A.R.

Question 1

Definition of pain

Answer 1

“unpleasant sensory & emotional experience with actual or potential tissue damage or described in terms of such damage” - IASP

Question 2

Nociceptive endings in the skin mostly have what type of fibres?

Answer 2

C or Aδ-fibres (a few have Aα/β-fibres)

*positions of endings largely speculative because they are very small. can see where main nerves go, but detection parts are tiny and buried in skin/cornea

Question 3

What are dorsal horn neurons?

Answer 3

Most are inter-neurones (both excitatory & inhibitory) - these are called dorsal horn neurons

Only a tiny minority are projection neurons (dorsal horn → thalamus etc.)

3rd class propriospinal neurons perform intersegmental communication the role of which is relatively poorly understood

Question 4

How can dorsal horn neurons be classified?

Answer 4

Based on input classification”

Type 1 = non-noxious (low threshold)

Type 2 = noxious and innocuous (multireceptive / wide dynamic range [WDR])

Type 3 = noxious only (high threshold / nociceptor specific)

• Can put electrode in spine and see that different stimuli activate different neurons e.g. electrode in lumbar spine, stroke paw, type I activated (only responds to non-noxious e.g. stroke, tickle, brush)
• generally Aβ carrying the non-noxious (and Aα fibres)
• Aδ/C carrying noxious

Question 5

How can different fibres in sensory neurons be classified?

Answer 5

C fibres (noxious): thin diameter axon (0.4 ± 1.2µm)

• unmyelinated
• slowly conduct (0.5 ± 2.0m/s)
• lots synapse in substansia gelatinosa (superficial layers [I + II] of dorsal horn)

Aδ (noxious):

• medium diameter axon (2±6µm)
• myelinated
• intermediate velocity(12±30m/s)
• also synapse in substansia gelatinosa and deeper within dorsal horn (lots in III, IV, V)

Aβ (MOSTLY innocuous):

• large axon (>10µm)
• myelinated
• fast (30±100m/sec)

Question 6

How have APs in nociceptive neurons been recorded?

Answer 6

Recorded from cell body within DRG because the nociceptive terminals are tiny (too small for an electrode)

Djouhri et al (1998): in vivo anaesthised guinea pig - electrodes into individual neurons knowing which segment/lamina - von Frey hair / heat impulse - APs of various sizes - use conduction velocity to determine fibre type (but only 30 degrees at recording site)

Yale et al: intact ganglion in vitro (only room temp): patch clamp recording - recorded current from cell bodies & stimulated dorsal root / injected current. In neuropathic pain model - hyperexcitability of neurons (bursts of firing)

Question 7

Why are APs in nociceptive neurons different shapes?

Answer 7

APs in different cells are underpinned by different voltage-gated ion channels - different combinations of sodium/potassium channels and different levels of the channels in different neurons

*Fang et al (2005): anaesthetised rats - different shapes/size of AP - recorded from DRGs in L3-L6 projecting neurons at 30 degrees - different rates of rise in C HTM, C LTM, A delta HTM, A delta D hair, Aa/B HTM, SA, G hair, MS

Question 8

What receptors exist in the synapse between nociceptive neuron and dorsal horn?

Answer 8

Acts on AMPA/NMDA

GPCRs also on terminals (Gi/Go) - inhibit the action of the VGCCs, particularly on the PRE-SYNAPTIC terminal

Not complete inhibition but make opening much less likely in response to an AP

Mu opioid receptor also Gi/Go - stops nociceptive signal from being transmitted along dorsal horn neuron by same mechanism (inhibits VGCC - reduces NT release)

Question 9

How is pain signal transmitted in the nociceptive neuron –> dorsal horn?

Answer 9

Depolarising wave carried by VGSCs (repolarised by VGPCs)

Depolarisation wave reaches pre-synaptic terminal, opens VGCCs (physically docked to vesicles containing glutamate)

(amino acid vesicles = glutamate
peptides vesicles = Sub P etc.)

