Anxiety clinical + neurobio

What is the evolutionary value of fear?

If you couldn’t experience fear, how would you learn what experiences would increase/decrease your chances of survival? You’d have to do trial and error which is dangerous and potentially lethal

• this is not beneficial evolutionarily

• fear allows us to generalize/group together dangerous things, which gives an advantage to the species

What are anxiety disorders? Which demographics are most affected by them?

persistent fear responses to stimuli that are NOT scary —> the threat is not real, or not imminent

• this is in contrast to fear, which is an emotional/physiological response to a real or perceived imminent threat that REINFORCES survival promoting behaviors (fight, flight, or freeze)

• anxiety is more prevalent in first world countries and are biased towards women, with the typical age of onset being young adulthood

• ¼ people experience anxiety in some form at some time

  • (generalized) anxiety disorders are higher in prevalence than specific phobias, social anxiety, or panic disorder

  • it is second only to MDD (unipolar depression), with rates only increasing

• fear is a sensory response to a perceived threat

In general, fear is an ACUTE response to an EXTERNAL threat (mainly emotional + physiological)

Anxiety is a CHRONIC response to an internal or external threat (that you can often expect ahead of time) —> mostly experienced as emotional and cognitive

• state that is maintained by cognitive processes predicting a future adversity

We know anxiety disorders affect the most amount of people second to MDD/unipolar depression, but what about PTSD?

PTSD is last (but it could be underestimated)

What are the diagnostic criteria for anxiety disorders?

• excessive fear/anxiety response to stimuli that do not normally evoke such a response. (anxiety leads to multifactorial effects)

  • physiological (autonomic arousal) —> activation of the HPA axis

  • emotional (fear, distress)

  • cognitive (anticipation, rumination, distractibility)

  • behavioral (avoidance of certain stimuli that, if unavoidable, is endured with distress) —> you want to AVOID the object(s) of your anxiety

• frequent and persistent

• INTERFERES with your role and (daily) functioning

What come under the umbrella of anxiety disorders? What is the common link between all of them?

• phobias

• separation anxiety disorder (seen in children)

• generalized anxiety disorder (GAD)

• panic disorder (triggered by panic attack)

• OCD - evoked by loss of order or control

• PTSD - evoked by reminders of specific past trauma

The common link between all of these is that we see EXCESSIVE (disproportionate to the situation) fear that can be specifically triggered + leads to avoidance of the object of the anxiety)

What are the characteristics of generalized anxiety disorder?

• essential feature: unrealistic OR excessive anxiety and worry about a number of activities or events

• diagnostic criteria: symptoms that persist for at least SIX MONTHS

  • excessive anxiety

  • worries that CANNOT be controlled

  • feeling on edge

  • poor concentration/mind going blank

• physical symptoms:

  • restlessness

  • fatigue

  • muscle tension

  • sleep disturbance

  • irritability/tendency to argue

What are the diagnostic criteria for a panic attack?

an episode of intense fear and discomfort, which peaks/approximately last for 10 minutes

it needs to include symptoms like:

• heart palpitations

• nausea/abdominal discomfort/chest pain

• sweating

• trembling

• a sense of things being unreal

• a fear of dying

Once these bodily cues become associated with panic or fear, a person might avoid activities that cause those sensations (since they have been conditioned to associate the stimulus with those things)

• over time, this learning process can make it so that even mild symptoms can trigger a full panic attack, because the body and mind now anticipate disaster BASED on those sensations

• ex. if the shopping mall makes your heart race, and you had a panic attack there, you associate the mall with that panic

What are the diagnostic criteria for social anxiety disorder?

this is kind of like an extreme case of stage fright - it is an intense, irrational fear of being negatively evaluated in at least 1 social or performance situation

• exposure to feared circumstance provokes an IMMEDIATE panic attack

• the mean age of onset is late teens

• the brain can end up perceiving social exclusion as a PHYSICAL pain in social anxiety patients

What are the diagnostic criteria for PTSD?

you have to have an instigating event for diagnosis (a severe threat like death, critical injury, violence)

you experience things like involuntary memories, dreams, flashbacks, psychological or physiological distress in response to reminders

• avoiding REMINDERS of the event

• cognitive and affective (emotion-related) alterations

  • amnesia for some of the aspects of the event

  • negative expectations about self and world

  • emotional changes (detachment, guilt/shame, loss of interest/positive emotions)

• some alterations in physiological arousal and reactivity

  • irritability

  • hypervigilance

  • startling easily

  • disturbance of sleep

  • poor concentration

  • reckless or self-destructive behavior

What are the diagnostic criteria for obsessive-compulsive disorder?

obsessions: persistent ideas, thoughts, impulses, or visual images that are INTRUSIVE and senseless

• they are a product of their mind

• you attempt to ignore or suppress them with another thought or action

compulsions: repetitive, PURPOSEFUL, and intentional behaviors that a person carries out in response to an obsessive thought

• performed according to certain rules or in a repetitive, stereotyped fashion

• this is designed to neutralize or prevent anxiety around some dreaded event or situation

• it is NOT always connected in a realistic way with what it is designed to prevent

The person recognizes the compulsions as excessive or unreasonable - and they do cause distress, especially if they are time consuming or interefere with normal routine and daily functioning.

