The Serotonin System and Fluoxetine

Question 1

Explain what 5-HT acts as, where it is found, and which molecule starts the biochemical synthesis pathway.

Answer 1

Serotonin = 5-HT
o These terms are interchangeable

Serotonin acts both as a neurotransmitter and a hormone
• 90% of 5-HT is found in the gut
o 90% of this comes from enterochromaffin cells; mainly in the stomach and the gut, interspersed with mucosal cells
o 10% comes from neurons
o Remaining 10% found in the CNS; particularly the midbrain

A lot of 5-HT is found in platelets – it accumulates here from plasma
o Released during agitation and vasoconstriction wherein it acts as a platelet agonist

Tryptophan is the start of the biochemical pathway – acquired from the diet in eggs, fish, soybeans, cheese, seeds

Question 2

With the use of a diagram to aid you, describe the biosynthetic pathway responsible for serotonin synthesis

Answer 2

• Tryptophan hydroxylase (TPH) begins the reaction, hydroxylating tryptophan to 5-hydroxytryptophan
o This is the rate-limiting step of the reaction
• L-aromatic acid decarboxylase (or dopa decarboxylase) decarboxylases 5-hydroxytryptophan into 5-hydroxytriptamine – this is 5-HT or serotonin
• Monoamine oxidase oxidises 5-hydroxytriptamine to an aldehyde
o This is then converted to 5-hydroxyindoleacetic acid (5-HIAA) by aldehyde dehydrogenase
o 5-HIAA is excreted through urine

https://www.google.com/url?sa=i&url=https%3A%2F%2Fwww.researchgate.net%2Ffigure%2FBiosynthetic-pathway-of-serotonin_fig1_332275176&psig=AOvVaw3LJHnhidEXpZ-LABxlmeGT&ust=1577283221793000&source=images&cd=vfe&ved=0CAIQjRxqFwoTCPjY4IO8zuYCFQAAAAAdAAAAABAD

Question 3

Briefly outline the projections of serotonergic neurons and their point of origin, as well as the implications this has on the roles of serotonin

Answer 3

The serotonin system projects from the midbrain to many other brain areas
• Originates in the Raphe nuclei
o 9 nuclei, B1-9; these contain most of the serotonin-containing neurons
o Are located along the midline of the brainstem and centre on the reticular formation
o The axons of these neurons reach almost every part of the CNS
 Axons of the lower raphe nuclei terminate in the cerebellum and the spinal cord
 Axons of the higher nuclei spread through the brain
 This explains the multifaceted roles of serotonin:
• Modulating cognition
• Reward
• Learning
• Memory
• Other physiological processes

Question 4

Explain the classification of 5-HT receptors; the 7 receptor subtypes, their location, function, and signalling pathway

Answer 4

o Sequencing
o Signal transduction mechanisms
o Pharmacology
o 5-HT receptor knockout mice
• 7 receptor subtypes:
o 5-HT1-7¬
o 6 of these are GPCRs, with classic GPCR structure – 7 TM domains
o 5-HT3R is ionotropic
 It’s a cation channel, facilitating movement of Na+ and K+
• (Mice have an extra gene – 5-HT5B)
• Many polymorphisms are present in these receptors, contributing to disease
• The relationship of receptors with pharmacological agents is promiscuous, meaning it’s hard to interpret data from studies
• KO mice have subtle phenotypes, suggesting the receptors fine-tune processes rather than directly regulate them

(table in lecture from 4/11/19)

Question 5

With the aid of a diagram, outline the structure and function of the serotonin transporter (SERT)

Answer 5

Serotonin transporter:
o Aka SERT
o Na+-coupled transporter
 Transports serotonin into the neuron alongside Na+
• Fluoxetine is an SSRI (selective serotonin reuptake inhibitor)
o Acts by blocking the serotonin transporter (SERT)
o Treats:
 Major depression
 Bulimia nervosa
 Obsessive compulsive disorder
o Fluoxetine blocks the serotonin transporter
 Therefore artificially increasing the availability of 5-HT in the synaptic cleft
 Fluoxetine is very specific – it doesn’t affect the availability of dopamine or noradrenaline
• SERT blockers are generally safe, and can’t be used for overdosing
o Unless these are combined with a monoamine oxidase inhibitor

Diagram: in lecture notes

Question 6

Outline the purpose, side effects, and pharmacology of SSRIs including their effects on receptors

Answer 6

Are used clinically to treat:

