119A M2

process of drug development and testing

- preclinical research (5 years) --\>
- investigational new drug application sent to FDA --\>
- clinical studies (phase 1: 1.5 years, phase 2: 2 years, phase 3: 3.5 years) --\>
- new drug application sent to FDA --\>
- FDA review --\>
- approval --\>
- postmarketing surveillance

preclinical research

- Discovery and early in vitro screening of compound
\--- screen for TIs and TDs (need at least 3 doses to make dose-response curve)

- Large-scale synthesis

- Animal testing

- At least 5 years

clinical studies

3 phases:
- phase 1: further evaluation of toxicity/pharmacokinetics in healthy controls; tens of people; 1.5 years
- phase 2: testing efficacy on targeted (pathological) group; hundreds of people; 2 years
- phase 3: scale up/large clinical trials, compare to current treatments; thousands of people; 3.5 years

7 years total

Postmarketing surveillance

monitor adverse reactions, product defects, long term side effects, drug interactions
- continuous and ongoing
- can patent for ~20 years; continuous surveillance and review

since we cannot completely replace animals, how do we use them appropriately?

refine (enhance wellbeing); reduce; replace

social defeat stress

mechanism to achieve a depression phenotype (very translationally-relevant for bullying)
- alpha male gets put into a cage with a non-dominant mouse; alpha will start to beat up on the submissive mouse

Tail-flick test of analgesia (pain-killing)

put light (heat) source under rat's tail where it will start to burn the tail; see how long until the animal flicks its tail away from the heat ("latency to flick")
- normal latency is under 10s, under 30s with analgesic

T-maze

assesses spatial and working memory
- hippocampus, PFC

Maze type that involves an alley ending in a "T" shape, giving the animal two path choices to reach food (sucrose pellets, froot loops) in goal box.
- rewards at arm A or B; if you get rid of A, should then go to B (shouldn't revisit A when restarting --\> taxing this memory)
- add delays between trials to tax spatial working memory

Effort T-maze

assesses cost-benefit decision making
- dopamine-implicated areas (hippocampus, PFC as well)

putting two rewards in both arms of the T-maze, but one arm containing the larger reward is blocked off by an obstacle

Radial Arm Maze

assesses working and spatial memory
- hippocampus, PFC

A maze for animals in which 8+ arms radiate from a central starting area, and certain arms may contain food at the end
- rats/mice are very good at this --\> can get them to go to a sequence of arms with no revisiting
- have to use allocentric cues (outside landmarks)

Morris Water Maze

assesses working and spatial memory
- hippocampus, could also be PFC
- place learning (platform location constant), matching-to-place learning (platform location moved), landmark learning (use cues in the pool)

rats dislike being in the water, have to find a submerged platform
- can have variable starting positions, in which the animal has to use allocentric cues (room cues around them)
- with constant positions, learning may be egocentric ("I go left")
- software can plot the animal movement over time

delayed-response task

A task in which information is provided, a variable delay is imposed, and then memory is tested.
- used to study working memory by testing monkeys' ability to hold information about the location of a food reward during a delay
- PFC (mainly), hippocampus
- instrumental
- could be cue/object-based learning or spatial (place)

ex. food put in one box, other box empty --\> boxes covered --\> variable delay --\> response (nonverbal, indicating where the food is through behavior)

Operant Learning

assesses the pursuit of goals (rewards)
- instrumental

using a Skinner box, animal has to do something to get a reward (goal-directed)
- over time, increase in rewarded lever pushing and decrease in non-reward lever pushing

Elevated Plus Maze

assesses anxiety
- rodents don't like open, high spaces, so prefer to spend their time in the two enclosed arms over the two open arms (species-specific)
- anxiolytics increase time in open arms

open field

assesses locomotor activity and anxiety

put into a box resembling an open field with edges and a least-covered center
- rodents tend to hug the walls (thigmotaxis), but will eventually venture out to the center
- can test time spent moving and times gone into center
\--- psychostimulants increase both (but binge doses do the opposite)