• stopping compulsions is anxiety-inducing, but performing compulsions is rewarding (meaning that it involves the VTA-nucleus accumbens, not the amygdala’s fear circuit)

Talk about the functional neuroanatomy of affectively (emotion) mediated behavior

LIMBIC SYSTEM:

• amygdala: detects threat, initiates fear responses, helps with HPA axis activation

  • damage leads to blunted fear, hyperactivity leads to anxiety disorder development

• hippocampus: contextual memory/encoding of fear-related environments (where and when something scary happened)

  • weak coupling with the amygdala leads to overgeneralized fear

NEOCORTEX:

• ACC: integrates emotion + cognition; puts fear in context of the environment

  • can be underactive in PTSD

BROCA’S: important for speech production and verbal expression of emotion

PFC: executive function, inhibition of responses (such as automatic fear), emotion regulation (can be hypoactive in PTSD)

What happens to PTSD patients who are exposed to emotionally negative images (who are then compared to people who are trauma exposed but are not PTSD)?

We see hyperactivation of the (right) amygdala (indicating a stronger response by the amygdala to the stressful stimuli) in the PTSD

• as in we see more activity for the PTSD than the trauma exposed people

What is increased amygdala responsiveness associated with?

• exposure to fearful faces

• diagnoses of separation anxiety disorder, PTSD, panic disorder

• panic attacks

• chemically-induced panic attacks in healthy controls

• people who are prone to having phobias

• being a carrier of catecholamine (dopamine, norepinephrine, epinephrine) polymorphisms

  • SERT promotor polymorphism (long or short allele)

  • COMT val158met (since COMT breaks down catecholamines like dopamine)

    • valine leads to higher COMT activity and faster dopamine breakdown, leading to lower levels of dopamine

    • met leads to lower COMT activity —> associated with better working memory, but greater sensitivity to stress

  • BDNF val66met: met involves impaired intracellular trafficking which REDUCES activity-dependent BDNF secretion; leads to LESS symaptic plasticity and hippocampal dysfunction

When we look at the SERT transporter allele, we see what?

When we compare those carrying the s allele (which leads to less serotonin reuptake —> chronic activation of autoreceptors —> lower serotonin release, as compared to SSRIs which lead to the initial suppression of NT release and eventually adaptation/downregulation of the autoreceptors) to those carrying the l allele

….we see a HEIGHTENED amygdala response (greater amygdala activation) when viewing fearful or angry faces, as compared to neutral ones

• these animals also might see ambiguous or neutral faces as more negative

• so basically the activation in the amygdala is increased in the short alleles compared to the long alleles

• this is similar to those with anxiety disorder

What happens with panic attacks?

We see decreased activity in the frontal lobe

• this leads to confusion and the greater capacity for IMPULSIVE behavior (since a big part of executive function is regulating impulses)

and increased activity in the thalamus (which acts as the brain’s relay center - it takes sensory information, except smell, and routes it to the APPROPRIATE cortical areas)

• the PFC helps to filter out irrelevant stimuli

• if the PFC isn’t working to its full capacity and isn’t shutting anything down, we can have an overwhelming sensory experience/the brain’s processing system gets overwhelmed

• this can lead to confusion in people

Which part of the brain is important for extinction (basically, learning a lack of association between the conditioned stimulus and conditioned response, when the conditioned stimulus is no longer paired with the usually negative unconditioned stimulus)?

the prefrontal cortex is important for extinction - basically, learning a lack of association between the conditioned stimulus and conditioned response (ex. freezing when you hear a bell chime that you have been “taught” pairs with an electric shock)

• lesions in the prefrontal cortex / disrupting protein synthesis critical for LTP = delay in extinction/this learning process

note: we can think of anxiety disorders as representing a DISRUPTION of the homeostatic balance between normal activation of adaptive fear (GOOD) and anxiety responses (BAD - OVERACTIVATION for non-threatening stimuli)

• we also see “malfunctions” in the frontal circuitry that chooses the appropriate and adaptive behavioral response by integrating (ADDING) your past experience (via hippocampal memory) + the current input (what is going on now) and looking at the PREDICTED OUTCOME of different response options

What are the neuroanatomical/functional changes in PTSD?