Calcium entry - vesicles fuses and releases content into synaptic cleft –> AMPARs and NMDAs activated by glutamate –> EPSP in dorsal horn

Question 10

What are possible methods for stopping nociceptors sending signals to CNS?

Answer 10

1) LA: block VGSCs (remove upstroke AP) - but blocks ALL APs so not systemic
2) Block VGCCs - but VGCCs responsible for NT at most other synapses (N/PQ type channels) - can be used like epidural in serious cancer pain
3) Block AMPA/NMDA receptors - but mediate neurotransmission at most fast excitatory synapses (ketamine can apply to spinal cord but can also impact motor neurons etc)
4) Morphine - acting on MOR - BUT MOR not only found on sensory nerve terminals (hence abuse / SEs)

Targeting sensory receptors / detecting molecules may be better / more selective target..

Question 11

What are the different sensory receptors found on primary afferent nociceptive neurons?

Answer 11

Transient Receptor Potential Family: TRPV1 / TRPV3/ TRPA1 /TRPM8

Ligand gated Family: ASIC (acid) / P2X3 (ATP - tissue damage) / 2PDKC / Piezo1/2 (mechanical)

GPCRs: Bradykinin-R (wasp stings), Histamine-R (itch/allergy)

Question 12

What is the role of TRPV1 in pain?

Answer 12

TRPV1 = capsaicin, noxious heat at 42C (also pH/chemicals). Selectively expressed in sensory receptors but found in other areas

Preclinical animal models: KO rats had less inflammatory hyperalgesia. Antagonists inhibit thermal and mechanical inflammatory hyperalgesia and in partial nerve injury models reduced tactile allodynia and mechanical HA.

Clinical trials: hyperthermia response e.g. Amgen terminates phase 1b trial for dental pain

CRUTCHLOW ET AL (2009) = MK-2295 (developed for osteoarthritis trials) for 14 days, immersed in 48 Degrees - perceived as innocuous.

Tried in every type of pain possible - all failed

Second generation TRPV1 antagonists that block capsaicin but not acid/heat responses : no hyperthermia response in rats. Not in human testing yet but lots of caution due to failures.

Question 13

What is the role of TRPV3 in pain?

Answer 13

Chemical or thermal stimuli (33-39 degrees in vitro): non-selective cation channel

Less known about functional role but sensitised upon repeat activation (whereas TRPV3 desensitises).

Preclinical models shows some efficacy in inflammatory models, 2010 - clinical trial for neuropathic pain begins?

Question 14

What is the role of TRPM8 in pain?

Answer 14

Cold / menthol activity

Relief of cold hypersensitivity in KO animals?

Question 15

What’s the role of TRPA1 in pain?

Answer 15

Possible role in chemotherapy induced peripheral neuropathy (cold allodynia common symptom)

Cisplatin increases trpa1 expression?

Question 16

Difference between acute and chronic noxious stimuli?

Answer 16

Acute noxious stimuli activate a specialised neuronal detection system that generates sensations of pain, and generally, adaptive behavioural responses

More persistent noxious stimuli, notably those involved with some chronic injuries and disease states, not only activate the pain-signalling system but also dramatically alter its properties so that weak stimuli produce pain

Hyperalgesic states arise from two mechanisms: peripheral and central sensitisation

Question 17

How does peripheral sensitisation occur?

Answer 17

Sensory nerve endings activated
Release cGRP and Sub P locally near terminals

Blood vessels then more permeable, plasma and leukocytes/monocytes escape

Inflammatory mediators from damaged cells attract leukocytes but can also act on the terminal and sensitise the terminals

Sympathetic post ganglion Neurons (NA and PG release) to sensitise neurone

Nerve growth factor also released by damaged skin, acts on the Trk A receptor to sensitise neuron

Question 18

What is peripheral sensitisation in terms of electrophysiology?

