Module 1 - Pain

Question 1

Describe the pathway from trauma to cortex:

Answer 1

Trauma is detected by receptors which release substance P/ histamine/ serotonin/ bradykinin/ prostaglandins/ NGF. The sensory neuron travels to the dorsal root ganglion. Once in the spinal cord, it synapses with an interneuron using neurotransmitters such as glutamate/ substance P/ GABA/ Enk. The interneuron travels up the spinothalamic tract to the cortex (mainly primary sensory and secondary sensory cortex and the anterior part of the insula and the cingulate gyrus.

Question 2

Give some examples of major nerves and their targets:

Answer 2

Median nerve (arms and hands)
Gastric nerve (stomach)
Sciatic nerve (legs and feet)
Trigeminal ganglion neurons innervate facial nerves (face)

Question 3

What is the difference in structure of the neurons involved in chronic and acute pain?

Answer 3

Small diameter, unmyelinated c fibre neurons for chronic pain (2mph). Medium diameter, thinly myelinated A-delta fibre neurons for acute pain (40mph). Large diameter, myelinated A-beta fibre neurons for touch (240mph). A-beta fibres (touch) can experience pain in some patients - connect to wrong lamina and then to a secondary neuron?

Question 4

What is the specificity theory of pain?

Answer 4

Descartes’ view: “the intensity of pain is directly related to the amount of associated tissue injury”
Generally applicable in acute pain, not chronic or phantom.

Question 5

What is gate control theory of pain?

Answer 5

Ronald Melzack and Patrick Wall 1960s:
“When the gates are opening, pain messages ‘get through’ more of less easily and pain can be intense. When the gate closes, pain messages are prevented from reaching the brain and may not even be experienced.”
Eg. rubbing area activates A-beta fibres which can override A-delta and c fibres because they’re faster.
There is also an inhibitory neuron from the brain to reduce the perception of pain.
(Diagrams include an interneuron between these fibres but it is imaginary but is still included because it explains some phenomena. The diagram also shows all the neurons synapsing to one secondary neuron but they connect to different neurons.)

Question 6

Where in the spinal cord do the different fibres terminate?

Answer 6

I-VI layers/laminae in dorsal horn, VII-IX in ventral horn and lamina X around central canal (additional column of cells).
C nociceptive afferents terminate mainly in laminae I and II.
A-delta afferents terminate mainly in laminae I, II and V.
A-beta afferents terminate mainly in laminae V, VI.
Interneurons in laminae I, II and III are GABA-rich and mediate gate control in the dorsal horn by synapsing on neurons that contain glutamate and substance P.

Question 7

Describe some of the biochemistry of bradykinin and its receptors:

Answer 7

It is a polypeptide made from kininogen by proteolysis in blood in response to tissue damage/blood coagulation.
Bradykinin receptors are B1 and B2, G-protein coupled receptors. Activation can lead to phospholipase or protein kinase C or arachidonic acid (from cellular phospholipid bilayer), and therefore prostanoids.

Question 8

Describe some of the biochemistry of prostanoids and its receptors:

Answer 8

Prostaglandins [PGE2] synthesised/released from nociceptive neurons in response to tissue damage. Binds to DP1, DP2, EP1, [EP2], EP3, EP4, FP, IP (G-protein coupled) receptor expressed at free nerve endings -> adenylate cyclases and increase intracellular cAMP -> protein kinase A activation -> phosphorylate channels.
Cyclooxygenase is rate-limiting enzyme of PGE2. COX-2 can be induced by proinflammatory cytokines.o

Question 9

How does NGF and TrkA affect nociceptive neurons?

Answer 9

NGF regulates peripheral sensitivity of nociceptive neurons - mechanical and thermal hyperalgesia. Also increases functional sodium channel expression.
TrkA is selectively expressed in unmyelinated nociceptive sensory neurons.

Question 10

Describe the biochemistry behind TRPV1:

Answer 10

A non-selective cation channel activated by capsaicin, noxious heat and low pH. Detects internal body temperatures as well so important for homeostasis and antagonists have not passed trials. Expressed exclusively in nociceptive sensory neurons. From cold to hot: ANKTM1 - TRPM8 - TRPV4 - TRPV3 - TRPV1 - TRPV2.

Question 11

Describe the biochemistry behind P2X3:

Answer 11

Non-selective cation channel activated by ATP. Responsible for ATP-evoked nociceptor activation.
Knockout mice show some deficiency in detection of some painful stimuli.

Question 12

Describe the biochemistry behind P2X4:

Answer 12

ATP activates microglial P2X4. Microglia release BDNF which acts on neurons to reduce expression of KCC2 (anion transporter) which increases intracellular Cl-. GABA is excitatory rather than inhibitory.

Question 13

Which parts of an action potential are mediated by calcium and which by sodium?

Answer 13

Sodium causes more depolariation.

Calcium causes more prolonged depolarisation.

Question 14

What is the structure of a voltage-gated calcium channel?

Answer 14

6 subunits x 4 domains: 24 alpha transmembrane for ion pore + accessory proteins of gamma (1-8) and beta (1-4)) and alpha2 and alpha2delta (1-4).

Question 15

What is the structure of a voltage-gated sodium channel?

Answer 15

6 subunits x 4 domains = alpha region. Forms channel, has accessory proteins such as 1 or 2 beta (1-4) associated with it to alter voltage dependence. Nav1.1-1.3 in brain and Nav1.7-1.9 in dorgal root ganglia.