• smaller hippocampus and anterior cingulate cortex (involved in cognitive functions + emotional regulation) volumes

  • the anterior cingulate communicates with the amygdala + PFC to regulate mood

  • it also decides where to focus your attention/whether or not you are distracted (cognitive role)

• increased activity of the amygdala (which can lead to hypervigilance)

• decreased functioning of the PFC and ACC

• increased cortisol levels and norepinephrine responses to stress (which kicks off the sympathetic/FIGHT OR FLIGHT nervous system)

What do effective treatments for animal studies of PTSD show?

• increase in neurogenesis (the birth of new neurons in the hippocampus)

• enhanced memory (as a function of the hippocampus)

• increase in volume of the hippocampus

In what disorder do we see hypoactivation of the PFC? In what disorder do we see hyperactivation of the amygdala?

hypoactivation of PFC = PTSD

• in critical areas for automatic, reflexive emotional regulation that are CRITICAL to the extinction of fear

• we also do see some increased activation of the amygdala, but social anxiety has this the most

hyperactivation of amygdala = social anxiety

What does the heritability of PTSD suggest?

You need a severe life event to trigger the development of PTSD/qualify for a diagnosis, but the heritability of PTSD suggests some biological vulnerability

• some gene variants may put you at more risk than others

What is a consequence of decreased functioning of the anterior cingulate cortex, which projects to the amygdala?

Remember that the anterior cingulate cortex is part of the brain’s emotional regulation network - part of its job is to send inhibitory signals to the amygdala to tell it to “calm down” and dampen EXCESSIVE fear and emotional responses

• this allows you to experience an appropriate level of fear and adapt your emotional reactions based on the context

• in PTSD, ACC hypoactivity/decreased activity = amygdala hyperactivity

  • this leads to heightened fear and anxiety as well as hypervigilance/exaggerated startle responses

  • we can see a reaction even to neutral or safe stimuli

• hypoactivity of the PFC is also important, because it sends signals to the ACC to coordinate the right emotional + autonomic responses

What provides a very simple and powerful explanatory model for many anxiety disorders?

Classical conditioning = pairing of an unconditioned stimulus (ex. food) with a conditioned stimulus (ex. a bell) to get a conditioned response (salivating in response to the bell chime) —> Pavlov’s dogs

We could also classically condition humans (ex. an infant named Albert learned to fear a white rat because every time it saw the white rat, a loud/scary noise played —> led to avoidance of the conditioned stimulus and a conditioned reaction (crying))

• the baby also showed generalization, which is “the ability to respond to stimuli which are qualitatively different but functionally equivalent” —> objects with a similar feature to the conditioned stimulus can evoke the same fear response

• Albert responded fearfully to other furry animals + a fur coat, showing generalization (where objects similar to the CS could elicit the CR)

When the CS is no longer paired with the US, we can have extinction where we see the conditioned response less (it decreases in frequency)

• this allows us to induce phobias for many objects and reverse them through extinction

We know that the ACC projects to the amygdala, but what else does?

The hippocampus, which sends inputs to the hippocampus

• basically, it provides the amygdala with detailed information about the context (where are you, what is your environment and surroundings like, what are the background cues)

• based on the context, the amygdala can generalize fear responses from one context to similar environments

  • the association of the conditioned stimulus with the unconditioned response (creating the conditioned response) is mediated by the amygdala

  • the central nucleus, which is part of the amygdala, is what mediates the fear response

  • other areas become involved in the fight or flight response, but the amygdala is central to the process

Though classical conditioning is great for modeling anxiety disorders, it isn’t a perfect model. What are some of its limitations?

• how different anxiety disorders are actually represented in the CNS

• the variable heritability (differences in heritability) between different anxiety disorders

• how the conditioned response arises with no history of specific CS/US pairing

• why we have a higher incidence of certain phobias (snakes, spiders, heights) and not with things like guns or automobiles which we are more likely to be exposed to and killed/injured by

  • this is likely because the former category is made of threats that would have been commonly encountered by our ancestors (so they have an evolutionary origin), but the latter category is made of modern objects/things

Also, classical conditioning experiments tend to treat organisms like a “black box”

• this means that we know what goes in (stimuli, reinforcements) and what comes out (responses, behaviors) but not the internal mental processes that would allow us to understand or predict their behavior

What are some risk factors for anxiety disorders?

