Neuropharmacology of G-protein coupled receptors (cannabis):

Question 1

Give an overview of cannabis, including the plants it’s derived from, industrial use, recreational use, and legal implications

Answer 1

Plant-based products derived from the flowers of Cannabis sativa (high Δ9-THC, low CBD), Cannabis indica (lower Δ9-THC, higher CBD), and Cannabis ruderalis (low Δ9-TBC, high CBD)

Industrial usage:
	Hemp
	Fibre used in paper and textiles
	Foodstuff
Has been used in religious rituals

Recreational drug:
	Dried flowers (marijuana)
	Resin (hashish)
	Liquid extracts (oil)
Usually consumed via smoking or ingestion

UK: Class B drug = possession results in fine or imprisonment

Question 2

Outline the psychotropic and somatic effects of cannabis use

Answer 2

Psychotropic:
General change in perception
Euphoria 
Increased appetite
Relaxation
Increased appreciation of music
Enhanced recollection (episodic memory)
Increased awareness of sensation
(At higher doses:)
Auditory and/or visual illusions 
Hallucinations
Ataxia (lack of voluntary coordination)

Somatic:
Increased heart rate
Dry mouth
Reddening of the eyes
Reduction in intra-ocular pressure
Muscle relaxation
Electroencephalography (EEG) – more persistent alpha waves

Question 3

What are the potential medical benefits of cannabis use?

Answer 3

• Bronchodilation – bronchial asthma
• Antiemetic effect – prevention of nausea/vomiting caused by anticancer drugs
• Appetite stimulation – palliative care for anorexia caused by opioids, antiviral drugs, AIDS-related illnesses, terminal cancer
• Analgesia – cancer pain, post-operative pain, phantom limb pain
• Decreased spasticity/ataxia/muscle weakness – multiple sclerosis, cerebral palsy, spinal cord injuries
• Decreased intraocular pressure – glaucoma

Question 4

What risks does cannabis use carry for mental and physical health? What about the societal risks?

Answer 4

Mental health:
Triggering psychosis
Increased risk of schizophrenia
Anxiety
Paranoia
Substance abuse disorders

Physical health:
Lung problems
Lung cancer (cannabis often combined with tobacco)
Increased heart rate/blood pressure  heart problems
Foetal developmental issues (if consumed when pregnant)

Legal/societal risks:
Prison/fine
Criminal record

Question 5

Briefly outline the chemistry of cannabis

Answer 5

Includes 483 known compounds
66 of these are cannabinoids – compounds only found in the cannabis plant, including:
Δ9-tetrahydrocannabinol (Δ9-THC)
Cannabidiol (CBD)
These two have received the most attention; both of them bind cannabinoid receptors

Question 6

Give an overview of the cannabinoid receptors - canonical and non-canonical

Answer 6

• 2 canonical cannabinoid receptors:
o CB1 receptor – GPCR; 7 TMDs connected by 3 intracellular and 3 extracellular loops
 Mainly expressed in the brain, kidneys, liver, and lungs
o CB2 receptor – GPCR; 7 TMDs connected by 3 intracellular and 3 extracellular loops
 Mainly expressed in immune cells
• Possibly non-canonical receptors:
o GPR55
o GPR119
o GPR18

Question 7

Outline the transduction mechanisms of CB1Rs. Use a diagram if you wish.

Answer 7

CB1R = GPCR
Gi/o-coupled – inhibits neurotransmitter release:

Positively regulate GIRKs (βγ subunit)
Negatively regulate Ca2+ channels (α subunit)
Inhibit adenylyl cyclase –> reduced i[cAMP] (α subunit)

Question 8

How does activation of CB1Rs lead to psychotropic effects?

Answer 8

Neocortex – responsible for higher cognitive functions and the integration of sensory information

Hypothalamus: controls appetite, hormonal levels, and sexual behaviour

Basal ganglia – involved in motor control and planning, as well as the initiation and termination of actions

Ventral striatum – involved in the prediction and feeling of reward

Amygdala – responsible for anxiety, emotion, and fear

Hippocampus – important for memory and the learning of facts, sequences, and places

Brainstem and spinal cord – important in the vomiting reflex, and pain sensation

Cerebellum – centre for motor control and coordination

Δ9-THC binds the CB1Rs found here, altering function and producing psychotropic effects

Question 9

Where are CB1Rs located? How was this mapped>?