Forced swim test

assesses behavioral despair, depression

placing animals in a cylinder of water from which they cannot escape and recording the time it takes for them to abandon attempts to escape (latency to float)
- controversial, but may be some predictive validity (effective antidepressants increase latency to float)
- chronic stress, go into float faster
\--- maternal separation as stressor: see high CORT (cortisol/corticosterone), high float (quicker latency to float, longer floating)

Conditioned Place Preference

assesses an indirect measure of how much an animal likes a drug

a rat repeatedly receives a drug in one of two distinctive compartments then on the test the tendency of the rat (now drug free) to prefer the drug compartment is assessed
- in training, rat is injected with saline or cocaine in compartment with grid floor
- in testing, rat is dropped in blank compartment, see where it goes

rats can move freely, see if it goes back to the room where it got the drug
- use the difference in time spent in one chamber or another (Rats will spend the most time where they derive the most pleasure)
- do 3 doses
- can also do CPA (aversion) using LiCl

Drug self-administration method

assesses Reward Learning / Addiction
- a catheter is implanted in the rat's body, can connect to an infusion pump of a substance attached to a lever the rat can freely push to self-administer substance
- the quantity of the drug is usually capped
- animals will self-administer many drugs readily
- can also have it lever press multiple times wherein the cost of drug increases each time (ex. the first time, get drug after one press, the second time, get drug after five presses, etc.)

stereotaxic surgery

brain surgery using a stereotaxic apparatus to position an electrode or cannula in a specified position of the brain
- done under anesthesia
- drill through skull, use needle (cannula) to put something into the brain

stereotaxic atlas

a map of a brain

Exist for various species

stereotaxic instrument

a device used for the precise positioning in the brain of an electrode, syringe, cannula, or other device

Intracranial self stimulation (ICSS) and recording

Self-administered stimulation to an area of the brain with an electrode implanted
- plug-in animals; can both record and stimulate from electrodes
- used in Parkinson's as self-controlled deep-brain stimulation (used in patients where pharmacotherapy has stopped working)

In vivo electrophysiological recordings

can do single-unit measurements (of one neuron) or local field potentials (a population of thousands of neurons)

can train AI network to track movements using many videos

"ICSS" experiments

Rats will press a lever to electrically stimulate the Nucleus Accumbens, causing a surge of dopamine release in this region that is very reinforcing.

- Many behaviors (often compulsive) stimulate dopamine release in the Nucleus Accumbens (Sexual excitement and orgasm, gambling, video games, shopping)

- can also optogenetically activate pathways (deliver bursts of light)

In Vivo Microdialysis

Collect and measure neurotransmitter samples periodically in the living animal brain.
- collecting tube (semi-permeable membrane) inserted into mouse brain, still allowed to move freely but with tube attached
- pump and collector remove samples of CSF; pushed through probe, collect at end of testing (segregated in time)
- neural correlate with poor temporal resolution

afterwards, subsequently run HPLC post-hoc

HPLC

way to separate molecules following (post-hoc) in vivo microdialysis
- ex. can differentiate norepinephrine and serotonin by size/charge

Fast-scan cyclic voltammetry

in-vivo technique for determining the identity/concentration of neurotransmitters in a part of the brain by inserting an electrode and rapidly changing the voltage applied to the electrode
- uses micro-electrode to measure neurochemicals in near perfect time (good temporal resolution)
- deliver charge --\> get real-time NT measure due to electron transfer

Oxidative/reductive cyclic voltamagram is the signature that identifies dopamine
- Change in current at oxidative peak is proportional to dopamine concentration
- redox reaction gives the profile of changes in dopamine
- now, there are biosensors for dopamine