• biology (genetics [which aren’t super strong, they overlap with depression risk factors], inhibited temperament, evolutionary history)

  • temperament: behavioral characteristics that are evident in the first two years of life, which are predictive of later personality features. For example, children rated as shy and inhibited at 21 months are more likely to develop phobias when they are older

• environmental (stressors, which also overlap with depression)

• living with someone with an anxiety disorder (which causes effects beyond genetic contribution, if you are related)

• trauma exposure (sexual abuse that can lead to the deveopment of social anxiety)

• accident, natural disaster, and combat

• adult violence/aggression

How can we answer the following - does the disorder run in families, how much do genetics contribute to the risk for this disorder, what genes are involved, what do the genes do?

• studies of incidence in families

• studies of incidence (concordance) between MZ/identical and DZ/fraternal twins

• linkage analysis and GWAS (genome-wide association study)

  • linkage analysis: family-based, tracks inheritance patterns across generations and focuses on rare, HIGHLY penetrant mutations (ex. Huntington’s) —> penetrant is if you have the allele, you likely have the disorder.

    • studies gene varients with BIG effects. Ex. we can study the PSEN1 mutation, which leads to the amyloidogenic pathway being favored and the development of early-onset Alzheimer’s

  • genome-wide association study: population-based, looks at single-nucleotide polymorphisms across the genome

    • studies gene variants with SMALL effects

• molecular biology, clinical research

What are the genetics of anxiety disorders?

• no autosomal genes that CAUSE anxiety have been identified in humans

• the inheritance pattern is non-Mendelian and complex

• when we do linkage/association studies (going from one generation of families to another), we see at least 12 loci for potential genes on various chromosomes

• However, most study have failed replication in independent samples —> shows that these results may be due to chance, or to other confounding variables like ethnicity and environment

  • because of this, we should focus on genes that affect fear circuitry/connectivity between fear regions (ex. the amygdala, ACC, and hippocampus)

  • FKBP5, FAAH, PACAP —> increased amygdala activation, reduced coupling to other regions like the hippocampus (meaning that regulation of emotion that is context-dependent will be impaired)

• we probably see interaction of the environment with multiple risk-increasing or predisposing genes, like the short-form version of the SERT gene

Which, when paired with the s/s SERT or 5-HTTLPR gene, lead to the development of mood/anxiety symptoms - early life stress, family history of mental health diagnosis, or general stressors such as poverty?

physical abuse, sexual abuse, and emotional abuse - when paired with the short-form version of this serotonin reuptake transporter (created by having two short alleles), lead to:

• reduced transcriptional efficiency (likely of BDNF) via less activation of signaling pathways (cAMP-CREB)

• increased cortisol response

• decreased inhibition of the amygdala (leading to overactivation)

Which disorders have the highest heritability?

bipolar, schizophrenia, substance abuse dsorders

panic disorders and depression (respectively) are more heritable than generalized anxiety disorder, which is consistent with the idea that these are ENVIRONMENTALLY TRIGGERED and more within the realm of environmental factors, even as substantial heritability is still present

• 30% risk of developing GAD because of genetics, 70% risk is due to environment/experiences

• we DO see a significant increase in risk for GAD when you have a first degree relative who also has it

• however, there is no prescribed “path” for anxiety disorder based on having a specific gene or trauma

How can we actually treat anxiety disorders?

• prevention —> we should treat people early in life to reduce the burden of anxiety disorders, since intervention = reduction in anxiety disorders while adulthood therapeutic treatments reduce their severity)

  • identification of populations at risk

  • intervention involving relaxation techniques, fear management, parenting advice

• behavioral

  • cognitive behavioral therapy (CBT)

  • exposure (phobias)

  • mindfulness and meditation, which allow us to focus attention

• pharmaceutical

  • anti-depressants of the SSRI and SNRI (less selective version since it is for serotonin and norepinephrine) class

  • benzodiazepines like valium

  • buspirone for GAD

  • B-blockers, which reduce voice tremor and pounding heart, for public speaking

How effective is cognitive behavioral therapy, which looks at identifying, grading, and reframing negative thoughts and cognitive distortions, in treating some forms of anxiety? What about exposure therapy or habituation, where a person is repeatedly exposed to the stimulus until their fear goes away

Can be pretty effective - when we did a scan of controls and people before and after CBT, we saw a reduction in the activity of the amygdala for spider phobia

How do benzodiazepines like valium work?

• when the benzo binds to a GABA receptor, it acts as an allosteric modulator

  • it won’t replace GABA by acting as a functional agonist, but it can bind to a separate site and enhance the binding of GABA to the receptor

• this leads to increased Cl- influx —> this is functionally the same as creating an efflux of K+ because it lowers the membrane potential

• the membrane potential increases

• excitability is reduced, meaning that the neuron fires less

How do barbiturates, which reduce anxiety/behave as anti-anxiolytics, work?

