Serotonin Transporter and Pharmacology

What does the serotonin transporter (SERT) do, and what does an SSRI do to it?

It takes 5-HT back into the presynaptic neuron; SSRIs block it, so 5-HT stays longer in the synapse

What is SERT encoded by?

SLC6A4, with S and L promoter variants (5-HTTLPR)

How was the SERT knockout rat made?

1. ENU alkylates DNA causing point mutations

2. Offspring are screened for mutations in the candidate gene

3. In SERT it was a C to A transversion at position 3924 (TGC > TGA), creating a stop codon

How was the SERT KO rat validated neurochemically?

1. Hippocampal microdialysis

2. Baseline 5-HT is much higher in SERT KO rats

3. Citalopram raises WT 5-HT to about the KO level

What did [3H]WAY-100635 autoradiography show about 5-HT1A receptors in SERT KO rats?

Lower 5-HT1A binding in SERT KO in cingulate cortex, CA2, CA3 and dorsal raphe, but not in septum, piriform cortex or CA1

What happens when a 5-HT1A agonist acutely stimulates the autoreceptor?

Inhibits firing, so less 5-HT released

What happens when a 5-HT1A agonist acutely stimulates the postsynaptic receptor?

Activation of the postsynaptic neuron

How does the 5-HT1A agonist have opposing effects at once?

It mimics 5-HT postsynaptically but shuts down the endogenous 5-HT supply through the autoreceptor

What happens to the 5-HT1A autoreceptor in a SERT KO rat, and why?

No transporter gives high synaptic 5-HT, continuous autoreceptor stimulation, desensitisation of the autoreceptor, and so continuous firing of the serotonergic neuron

When are the postsynaptic receptors also less sensitive?

When long-term high 5-HT desensitises receptors on both sides of the synapse

Outline a pharmacological experiment aimed at one specific receptor (the lecture's 5-HT1A example)

1. Choose selective agonist and antagonist for the receptor

2. Choose a quantifiable read-out linked to the receptor

3. Use unstressed measurement (telemetry)

4. Use dose-response with a vehicle control; agonist alone, antagonist alone, then antagonist + agonist

5. Compare genotypes

What does it mean when the antagonist blocks the agonist effect?

The effect is specific to that receptor

In the 5-HT1A body-temperature experiment: what was measured, how, and which doses of flesinoxan?

1. Body temperature via implanted radiofrequency telemetry transmitters

2. Flesinoxan 0, 0.3, 1, 3 and 10 mg/kg

3. 60 min baseline then 4-5 h recording

What do 5-HT1A agonists cause in terms of body temperature?

Hypothermia

Wild-type rats given flesinoxan: what happens to body temperature?

1. Saline causes stress-induced hyperthermia (SIH)

2. Flesinoxan reduces SIH dose-dependently and at higher doses causes hypothermia (about 34.6 C at 10 mg/kg)

This concludes that 5-HT1A receptor stimulation lowers body temperature

SERT KO rats given flesinoxan: what happens?

SIH is reduced and there is no hypothermia

WAY100635 (5-HT1A antagonist) alone: effect on body temperature in WT vs SERT KO?

WT: silent antagonist, no effect on temperature

KO: increased and prolonged hyperthermia

WAY100635 + flesinoxan together: what does it show?

WT: WAY100635 blocks flesinoxan's hypothermia, confirming it is 5-HT1A-mediated

KO: it blocks flesinoxan's effect on SIH and shows increased hyperthermia

Which 5-HT1A receptor populations are linked to hypothermia?

Probably via postsynaptic 5-HT1A receptors

Which 5-HT1A receptor populations are linked to stress-induced hyperthermia?

Probably via presynaptic 5-HT1A autoreceptors

Cocaine-induced locomotor activity: SERT KO vs WT?

Much higher locomotor activity in SERT KO than WT after 20 mg/kg cocaine (little response at 10 mg/kg in both)

What did 8-OH-DPAT and WAY100635 show about cocaine hyperactivity?

• 8-OH-DPAT induced more activity in WT (KO less sensitive)

• Cocaine hyperactivity was prevented by WAY100635 in both genotypes, so it is 5-HT1A-dependent, and stronger in KO

Reinforcing properties of cocaine in SERT KO rats: what did self-administration (FR, PR), extinction and CPP show?

• FR: more active responses; PR: higher breakpoint (works harder, clearest at 0.9 mg/kg/infusion)

• Extinction: keep responding to cues without cocaine

• CPP: larger place preference

Microdialysis in nucleus accumbens and hippocampus (Verheij et al. 2014): what differs in SERT KO after cocaine?

• Basal 5-HT is higher in KO but the cocaine-evoked 5-HT increase is much smaller

• Dopamine and noradrenaline (basal and evoked) are unchanged, in both nucleus accumbens and hippocampus

Summarise how neurotransmitter systems influence each other in the SERT KO story

• 5-HT levels set 5-HT1A sensitivity (desensitisation)

• 5-HT1A receptors are needed for cocaine hyperactivity

• A blunted serotonin response to cocaine (with normal dopamine) accompanies enhanced cocaine reward