1. Major depressive disorder – citalopram, fluoxetine, fluvoxamine, paroxetine, sertraline
2. Generalised anxiety disorder – escitalopram (stereoisomer of citalopram)
3. Obsessive-compulsive disorder – escitalopram, paroxetine
4. Post-traumatic stress disorder - paroxetine
5. Social anxiety disorder - paroxetine
6. Bulimia nervosa – fluoxetine, sertraline

Side effects include:

1. Nausea
2. Vomiting
3. Constipation
4. Diarrhoea
5. Insomnia 

1-4 are predominantly related to the expression of 5-HTRs in the gut

SSRIs take ~14 days to work, this is due to the effects on receptor localisation:
5-HT(1A)R is an inhibitory pre-synaptic receptor
- Important for mood and behaviour
- Found in the brainstem (in the Raphe nuclei)
- Hippocampus
- Hypothalamus
- Amygdala
- Entorhinal cortex
It acts as an autoreceptor and decreases 5-HT release – working against the intended effects of SSRIs
Eventually, this decreased with chronic SSRI treatment
Chronic treatment –> desensitisation of the 5-HT1AR –> reduced inhibition of 5-HT release ∴ more 5-HT is released, magnifying the effects of the SSRI

This is known as the therapeutic threshold and takes ~14 days to reach

Question 7

What are other uses for 5-HTR modulating drugs?

Answer 7

Migraines are thought to be caused by cranial vasodilation
o 5-HT1B/D/FRs are expressed on cerebrovascular smooth muscle
 Agonists can induce cerebral vasoconstriction
o 5-HT1B/D/FR agonists include:
 Ergot alkaloids
 Triptans
• 5-HT(1B)R is also found in peripheral smooth muscle, and is contraindicated in cardiovascular/cerebrovascular disease and hypertension
 These inhibit the release of vasoactive peptides, preventing vasodilation

5-HT2R antagonists are used as anti-hypertensives, e.g. ketaserin

5-HT3R antagonists are used for the prevention of nausea and vomiting, e.g. ondesetron and tropisetron

5-HT2CR agonist – lorcaserin; used to treat obesity in the USA
o 5-HT2CRs are located on the pro-opiomelanocortin (POMC) neurons in the hypothalamus
o Activating POMC neurons supresses appetite
o Links to Prader-Willi syndrome – a genetic form of obesity, caused by truncation of the 5-HT2CR and reduced expression of this receptor on the cell surface

Question 8

Give an overview of why there are pre-clinical methods for assessing drug interactions with CNS reward pathways, give examples of 4 of these tests and explain what they show and how drug administration affects the assay

Answer 8

Most studies have used rats:
	Wistar
	Sprague Dawley
	Long-Evans
	Some use mice, or:
	Squirrel monkeys
	Rhesus monkeys

Fluoxetine has been used in many pre-clinical studies to investigate reward-related behaviours, including drug addiction (reinforced behaviours)

Forced swim test:
Place rats in container with water
Rat will try to swim/climb to safety
This continues until the rate stops swimming and becomes immobile – it’s then said to be in a state of despair
The more time the rat spends in an immobile state, the more depressive-like its behaviour is said to be
Anti-depressants reduce immobility time (prolonging the escape response)
Latency = time until the first period of immobility; antidepressants increase latency

Sucrose preference test:
Depressive-like behaviours are induced by (e.g.) chronic stress
Restraint stress
Early-life stress
Intruder stress
Animals have access to water, and water+sucrose
Reduced preference for sucrose = depressive-like behaviour
Antidepressants increase the sucrose preference relative to depressive animals, but not in healthy animals (doesn’t induce a sweet tooth)

Self-administration:
Cocaine:
Control = saline solution, this separates out random pressing
This is often combined with a light/sound to indicate drug availability
Reinforcing drugs = animals want to repeat the administration
Pre-treatment with fluoxetine reduces cocaine self-administration ∴ reduces reinforcing

Electrical:
Stimulation of lateral hypothalamic neurons (these project to the VTA and then the nucleus accumbens) –> increase in dopamine release = reward stimulus

Conditioned place preference:
3 distinct chambers with a different colour/texture
‘conditioned’ with positive/negative reinforcement e.g. cocaine (positive), pain (negative)
The animal is allowed to explore freely, and the effects of the test drug are evaluated

Progressive ratio test:
Rats are trained to press a lever for a reward
To get the reward they have to work progressively harder – press the lever more
This continues until the ‘breakpoint’
Reward isn’t worth effort
Increased breakpoint (n) = more motivated
Decreased breakpoint = less motivated
E.g.:
5-HT2cR inverse agonist
Mice trained to press levers for evaporated milk
Treated with/without 5-HT2C modulator SB242084
Modulator seemed to increase motivation