Barbiturates bind GABA receptors just like benzodiazepines/valium does, but at a separate site from either benzos or valium. So they are also allosteric.

• they increase the DURATION of the opening of Cl- channels when GABA is bound (while benzodiazepines like valium increase the frequency)

Talk about the structure

hippocampus supplies amygdala with context (from previous memories): CONTEXT

• you could feel fear in response to non-threatening stimuli, like in a movie

medial prefrontal cortex regulates the amygdala so that its output generates less fear/anxiety: REGULATION

• dysfunction results in an inability to regulate the fear response

• inability to make appropriate decisions or actually take action when confronted with the fearful stimuli

the amygdala itself receives emotional stimuli, and outputs an emotional response

• dysfunction can lead to enhanced sensitivity to fear, integration of a non-threatening stimulus with memory/context information in a way that treats it as DANGEROUS when it isn’t (maybe because the hippocampus can’t provide the right contextual input due to STRESS or UNDERDEVELOPMENT)

fear and anxiety can result from dysfunction of the PFC (lack of emotional regulation), improper stimulus (as in, the stimulus isn’t truly dangerous), faulty memories (arising from hippocampal dysfunction, potentially leading to overgeneralization towards stimuli that resemble the conditioned stimulus)

What are the neurotransmitters of anxiety?

• glutaminergic communication between the thalamus and cortex

  • thalamus sends sensory information to the sensory cortices and to the prefrontal cortex

  • cortex sends top-down feedback to MODULATE and FILTER input

    • because glutamate is the excitatory transmitter here, excess glutamate can lead to overexcitation = overwhelm of the prefrontal cortex

We also see glutaminergic activity in the amygdala, because this is what pairs the unconditioned + conditioned stimulus in the brain and is responsible for that “learning”

• depends heavily on NMDA receptor activity - when the NMDA receptors are activated by glutamate we see increased gene expression and AMPA receptor insertion, leading to LTP/synapse strengthening

• serotonergic communication between the raphe nucleus and the amygdala, which regulate fear responses (can be excitatory or inhibitory, depending on the receptor subtype)

• noradrenergic communication between the locus coeruleus and norepinephrine

  • when the LC is activated, it sends norepinephrine to MULTIPLE regions of the brain

  • hypothalamus is most important - when it sends norepinephrine to the hypothalamus, the HPA axis is activated (CRH —> ACTH —> cortisol)

  • amygdala activity is also enhanced via NE

• GABAergic communication happens between “all” of these regions and helps to reduce anxiety levels throughout the circuit

What is the role of social neuropeptides?

oxytocin - made by and released by the PVN of the hypothalamus; responsible for social bonding and stress buffering (can reduce anxiety and fear)

• we can use oxytocin agonists to treat fear/anxiety

vasopressin - also made by and released by the PVN of the hypothalamus; responsible for modulation of stress and anxiety (but is PRO anxiety)

• so we can use vasopressin antagonists to treat anxiety symptoms

What are the symptoms observed in animal models of fear and anxiety?

• spontaneous avoidance of bright, open environments (which may be because these environments give them the feeling of being vulnerable)

• social avoidance of conspecifics (more dominant members of their species)

• response to predators, predator odors

In experiments, we can have mice drink sugar water in exchange for being shocked occasionally. We use this to test for whether the mice can endure the aversive stimuli to receive the pleasant stimuli.

• the more anxious mice have a lower threshold for the shock stimulus

Controls may show some fear to these things, but the anxious animals will show EXAGGERATED fear responses

What is the elevated plus maze - how does it capitalize on the anxious animal avoidance of bright, open areas? What is QTL mapping? What is the open field test?

This test allows us to compare different mice strains (and thus genetics) in terms of where they are on the “scale” between fear and exploration

• which strains have high activity in the elevated maze? which strains have low activity

• QTL (quantitative trait loci) mapping allows us to find correlations between genetic markers, or the genotype, and trait values, or phenotypes

The open field test allows us to look at total time spent along the edge, as well as total locomotion that the mice exhibit.