Imaging calcium in vivo, in freely-moving animals

- use fluorescent tag to calcium and some kind of imaging probe
- can be a proxy for action potentials (neural correlate)
\--- 3-5 spikes per Ca2+ trace
- has a single cell resolution and can track the same cells over time (can track single cells)
\--- but, can't know it's the same neuron
- can study something like learning as well as cells changing

get into the brain using a stereotaxic instrument (ex. cannula) under anesthesia

Ionotropic receptors

open when bound by a transmitter (also called a ligand-gated ion channel)
- ligand-gated
- very precise in time
- basis of optogenetics

Metabotropic receptors

recognize the transmitter but instead activate G proteins (usually activate longer-term cell cascade)
- transmitter binds --\> G protein released --\> activates second messenger
- longer in time scale
- basis of chemogenetics (DREADDs)

Neural correlates

fiber photometry (measuring GCAMP: bulk activity in 1 brain region), calcium imaging, in vivo microdialysis (phasic v. tonic dopamine), fast-scan cyclic voltametry

DREADDs

engineered G-protein coupled receptors which are activated by otherwise inert ligands
- have to mutate a receptor (point mutation on excitatory hM3Dq receptor, for example) and add fluorescence --\> introduce them with stereotaxic instruments into specific neurons --\> exogenous ligand cannot bind (null effect) --\> instead, respond to exogenous ligand (CNO)
- can do the same with hM4Di, just inhibitory
- need mutated receptors, fluorescence, and promoter tags/plasmid
- can target specific cell types/areas and introduce receptors into just those neurons (very precise)
- can have Cre mice (already mutated), but still have to introduce receptors

how do you introduce DREADDs?

DNA coding for the DREADD (hM3/hM4) and a fluorescent tag (GFP) are inserted into a viral vector --\> virus containing the DREADD DNA is injected into a specific brain region, where it begins expressing the DREADD as well as the fluorescent tag --\> CNO is administered (systemically: orally or via injection like IP or IC) and binds to the DREADD to selectively activate or inhibit the brain region depending on receptor type
- 90 minutes orally; hard to control plasma levels

not instantaneous (takes 3 weeks after surgery for receptors to express)

hM3Dq

Excitatory DREADD (mutant cholinergic receptor)

hM4Di

Inhibitory DREADD (mutant cholinergic receptor)

how can DREADDs be used?

can be used to map out circuits (ex. determine whether afferents from target region A are really involved in behavior)

Cre systems can be used to restrict DREADD expression to genetically defined neuronal populations
- can insert Cre-dependent DREADDs at a particular location, then insert the Cre in another location
- this Cre with travel in the opposite direction (retrograde - from the axon to the soma) and, if two locations are connected, cause Cre expression in first location (looking at a specific pathway)

receptors stay around for very long time

what receptor type does DREADDs work with?

metabotropic

what are the issues with DREADDs?

CNO may not be completely inert --\> reverse-metabolized to clozapine (an anti-psychotic) that may produce behavioral changes of its own
- may reactivate DREADDs or bind to endogenous receptors
- not insurmountable; need appropriate controls

Clozapine

atypical antipsychotic

blocks serotonin 5HT receptors and muscarinic ACh receptors as well as dopamine D2 receptors
- Fewer motor control effects

DCZ sometimes used instead of CNO in monkeys
- need monkeys to work before humans (targeting pathways)

What controls do you need for an inhibitory DREADDs experiment?

1. hM4D(Gi) + CNO
- experimental group (inhibitory receptor, systemic CNO)
2. hM4D(Gi) + vehicle
- group that expresses DREADD but no CNO
3. EGFP + CNO
- green fluorescence (control for expression of virus)
4. EGFP + vehicle
- null-null (not always used)

effort to do more within-subjects experiments

how do you validate your DREADDs technique?

can look through brain sections to see where you saw expression
- ex-vivo validation by activating in CNO bath

Optogenetics

uses genetic tools to insert light-sensitive ion channels into neurons to manipulate the activity of individual neurons
- have to be tethered to something that delivers light
- stereotaxically introduce opsins via a virus

what receptor type does optogenetics work with?