Answer 9

• CB1R locations were originally mapped with [3H]-CP-55940 (a tritiated CB1 agonist) autoradiography; then with in situ hybridisation (RNA locations)
• CB1Rs have the highest density of any GPCR
• The basal ganglia and cerebellum are among the highest-labelling areas; though the number of CB1Rs is high in cortical and hippocampal areas, too
• CB1Rs are found presynaptically, and inhibit neurotransmitter release

Question 10

Outline the tritiated ligand-labelled assay (binding assay) for measuring receptor pharmacology. Use a diagram if you wish

Answer 10

Ligand binds receptor ∴ you can measure its radioactivity
Then expose an unlabelled ligand
You can measure the loss of radioactivity that occurs
Can be done in brain tissue/membranes generated from cells
Allows you to measure the binding properties of receptors by trying to displace the tritiated ligand with other ligands

Question 11

Outline the functional assay for measuring receptor pharmacology. Use a diagram if you wish

Answer 11

• Normally: agonist binds –> G-proteins bind receptor –> GDP release
• Radiolabelled GTP binds the receptor –> measure the amount of radioactivity that’s bound; tells you if G-protein has been activated, and this is activation/inhibition – shows the difference between an agonist and an antagonist
• More of a functional assay than the tritiated assay

Question 12

Outline the pharmacology of THC

Answer 12

Binding affinity (Ki) is usually reported to be around 10-50nm

Inhibitory EC50 values are in a similar range – 10-50nm

Evidence suggests that Δ9-THC is a partial agonist compared to synthetic ligands; this means it has a lower efficacy than the synthetic ligands

Question 13

Outline the pharmacology of cannabidiol

Answer 13

• Acts as a functional antagonist at CB1Rs

* There’s evidence that cannabidiol acts as an inverse agonist at CB1 receptors

Question 14

Explain the possible mechanisms by which CB1 agonists may inhibit GABA-mediated synaptic transmission

Answer 14

• Possible mechanisms for this:
o The CB1 agonist reduces the postsynaptic sensitivity to GABA (this would be a postsynaptic effect)
o The CB1 agonist reduces presynaptic release probability (this would be a presynaptic effect)
 Thereby:
• Reducing the probability of an action potential firing
• Inhibiting release mechanisms
• Inhibiting action-potential (voltage)-dependent Ca2+ channel activity
• Inhibiting action-potential (voltage)-independent Ca2+ channel activity

Question 15

What evidence is there for CB1 agonists inhibiting GABA-mediated synaptic transmission?

Answer 15

• Miniature synaptic currents can be used to determine the synaptic locus of drug effect:
o Measures spontaneous quantal release – each event corresponds to the effect of a single vesicle (quanta)
 If:
• The amplitude of the events is reduced – this is a postsynaptic effect
• The frequency of events is reduced – this is a presynaptic effect
• No effect – this is presynaptic and the effect is action potential-dependent

Question 16

What does the evidence for CB1 agonists inhibiting GABA-mediated synaptic transmission tell us?

Answer 16

• CB1 agonists do not affect:
o Miniature IPSC frequency
o Miniature IPSC amplitude

But, they do reduce the evoked IPSCs.
Therefore, they reduce action-potential-dependent GABA release through inhibition of Ca2+ channels; therefore the effect is on the presynaptic neuron

Question 17

Briefly outline the 2 main endocannabinoids

Answer 17

• Anandamide (AEA) and 2-arachidonylglycerol (2-AG) are both endogenous agonists at CB1 receptors – they are endocannabinoids
• 2-AG is more abundant in the human brain and has a higher efficacy, but similar potency, to AEA
• Neither appear to act as classical neurotransmitters; they’re lipid soluble so aren’t stored in vesicles

Question 18

What does DSI stand for?

Answer 18

Depolarisation-induced Suppression of Inhibition

Question 19

What causes DSI? Which cells has it been observed in?

Answer 19

•	The depolarisation of a postsynaptic cell results in a transient reduction in GABAergic transmission
•	This has been observed in:
o	Cerebellar Purkinje cells
o	Medium shiny neurons of the striatum
o	Hippocampal pyramidal cells

Question 20

What is the evidence for DSI being mediated by a retrograde messenger?

Answer 20

• Evidence that DSI is mediate via a retrograde messenger:
1. The increase in postsynaptic i[Ca2+] is necessary and sufficient for the induction of DSI
a. Blockers of Ca2+ entry block DSI
b. Postsynaptic Ca2+-chelating agents prevent DSI
c. Artificially raising i[Ca2+] (AKA, uncaging) induces DSI
2. The frequency (number) of spontaneous inhibitory synaptic events is reduced – fewer vesicles are released presynaptically
3. Postsynaptic sensitivity to exogenously applied GABA is not affected by depolarisation
• 1 = postsynaptic trigger, 2+3 = presynaptic effect
o Postsynaptic trigger  presynaptic effect
4. DSI isn’t blocked by classical neurotransmitter receptor antagonists
5. DSI is not synapse- or cell-specific
• A factor released from cell 1 suppresses inhibition on cell 1 and 2

Question 21

What is the evidence that endocannabinoids mediate DSI?

Answer 21

Evidence for this:
o The synthesis and release of endocannabinoids is dependent on Ca2+
o CB1 agonists depress GABAergic transmission
o DSI is blocked by the CB1 antagonists
o DSI is mimicked by blocking endocannabinoid uptake
o DSI is absent in the CB1-knockout mouse

Endocannabinoids are released in response to postsynaptic depolarisation (action potentials) –> suppression of GABA release