• if the mice spend more time on the edge (close to the walls), because that feels safer, that can serve as a measure of fear (as opposed to more exploratory behavior that would have them in the center area)

What are some tests within the two paradigms (unlearned, learned) that we can use to study anxiety in animals?

unlearned (which measure fear and anxiety):

• elevated plus maze

• open field test

• light-box dark exploration

learned (which measure PTSD symptoms):

• fear conditioning

• reflex potentiation (measuring the strength of a reflex following an activity) —> people with PTSD tend to startle more easily/aggressively

• conditioned place aversion (which is similar to conditioned place preference that was discussed within addiction, but this is the opposite of that)

• punishment-induced conflict (ex. mouse being shocked but getting to drink sugar water —> the mouse experiences a conflict because an action can lead to both reward and punishment)

What are some characteristics of the US (unconditioned stimulus) in the conditioned fear model (ex. when we pair a neutral conditioned stimulus, like a bell chime, with a negative stimulus like a foot shock)?

• freezing

• autonomic changes (increase in heartrate)

• decreased sensitivity to pain

• potentiation of reflexes (startling, blinking)

• release of glucocorticoids (cortisol in humans, corticosterone in rats)

Why is extinction (where the conditioned stimulus is no longer paired with the unconditioned stimulus) not synonymous with forgetting?

over time, the freezing behavior (the conditioned response) in response to the now-unpaired conditioned and unconditioned stimulus will decrease

• this is not FORGETTING —> this is the animal RELEARNING. It is now understanding that the conditioned stimulus is no longer dangerous.

Talk about the amygdala.

the lateral amygdala receives information about threats from:

• thalamus (ROUGH sensory input)

• hippocampus (contextual information - what happened in the past, from similar threats)

• cortex (FINE-TUNED sensory processing)

the lateral amygdala will then INTEGRATE these inputs (ex. integration of the conditioned and unconditioned stimulus) and then send these to the central amygdala. The central amygdala is what actually OUTPUTS to other brain regions:

• hypothalamus (to activate the HPA axis)

• periaqueductal gray (freezing response)

What can BLOCK or AFFECT fear conditioning?

lesions of central or lateral amygdala can ABOLISH fear conditioning or pairing of the US and CS

• lesions of smaller regions in the amygdala can be lesioned without losing fear conditioning (measured through a freezing response - since that is what shows that the pairing of the conditioned and unconditioned stimulus has successfully happened), but lesioning the ENTIRE amygdala leads to no freezing response

• hippocampal lesions don’t abolish fear conditioning, but remove conditioned place aversion (which makes sense - the hippocampus is what situates what is happening now in the context of what happened in the past)

NMDA receptors (in the amygdala) being blocked also blocks fear conditioning for BOTH short and long term memory

• this makes sense since NMDA receptors are the learning and memory receptors (increase synaptic strength by inserting more AMPA receptors)

Are NMDA receptors (ex. in the lateral amygdala, which integrates the conditioned and unconditioned stimulus) necessary for fear conditioning? By extension, is LTP most important in the lateral amygdala?

Yes and yes. If we put an NMDA receptor antagonist in thee areas, we see a SIGNIFICANT decrease in the freezing response.

How does the extinction of fear conditioning memory work?

Just like fear conditioning needs the lateral + central amygdala (and specifically the NMDA receptors in the lateral amygdala) to work, extinction NEEDS the prefrontal cortex to work.

—> this is because, once the lateral amygdala excites it (via glutamate), the PFC INHIBITS the lateral amygdala via GABAergic projections. This basically means that it is telling the lateral amygdala to STOP integrating the US + CS

• lesioning the PFC, or inhibiting protein synthesis (needed for LTP), will also block extinction

• this is another signal that the extinction process is less like forgetting and more like relearning (“forgetting” is stored like a memory)

If we don’t have a high activation of the prefrontal cortex (specifically the medial prefrontal cortex), we can keep having MALADAPTIVE and PERSISTENT/LONG-LASTING fear responses that aren’t appropriate to the situation

What is some important evidence that the prefrontal cortex is important for extinction?

• people with PTSD have REDUCED prefrontal activation with autonomic arousal when viewing trauma-related stimuli

• in animal models, we see that stress can REDUCE the functional activity of the prefrontal cortex, which is critical for fear extinction

  • this applies for both acute and chronic stress

• stress also reduces the lengths and density of dendritic trees (meaning we see less arborization, which shows how there are fewer synaptic connections here)

PFC inhibition of the amygdala (especially the lateral amygdala - they talk to each other) can be weakened via stress

• when conditioned to fear a new stimulus, chronically stressed animals show a reduced ability of the PFC to inhibit the amygdala compared to mice that have not been chronically stressed

researchers did an experiment (ex. conditioned place aversion), and then immediately infused norepinephrine into the lateral amygdala into the brain after the training

• the mice showed enhanced retention (a stronger memory for the aversive experience) the next day

• even though the amygdala is not the primary site for memory retention, it influences the strength of memories in other brain regions

remember that the endogenous source for NE in the body is the locus coeruleus, which projects to a lot of regions, but also to the amygdala to enhance its activation.