ionotropic

opsins

Some algae and bacteria produce light-sensitive proteins called opsins, which resemble the mammalian opsins found in light-receptor cells in our eye

Channelrhodopsin

responds to blue light by allowing Na+ions to enter the cell, depolarizing it (excitatory)
- can sometimes excite beyond normal physiological limits
- more action potentials when blue light is on

Halorhodopsin

responds to yellow light by allowing Cl-ions into the cell, hyperpolarizing it (inhibitory)
- usually more within physiological limits
- less action potentials when yellow light is on

how do you use optogenetics?

neurons are modified to express light-sensitive ion channels, enabling them to be specifically controlled using light (injected into brain)

- then, stimulating the brain with light, delivered by fiber-optic cables, can excite or inhibit those targeted neurons (expressing ChR2 or NpHR)

drawback of optogenetics

need behavioral controls to inserted light

how do you validate optogenetics?

in vivo electrophysiological recording or ex vivo.
- electrode records from single unit as evidence of activation
- Both the optical fiber and the electrodes are placed several hundred micrometers from the original virus injection site

In vivo micro positron emission tomography (microPET)

gives a radioactive footprint of drug binding
- can record over lifetime of receptor expression/binding (can do developmental studies)
- measures metabolic activity of the cells of body tissues (blood flow, NTs, radiolabelled drugs)
- but, risk exposure to radioactivity, so not used as much anymore

Radioligand Binding

Technique used to measure the affinity and relative density of receptors in a particular brain area by using a radioactively labeled ligand for the receptor.
- exposure to radioactive isotope to see how tightly bound to the receptor the ligand is (density/health of receptors)
- Brain region of interest is dissected out; a ligand is radioactively labeled and incubated with the tissue
- unlike microPET, have to use micro-punches of tissue to expose to radioactivity and look at binding (homogenize tissue)
\--- so, don't know the distribution)

radioligand concentration has a curve with a Kd and Bmax
- specific binding (% of Bmax) on y-axis

Drawback of Radioligand Binding

since you have to take micro-punches, you have to know where to go

Autoradiography

allows visualization of distribution of receptor bindingin slide-mounted brain sections
- take a coronal section --\> expose to isotope --\> look at the distribution of those receptors in the brain
- good for if you don't know where to look, but have to expose yourself to radioactivity

Kd

dissociation constant (rate at which ligand dissociates from receptor)
- measure of affinity
- concentration where half the ligand binding sites on the protein are occupied

Bmax

maximal number of receptors bound
- binding saturation; efficacy
- point at which all of the receptors are bound

Immunofluorescent staining

can detect protein in tissue:
- An antibody binds to the protein.
- Chemical treatments make the antibody visible.

type of IHC
- have to do microscopy

antibodies generated to bind to specific proteins and fluoresce (glow)

In situ hybridization

uses radioactive (or not) probes to detect neurons with a specific RNA or DNA (genetic material), identifying neurons where the gene has been turned on
- uses probes to label specific RNA or DNA

Immediate early genes (IEGs)

A class of genes that show rapid but transient increases in expression in cells that have become activated.
- IEGs such as c-fos are expressed when cells first become active.
- Visualized with immunocytochemistry, which capitalizes on the affinity of antibodies for specific proteins

ex. cells that I'm turning on with CNO are same ones activating with behavior

ex-vivo techniques

(outside the body) radioligand binding, autoradiography, immunofluorescent staining, in situ hybridization, IEGs

neuropharmacology techniques

stereotaxic surgery, ICSS, in vivo electrophysiological recordings, in vivo microdialysis --\> HPLC, fast-scan cyclic voltammetry, in-vivo calcium imaging, DREADDs, optogenetics, microPET

psychopharmacology/behavioral techniques

tail-flick test, T-maze, Effort T-maze, Radial Arm Maze, Morris Water Maze, delayed-response task, operant learning (Skinner box), elevated plus maze, open field, forced swim test, conditioned place preference

synthesis of dopamine

Tyrosine --\> (TH converts to) DOPA --\> (AADC) Dopamine

- dopamine can be metabolized into metabolites that can be assayed ex-vivo as a proxy for DA
\--- degradation proteins like COMT or MAO can also be assayed as DA proxy (turns into HVA)

rate-limiting enzyme of dopamine synthesis

tyrosine hydroxylase
- can increase/decrease with demand
- can label with Cre to get fluorescence (visualize DNA)

how is norepinephrine related to dopamine?

