in the dentate gyrus ▪ LTP decays/runs down after ~4 days o Induce LTP, but administer an NMDAR antagonist, CPP (black) ▪ Decay/run-down is prevented ▪ LTP is maintained - - Train rats in a memory task, test them 5 days later o Daily systemic administration of NMDAR antagonist (CPP) vs. vehicle (saline) ▪ Vehicle rats perform poorly on the task after 5 days = forgetting ▪ NMDAR antagonist rats perform well on the task after 5 days = forgetting was inhibited  o Suggests that NMDA receptors are important in the active process of forgetting - Activation of NMDARs is important in forgetting – why might that be so? o CALCIUM - NMDARS regulate forgetting via Ca2+ influx o Blocking both NMDARs and voltage-dependent Ca2+ channels (vdCCs) prevent forgetting of an object-location memory o GluN2B-containing NMDARs is the key NMDAR subtype mediating the decay of LTP/Forgetting ▪ GluN2B subunits stay open longer, allowing more Ca2+ influx - 7 day retention interval: NMDAR agonist infused daily into dorsal hippocampus - 14-day retention interval: GluN2B selective inhibitor infused daily into dorsal hippocampus - - Selectively blocking GluN2B-NMDARs prevent forgetting - NMDAR agonist promotes rapid forgetting - NMDARs bidirectionally regulate forgetting - - Calcineurin = phosphatase in the brain that is activated by moderate Ca2+ o Phosphatase remove phosphate groups when they are activated - Calcineurin dephosphorylates stargazing - Stargazin-PSD-95 complex is destabilized - AMPARs are freed from PSD-95 - AMPARs undergo endocytosis - Inhibiting calcineurin results in AMPARs present in the PSD would remain there - o Activation of NMDARs and activation of calcineurin contribute to forgetting - Rac1 contributes to forgetting by regulating actin dynamics o Rac1 = member of the Rho family of GTPases that contribute to actin dynamics ▪ Rac1 exists in either an activae or inactive state Active Rac1 = rapid forgetting Inactive Rac1 = rate of forgetting is much slower o Evidence for Rac1 involvement in forgetting: ▪ Overexpressing Rac1 in hippocampus of rats accelerates forgetting in object-recognition memory ▪ Reducing Rac1 in hippocampus of rats enhances retention of objection- recognition memory - - Rac1 contributes to Forgetting: inter-trial intervals (ITI) o When there are multiple trials, spacing of the trials results in better retention o Contextual fear conditioning studies: ▪ 12-seond ITI also resulted in more rapid forgetting compared to a 120- second ITI ▪ Rac1 inhibitor prevents forgetting ▪ Rac1 activator promotes forgetting  o - Summary o Maintenance ▪ PKM-zeta prevents the endocytosis of AMPA receptors o Forgetting ▪ NMDA receptors responding to low glutamate input: allow lower concentrations of Ca2+ into the cell ▪ Calcineurin: dephosphorylates stargazing ▪ Rac1: affects actin o Synaptic plasticity (bidirectional modification of synapses) evolved as a homeostatic mechanism to properly balance excitation and inhibition needed to maintain neural circuits 11/11/2024 Skipping first 20 minuts - Engram exists dormant in between encoding and retrieval phases where it is active - Encoding -> engram dormant state - Retrieval -> engram activated -> ecphory memory - Engram has four properties o Persistence, ecphory, content and dormancy o Engram is a persistent change in the brain that results from a specific experience or event o An engram has the potential for ecphory-that is, an engram may be expressed behaviorally through interactions with retrieval cues, which could be sensory input, ongoing behavior, or voluntary goals. The term ecphory can be thought of as roughly equivalent to retrieval o The content of an engram reflects what transpired at encoding and predicts what can be recovered during subsequent retrieval - Until recently the hunt for engrams was restricted by availabled methods o Ablation of a large cortical area  o Ablation of a smaller cortical area o The use of stereotaxic surgery to target more specific regions such as the basolateral amygdala (BLA) or hippocampus for electrolytic or neurotoxic lesions Key LA = Lateral nucleus, BA = Basal nucleus, CE = central nucleus o The best one could hope for with such approaches was to find brain regions that contain cells that may be necessary for the behavioral expression of the engram o In spite of this limitation these approaches, combined with the work of neuroanatomists, have proven enormously valuable in generating hypotheses about where to look for engram cells and identifying the neural components of the various “memory systems” that are discussed in later chapters - Memories consist of collections of interconnected neurons that can be distributed widely across brain regions - But can