Question 16

Which sodium channels can’t be blocked by tetradotoxin? Which amino acids confers TTX sensitivity?

Answer 16

Nav1.5, 1.8, 1.9. Phenylalanine and tyr(osine?)

Cysteine and serine do not confer TTX sensitivity. Nav1.8 Ser-> Phe increases TTX sensitivity by 21,000.

Question 17

How does the gating mechanism work?

Answer 17

Sliding helix model. Paddle model. ?

Question 18

Describe the difference between the sodium currents in Nav1.7-1.9:

Answer 18

Nav1.7: fast TTX-S, steep slope, doesn’t last very long - slowly inactivates and sets threshold for generation of action potentials. KO mice show reduced responses to mechanical, not thermal, stimuli (Nassar 2004)
Nav1.8: Slope is just as steep but it lasts longer - re-primes rapidly and contributes to continuous firing activity during sustained depolarisations.
Nav1.9: Much smaller slope but lasts a lot longer - activates at around -70mV and the persistent current likely has a role in setting resting membrane potential. KO mice show normal responsiveness to mechanical and thermal stimuli (Priest 2005)

Question 19

What symptoms are associated with mutations in Nav1.1 and Nav1.2?

Answer 19

Generalised epilepsy with febrile seizures with severe myoclonic epilepsy in infancy. Familial hemiplegic migraine and familial febrile convulsions.

Question 20

What symptoms are associated with mutations in Nav1.4?

Answer 20

Paramyotonia congenita, myotonia congenital, myotonia fluctuans. Hyperkalaemic periodic paralysis. Myasthenic syndrome.

Question 21

Which channel is associated with the complete inability to sense pain and paroxysmal extreme pain disorder and inherited erythromelalgia?

Answer 21

Nav1.7 (deletion or translocation for inability)
Paroxysmal extreme pain disorder is autosomal dominant, associated with autonomic dysfunction, skin flushing.
Inherited erythromelalgia is erythema and burning pain triggered by mild heat.

Question 22

What factors affect Nav1.8 currents and how?

Answer 22

PGE2 increases TTX-resistant Na+ current and causes a hyperpolarising shift of its activation curve. They modulate these currents in sensory neurons via protein kinase A cascade (England 1996)
Intracellular cAMP increase induce currents in Nav1.8 by PKA phosphorylation.
Bradykinin excites TTX-resistant primary afferent fibres in a Na+ dependent manner.

Question 23

What is the function of Nav1.8 and where is it found?

Answer 23

KO mice have diminished mechanosensation and delayed inflammatory pain thresholds - partial analgesia to noxious thermal and mechanical stimulation. Gain of function mutations cause painful neuropathy.
Expressed exclusively in nociceptive small diameter c fibre sensory neurons. TTX resistant.

Question 24

What is the relationship between p11 and Nav1.8?

Answer 24

p11 (S-100 related calcium binding protein) is localised at cytoplasmic surface of plasma membrane and plays role in membrane trafficking, eg. exocytosis, endocytosis, cell-cell adhesion and controls translocation of Nav1.8 to plasma membrane (its mRNA coexpresses with Nav1.8 in small diameter DRG neurons). p11 binds N-terminal intracellular loop of Nav1.8. p11 KO mice have reduced TTX-resistant Na+ current and reduced mechanicaly and thermally-evoked spinal cord neuronal activity (Foulkes 2006). p11 binds specifically to Nav1.8 )not other voltage-gated sodium channels) (Poon 2004).

Question 25

What are lipid rafts?

Answer 25

Made of cholesterol and sphingolipid, they are enriched microdomains of the membrane. Implicated in signal transduction and endocytosis, act as a platform on membrane where proteins are sorted and functionally localised. Proteins involved in cell adhesion and axon guidance, synaptic activity and growth factor receptors. Nav1.8 colocalises with it. Disruption of lipid rafts in sensory neurons by MbetaCD -> unresponsiveness to mechanical stimuli, disruption in middle part of axon is sufficient to block action potential propagation.

Question 26

How does the action potential propagate in an unmyelinated axon with regards to Nav1.8?

Answer 26

Nav1.8 on lipid raft opens and there is a local influx of Na+ -> action potential initiation. The depolarisation spreads due to the absence of myelin insulation. Before the depolarisation is attenuated to sub-threshold level it reaches the adjacent Nav1.8 cluster and triggers subsequent Na+ influx.

Question 27

How do some current analgesics work?

Answer 27

Aspirin - irreversible acetylates COX.
Ibuprofen and mefenamic acid -> reversible competitive inhibition of COX.
Paracetamol - scavenges hydroperoxides which stimulate COX (no anti-inflammatory effect).
NB: COX-1 protects stomach and maintains renal blood flow.
COX-2 mediates pain inflammation by sensitising peripheral nociceptors. No COX-2 inhibitors on market because cardiac problems.
Morphine activates mu and delta receptors -> decrease of cAMP production, hyperpolarisation of membranes by stimulating an inward rectifying potassium channel. Hyperpolarisation -> decreases release of NT from nerve cell.
Gabapentin, pregabalin reduce calcium currents after binding to alpha2delta subunit of voltage-gated calcium channel and prevent translocation of alpha1 subunit to plasma membrane.