it is a metabolite of DA (through DBH)
- and epinephrine is a metabolite of NE

where do many precursors come from, generally?

diet (but limit to how much crosses the BBB)

what are dopamine and norepinephrine?

catecholamines (due to catechole nucleus) and monoamines (have an amine group)

- Neurotransmitters such asthese are taken back up from synapse and recycled

how does dopaminergic signalling work?

synthesis of dopamine --\> VMAT2 packages into vesicles (as VMAT2 increases, DA exocytosis increases) --\> binds to D1,D2 receptors --\> DAT reuptakes DA --\> VMAT2 repackages

DAT

transporter responsible for DA reuptake, can prepare it for ready release again

inhibitors of DAT (dopamine transporter) would acutely increase DA in synapse

VMAT2

protein that packages neurotransmitter into vescicles

inhibitors of VMAT2 will decrease DA in synapse

D2 receptor family

G-protein/metabotropic receptors (D2, D3, D4)
- activate Gi, decrease cAMP, decreases DA
- Also activates a G protein that opens K+which decreases excitability

can also occasionally be found on the presynaptic neuron (somatodendritic)

autoreceptors in substantia nigra, VTA (modulatory, negative feedback role)

postsynaptic receptors in areas dealing with motivated behaviors and learning (cerebellum thalamus, hypothalamus, hippocampus, etc)
- same as D1

D1 receptor family

G-protein/metabotropic receptors (D1, D5)
- active Gs, increase cAMP, increases DA

never found on presynaptic neuron

postsynaptic receptors in areas dealing with motivated behaviors and learning (cerebellum thalamus, hypothalamus, hippocampus, etc)
- same as D2

Dopamine and Effort-based Decision Making (Kurniawan): what are the roles of dopamine?

DA and (motivated) behavior:
- DA involved in a lot of cognition (as well as investment of effort and motivated behavior)

1. vigor control
- reaction time to get reward (effort is more sustained); faster RT
- "wanting"
- high effort response and choice

2. learning through reward prediction errors
- difference between expected and actual reward outcome
- DA is more involved in mismatch--when you expect one thing and get another (ex. anticipating when you'll get a reward and when you won't)

3. flexible approach behavior
- ability to flexibly re-engage to reward-relevant behavior after being distracted

4. salience (motivationally-significant)
- biologically salient events following an action
- formation of action-outcome association

Nigrostriatal DA Pathway

vital to motor movement
- start in substantial nigra (A9 cell cluster), goes through globus pallidus (voluntary movement), terminates in caudate-putamen (striatum)

very involved in Parkinson's

Mesolimbic DA Pathway

important in emotion, memory and reward processing
- start in ventral tegmental area (A10 cell cluster, midbrain), terminates in limbic system (amygdala, hippocampus, nucleus accumbens)
- in rodents, terminates in olfactory tubercule (important for rodents)

Mesocortical DA Pathway

important in cognition and decision making
- start in ventral tegmental area (A10 cell cluster, midbrain), terminates in cortex
- terminates in hippocampus, lateral septum (input to hippocampus), anterior olfactory nucleus (neocortex)

main circuit of prediction (conserved across species)

Dopamine and Effort-based Decision Making (Kurniawan): what are dopamine's main pathways? (go/no go)

D1 "DIRECT" Inhibition of Inhibition, Promotes behavior; GO (striatonigral)
- D1 striatum excited by cortex --\> inhibits Gpi/SNr (normally inhibitory), thereby exciting the thalamus