we determine if a neuron(s), cells belong to the engram supporting a neuron o To prove a cell is part of engram it has to be active at encoding and active at time of retrieval o o Cell also should be able to be directly activated of the cell to generate appropriate behvaior o Cell should also be able to be inhibited or ablated to prevent the occurrence of the appropriate behavior - Neurons activated by the first experience would express Arc in the cytoplasm, whereas neurons activated by the second experience would express Arc in nucleus o When transcribed in the nucleus, Arc very quickly translocate to the cytoplasm. John Guzowki took advantage of this to successfully determine the correspondence between neurons activated when the rat explores the same environment on two different occasions. Thus, if the same neuron is activated by both experiences Arc should be present in both its nucleus and the cytoplasm, whereas cells activated by only the first or second experience would have Arc expressed only in either the nucleus or the cytoplasm 11/13/2024 - Creating lasting neuronal tag: The TetTag Mouse o The TetTag mouse is a genetically engineered so that a reporter gen, tau,Lacz will be expressed only in cells that are active at a particular time  o o When active Lac will remain in the system for a months unlike Arc, o tTa system would be controlled by antibiotic in the drinking water, doxicillin in water leaads to DOX state where marker is not activated o to be memory cell has to have two markers ▪ one marker for when cell is initially activated ▪ marker for when cell is undergoing retrieval o - o Experiment design, mice were habituated to context A with Dox in drinking water, then conditioned to context B with Dox removed. This allowed the tTa system to drive the expression of ChR2 in cells that were active during contextual fear conditioning. Mice were then tested in Context A where they had never been shocked o Photostimulation of rhodopsin channels in mice in habituated environment triggers freezing response  o - Kaoru Inokuci o A mouse with a head-mounted miniature fluorescent microscope o Mice were genetically engineered so that cells activated by context exploration switched from fluorescing green to fluorescing red. Context exploration activated subassemblies of neurons. Each number represents a specific subassembly o During sleep some but not all of theses assembles replayed. In contrast, neurons that continued to fluoresce green did not display coherent activity patterns of replay o During the retrieval period when the mice were returned to the context, oly subassemblies that were replayed during sleep were activated o 11/18/2024 - The fate of retrieved memories o The existence of an aengram can only be confirmed when the process of ecphory/retrieval processes influence memory o Two related effects on retrieval functions ▪ Destabilization function – the claim is that retrieving a memory returned it into a labile state that is vulnerable to disruption ▪ Integrative function – old memory can be integrated into creating new memory, which results in a new engram that contains botho the retrieved and new information o Two Italian psychiatrists, cerlettia dn Bini applied electrical current across the brain to treat severe psychiatric disorders called electroconvulsive shock therapy ▪ Noticed that ECS patients had impaired memories ▪ Era was unsuccessful because was amnesia due to disrupted memory encoding or because retrieval failure o A reactivated fear memory is vulnerable to disruption by ECs  ▪ Lewis used a fear-conditioning experiment to study the vulnerable of a reactivated memory—a noise CS was paried with a shock US. In one condition, 24 hours after fear conditioning , the CS was presented to reactivate the fear memory. Some animals received electroconvulsive shock (ECS), while other did not. In the second condition, the fear memory as not reactivated, but all animals received either ECS or no ECS. All animals ▪ o o Does not matter how memory entered active state, they are vulnerable to disruption whether or not the active state is induced by novel experience or retrieval cues - - - Memory Theory o When a retrieval cue activates a well consolidatead but inactive memory trace (A) the synaptic connections linking the neurons involved in the trace become unbound  o However retrieval also initiates protein synthesis and the memory trace is reconsolidated. Thus, when it returns to inactive state it will be stable - What destabilizes memory in recall o Ubiquitin proteasome system o Glutamate -> calcium -> o o Proteasome degrades “master” scaffolding protein called shank that is believd to be critical to the scaffolding proteins o