• in turn, the amygdala can modulate other structures, like the hippocampus (relevant for memory)

• what is the locus coeruleus activated by? stress signals in the periphery, such as inflammatory cytokines

• the locus coeruleus, like many other regions of the brain, is regulated by GABA (or endogenous opioids)

Where does acetylcholine come into the picture? What is its source?

Eventually norepinephrine from the locus coeruleus will hit its peak and become less effective, meaning that the brain will increasingly rely on acetylcholine instead

• NE is released in short bursts, and is intended for immediate threats

• ACh is relevant for situations that require MAINTAINING vigilance, such as for a sustained threat/when the threat persists

  • this is really helpful because under high NE the prefrontal cortex can “shut down”

The ACh in the fear/stress circuitry comes from the cholinergic neurons in the basal forebrain

• these are connected to the hippocampus

• the hippocampus is the obvious brain structure that is relevant for memory, but the basal forebrain cholinergic neurons are CRUCIAL for memory formation and storage because they regulate attention + arousal

  • this is critical for encoding NEW information

  • lesions in this region are linked to memory impairments

How is fear circuitry modulated by stress? By what brain region do these three modulate the response by?

• hippocampus attenuates (reduces) the glucocorticoid response to stress by creating a negative feedback loop (via glucocorticoid receptors)

• the amygdala facilitates (enhances) the glucocorticoid response to stress by activating + amplifying the HPA axis

• the PFC regulates the HPA axis in emotional/physiological stress, but not other stress (depends on whether the stress is cognitive or more physical)

ALL THREE modulate the response via the PVN (in the hypothalamus) which make CRH/CRF (corticotropin releasing factor/hormone), signaling to the pituitary to make ACTH

What is the connection between CRH/CRF and consolidation? How does this process actually occur? What is the connection between mild/moderate stress and memory - is it impaired or enhanced?

glucocorticoids can enhance memory consolidation in the short term (ex. moderate acute stress) → your brain will mark certain memories as important

• this applies to emotional or stressful memories and allows for long-term consolidation (via interactions between the amygdala and hippocampus)

  • chronic/excessive stress will end up impairing memory

• the lateral amygdala has CRF receptors —> it can actually be released locally as a neuropeptide in the amygdala, not just from the hypothalamus to the pituitary gland

This is part of the reason that stressful events are remembered more vividly/with more detail.

How can we block the effects of CRH (which can help to consolidate memories, for mildly/moderately stressful events)?

We can block it with propranolol, which doesn’t block CRH directly but rather blocks its downstream effects

• we use propranololfor social anxiety because it BLOCKS stress-induced fear memory enhancement

What are the two CRF receptor subtypes, and what are their functions? Where in the brain are they found?

• CRF-1 is the excitatory one (ANXIETY-PROMOTING). If you knock it out in mice, they will show decreased anxiety and IMPAIRED fear responses

  • found in the amygdala (to amplify the emotional URGENCY of stress) and the LC (which increases NE release to multiple regions of the brain and the “fight or flight” response)

• CRF-2 is inhibitory. If you knock it out, these mice will be hypersensitive to stress/anxiety and show exaggerated responses.

  • found in the hypothalamus, acts as a BRAKE to prevent excessive activation. —> promotes recovery and reestablishment of homeostasis

So basically these two have opposing effects on stress/anxiety

How is the nucleus accumbens/VTA (reward pathway) connected to the CRF?

Not only do both the VTA and the nucleus accumbens play a role in the reward circuit, but they are key for CRF

• the VTA contains a lot of CRF receptors, and when CRF binds there, it stimulates dopamine release

  • so short term/acute stress is reinforcing (provides motivation to keep taking the drug)

  • chronic stress and repeated activation of CRF receptors lead to a decrease in dopamine release (in the nucleus accumbens) over time

However, we can block the aversive effects of CRF and stress in the nucleus accumbens with a glucocorticoid receptor antagonist (to minimize the effects in dopamine release reduction)

How do other neurotransmitters play a role in generalized anxiety disorder?