D2 "INDIRECT" Excitation of inhibition; Suppresses behavior; No Go. (striatopallidal)
- D2 striatum excited by cortex --\> inhibits GPe (normally inhibitory), thereby exciting Gpi/SNr (inhibitory) --\> thalamus inhibited

thalamus can feed back to frontal cortices, modulate pathways

Direct and Indirect DA Pathways

shows that dopamine is important in every motivated behavior (and impulse-control disorders like Tourette's and OCD)

newer version shows much more interconnectivity

DA D1 agonists and antagonists in Working Memory (Floresco: moving away from inverted U)

A: During training the rat retrieves food from four randomly selected arms.
- uses radial arm maze, rat should remember where it's been
- During the test phase after a delay, the blocked arms are now open and rewarded.

B: Both D1 antagonists and agonists (IC infusions) dose-dependently impaired performance (working memory, \# of errors)
- too much or too little D1 activity (there is optimal D1 activity \-- important in misuse of ADHD medication)

C: Extending the delay results in poorer performance and reduced PFC DA efflux.
- found, after various delay lengths, errors were increased and dopamine was decreased (may be moving away from optimal level on curve)
- however, D1 agonist can rescue performance acutely

DA D2 agonists and antagonists in risky decision making (Floresco: moving away from inverted U)

discriminating probabilities of reward (D2 also does motor engagement/inhibition)

A: Rats had to choose between a small/certain reward or a large/risky one.
- certain lever delivers 1 pellet per press
- risky lever may deliver 4 pellets, but probability decreases each time (ex. 100% --\> 50% --\> ...)
- generally, choice of risky lever goes down over time

B: Blocking D2 receptors in PFC increased risky choice
- increased risky choice at the most uncertain levels/lowest probabilities

C: Blocking D1 receptors reduced risky choice

D: D2 agonist abolished risky choice during the initial block and increased risky choice during the final block (interaction).
- flat curve (animals cannot discriminate risk)

E:D1 agonist induced nonsignificant increases in risky choice

these data show that reduced or excessive DA can affect different cognitive functions
- D2 receptor --\> risky choice
- D1 receptor --\> working memory

drugs that affect the dopaminergic system

- DOPA: Converted to DA in the brain
- Phenelzine: Increases catecholamine levels by inhibiting MAO
- AMPT: Depletes catecholamines by inhibiting tyrosine hydroxylase
- Reserpine: Depletes catecholamines by inhibiting vesicular uptake
- 6-OHDA: Damages or destroys catecholaminergic neurons (no therapeutic effect, model of Parkinson's)
- Amphetamine: Releases catecholamines
- Cocaine and methylphenidate: Inhibit catecholamine reuptake
\--- both these and amphetamine have a fast speed of onset (plays a role in addiction) and are two ways of increasing DA (and NE) in the synapse
- Apomorphine: Stimulates DA receptors generally (agonist)
- SKF 38393: Stimulates D1 receptors (agonist)
- Quinpirole: Stimulates D2 and D3 receptors (agonist)
- SCH 23390: Blocks D1 receptors (antagonist)
- Haloperidol (Haldol): Blocks D2 receptors (antagonist) (used in treatment of psychosis)

DAT gene knockout results

Animals lacking the Dopamine Transporter (DAT) (DAT -/- ) are hyperactive in an Open Field test.
- DAT involved in suppressing locomotion

Y-axis shows "locomotoractivity" (by breaking infrared beams in the Open Field test) over time (X-axis)
- could be hyperactivity to novel situations, as it decreases over time

D1 receptor knockout results

Mutant mice lacking D1 receptors do not show hyperactivity after cocaine
- mutants don't have cocaine-induced hyperactivity (maybe even a decrease)
- D1 could be modulating catecholaminergic-related locomotion

norepinephrine metabolized by...

MAO and COMT

roles of NE

important in arousal and attention

NE pathway

The locus coeruleus contains a dense cluster of norepinephrine neurons with widespread innervation
- starts in LC (A6, largely-conserved blue spot, soma of system), innervating the hippocampus and cortex (as well as many other areas like the thalamus)
- PFC --\> prediction

what three brainstem regions is NE released in?

Locus coeruleus (pons)

Lateral tegmental system (midbrain)

Dorsal medullary group

NE receptors

Noradrenergic fibers from the locus coeruleus project broadly to cortex
- five subtypes, all metabotropic (alpha and beta); both excitatory and inhibitory, depending on receptor subtype
- NE systems modulate processes including mood, arousal (attention), and sexual behavior (readiness/preparedness to engage with environment)

possible role of LC in attention results

shows that performance is best at some "optimal" tonic LC activity level
- regulates balance of focused and flexible behavior
- inverted U dose-response curve
- phasic/low tonic LC activity associated with drowsy and non-alert
- high tonic LC activity associated with scanning labile attention ("everything is important")
- optimal level at top of curve associated with focused attention; balance of phasic and tonic activity (PFC)

tonic \= baseline (opposite is phasic --\> bursts)
- want both tonic and phasic for signal-to-noise ratio

this is a Yerkes-Dodson Relationship (also seen for stress and other drugs like caffeine)

LC & attention study with DREADDs

Chemogenetically stimulating LC tonic activity impaired task performance, increased response times and omission (opting-out) rates in rats
- increase tonic activity, decrease signal-to-noise ratio, impair performance
- pupil dilation could mean more LC activity (paying more attention)

what disorders is serotonin implicated in?

Affective Disorders (depression & bipolar), Anxiety Disorders, Circadian Rhythm Disorders, Eating Disorders, Migraines, OCD, Premenstrual Syndrome (PMS), PTSD, Schizophrenia, Sexual Disorders, Sleep Disorders, Stress, Substance Abuse Disorders, Aggression (Conduct Disorder, Impulse Control Disorder)

SSRIs have off-label use for some of these
- stress regulation, metabolite of 5HT is melatonin, pain/chronic fatigue
- not one single behavior/cognition

how does serotonin signalling work?

tryptophan --\> (TPH enzyme) serotonin --\> VMAT2 packages into vesicles --\> binds to postsynaptic (2A) or presynaptic (1A) receptors --\> reuptake by SERT --\> reuptake or breakdown by MAO

serotonin rate limiting enzyme

tryptophan hydroxylase (TPH)

SERT

serotonin transporter (reuptake)

Serotonin precursor

Tryptophan

chemical characteristics of 5HT (serotonin)

indoleamine and monoamine

serotonin roles

enigmatic --\> responsible for everything, responsible for no one thing
- very important in mood and cognition (a variety of functions within those domains)
- LSD resembles serotonin (5-HT) and 5-HT itself is a psychotropic agent/can produce hallucinogenic effects (acts on 5HT2A receptors)

serotonin receptor subtypes

13 second-messenger "metabotropic" receptors (of which 1A and 2A are most prominent)
- 1 ligand-gated "ionotropic" (not much known)

- 5HT1A - GPCR autoreceptors; regulate release
- 5HT2A - GPCRs; signal transduction and plasticity

PCPA

parachlorophenylalanine

selectively blocks 5-HT by irreversibly inhibiting tryptophan hydroxylase (blocks 5HT production)
- One or two high doses of PCPA can reduce brain 5-HT levels in rats 80-90% for a long time
- experimental drug administered systemically to selectively decrease 5HT (never given therapeutically)
- can study what 5HT is important (and not important) for

serotonin depletion & effects on reward learning and motivation

impaired reward learning and intact motivation after serotonin depletion in rats
- a large dose of PCPA impaired approach to conditioned stimuli (autoshaping); not learning
\--- but, not impaired at effort-based decisions (no DA changes), but slowing of response
\--- correlated with decrease in frontal cortex 5HT

Tryptophan entry into the brain

dietary tryptophan can sometimes cross BBB, then be converted to serotonin