• norepinephrine tends to increase anxiety (too much norepinephrine = autonomic nervous system is hypersensitive + overreacts to various stimuli)

  • so the LC, which is the source for the norepinephrine in the body, might play an important role in anxiety regulation

  • norepinephrine stimulates the sympathetic “fight or flight” nervous system, which is associated with stress + fear stimuli and leads to the physiological changes we experience (ex. faster heart rate)

  • panic attacks and insomnia are also due to an increase in NE

    • a lot of people with anxiety medicate (or self-medicate) via alcohol, opiates, or benzos/valium to REDUCE NE firing (which can indirectly lead to more CRF expression)

• GABA: anxiety might be linked to underactive GABA signaling (leading to overactivation of the HPA axis) or downregulated receptors for benzodiazepines (benzos)

• serotonin in excess can cause GAD (but there are mixed effects depending on which receptor subtype is activated)

  • 5-HT1a receptors in the PAG —> inhibit defensive behaviors (fight, flight, or freeze) —> AXIOLYTIC or anxiety-”breaking”, because this is an INHIBITORY receptor

  • 5-HT from the dorsal raphe nucleus (released in the amygdala + PFC) can regulate threats/anxiety responses. AXIOGENIC/anxiety causing because of the 5-HT2a receptor

How does norepinephrine play a role in CREB phosphorylation/activation, and the increase in levels of CRH/CRF? What target gene is of interest here?

NE binds to beta-adrenergic receptors, which are G-protein coupled (Gs) and will lead to the phosphorylation of CREB via signal cascade

• important to remember that CREB does not only play a role in BDNF expression, but also in CRF expression (it’s a transcription factor for multiple genes)

So a stressful experience may indirectly lead to more CRF expression (in a kind of cycle) via an increase in norepinephrine

• which might further activate the HPA axis

Why are NE levels elevated in the first place? hint - it has to do with the inhibitory NE receptor, which we knocked out in mice to observe the effect on anxiety levels

This might be due to an INCREASED responsiveness of LC (locus coeruleus) NE neurons to stimuli

• maybe the a2AR inhibitory autoreceptor, which limits norepinephrine release from the presynaptic neuron, is the reason why

• if we block (via knockout or antagonist) or reduce the activity of this autoreceptor, we see an increase in the production/release of NE and increased anxiety symptoms (ex. in a2AR knockout mice)

  • these mice spent more time in closed arms of the mice during the elevated-plus maze test, relative to the control mice

What is GAD67, and what effect would knocking it out have on inhibitory signaling in the brain, as well as on fear extinction?

this is an enzyme that converts glutamate into GABA

• by knocking it out, we decrease GABA release and signaling, which is especially important when looking at PFC inhibition of the lateral amygdala (which it outputs to)

• if PFC cannot successfully inhibit the lateral amygdala, as seen in GAD67 knockout mice, then the amygdala will be overactive

  • GABA also plays a role in fear extinction, or relearning that a stimulus is not harmful. If we decrease GABA signaling (ex. via GAD67 knockout), we are less likely to extinguish the fear response (measured via a CR in mice)

  • GABA signaling DECREASES excitatory output from the amygdala to the prefrontal cortex (when alerting it to a threat/sending emotionally relevant signals)

Why are the interneurons in the amygdala GABAergic, and not the prefrontal cortex neurons (which inhibit the lateral amygdala)?

Because the prefrontal cortex is sending excitatory signals to these GABAergic neurons, which will LOCALLY inhibit the amygdala (but on the orders of the PFC)

• the connection between the PFC and amygdala is glutaminergic, the GABA signaling comes into play via a local circuit

We know that the 5-HT2a = pro-anxiety receptor, while the 5-HT1a is anti-anxiety (serotonin autoreceptor). How does stress affect the levels of 5-HT1a?

• decreased levels after stress or exposure to CRF

  • this leads to a lower threshold for stimuli to lead to anxious responses

• we see decreased binding of serotonin to these receptors in multuple parts of the brain (ex. amygdala, cortex, dorsal raphe nucleus)

5-HT1a knockout mice show increased anxiety (less time in the open arm of the elevated maze or in an open field) —> overexpressing it leads to the opposite (reduced anxiety from elevated serotonin release)

• if we shut down these receptors shortly after birth, we can’t rescue the phenotype in adults because the effects on neuronal function are very long-lasting because the circuit CAN’T perform a normal fear response anymore

• we can rescue the phenotype assuming that the 5-HT1a receptors were present during development and only knocked out/blocked during adulthood

We know that anxiety is accompanied by physiological symptoms (ex. sweating, faster heart rate) but this is a chicken and egg issue - which comes first? What if the body controlled the brain, similar to how the gut microbiome can affect brain function?

Optogenetic (light-sensitive ion channels) activation of heart muscle cells produces anxiety —> so when the heart rate of mice was increased independently of whatever they were doing, we saw greater anxiety

• the heart activation lead to insula activation/increase in firing rate of neurons in the insula, which is associated with pain and anxiety

• if these ion channels are inhibited (“decoupling” heart rate from anxiety), we see that heart rate NO LONGER leads to a change in behavior

  • the insula is part of the limbic system and provides an awareness of bodily states/acts as a bridge between the brain and the body