Answer 18

More APs for a given stimulus - or stimulus-driven spiking when it didn’t occur before

APs arise in initial segment where very high conc of VGSCs

Excitability = stimulus response measure for APs

Stimulus must be enough to move membrane potential from Vrest to AP threshold

Thresholds difference in difference cells: rheobase = amount of current required to produce 1 AP

Varies between cells within individuals and also between individuals - pain thresholds likely both nature and nurture (used human nociceptor like neurons from stem cells - note stem cells tend to strip epigenetics so difficult to study nurture)

Question 19

How can you make cell more likely to fire without changing the stimulus?

Answer 19

Depolarise resting potential (close resting potassium channels or Open cation channels)

Increase resting membrane resistance (close channels open at rest like potassium leak channels)

Change activity of sub threshold VG channels eg increase the opening of voltage dependent T TYPE CALCIUM CHANNELS (low threshold), or decrease opening of low threshold potassium channels

Make action potential more hyperpolarised - lower threshold at which VGSCs activate - change their hating properties or just put more in the cell to lower the functional threshold behind AP firing

Question 20

What is the problem with recording from nociceptor To study how action potentials are initiated

Answer 20

The AP initiates adjacent to the sensory terminal so changes determining whether AP fires should occur in this region

Can’t IC RECORD directly from sensory terminals as too small and difficult to live image

Cell bodies used as a surrogate for the terminal

Vrest of cell body is ~55mV, are they the same? Eg Sally Dawson’s (Bristol) recordings from cell body

Question 21

Describe the VGSCs that make the upstroke of the AP

Answer 21

Membrane channel with core alpha outer subunit which is where most of the voltage sensors are (also has pore in middle and drug binding site).

The alpha subunit is one protein that has 4 repeats of 6 transmembrane segments, forms loop around central pore

Beta subunits are smaller and subtly influence voltage dependence - also involved in trafficking

There are 9 different VGSCs as there are 9 different alpha subunits

Question 22

Which sodium channel subtypes are found in the DRG?

Answer 22

Nav1.6 (also CNS)
Nav1.7 (also SCG)
Nav1.8 - NOT blocked by TTX*
Nav1.9 - NOT blocked by TTX*

(Nav1.3 - found in brain and foetal DRG but usually disappears in adult - may reappear in certain pain states)

(1-4 and 6-7 all TTX sensitive)

Question 23

Gating properties of VGSCs?

Answer 23

Voltage clamp - turn on at around -50mV

Turn on very fast and turn off during depolarising step: SELF INACTIVATION (instrinsic ball and chain mechanism)

As voltage steps increase, peak current gets smaller - not because channels are closing but because driving force for sodium changes (same number of channels are open) - sodium efflux with continued depolarisation (but at first, current rises because more channels are opening)

Question 24

What is steady state inactivation?

Answer 24

If use voltage clamp, always stepping to same place (point with largest current - max channel activation) but diff starting point: currents biggest when starting at very negative potentials (if you start from -60/-50, channels are already inactivating so the current is smaller)

*note: cell body recordings, don’t know if true of nerve terminal as not recorded there

Repeat experiment after waiting 10s, get exact same
If step up then back down then back up immediately - nothing chappens (inactivation)

AFTER 50-60MS most current returns through VGSCs - recovery important for cells to repeatedly fire; fundamental property of the channel

Question 25

How do recovery times differ in VGSCs?

Answer 25

Nav1.7 (TTX sensitive): paired pulse experiment at physiological temperature:
50% recovery ~1ms
100% recovery at 1000ms (similar to most VGSCs)

Nav1.8 (TTX insensitive): at room temperature: inactivation relatively slow compared to other sodium channels (10s of ms rather than ms)

Data is from HEK cells expressing cloned recombinant sodium channels

Question 26

What are inactivation / activation curves

Answer 26

Inactivation curve shows amount of peak current you get depending on starting voltage

Activation curve shows number of open channels (not the current flowing through them)

Question 27

What is the significance of TTX?

Answer 27

Tetrodotoxin

TTX-insensitive channels (1.7, 1.8) important in sodium current in sensory neurons

500nM TTX will block all sensitive channels; some current lost when block these channels but many are resistant

Drug blocking TTX-INSENSITIVE channels (blocks current better) developed as pain therapy

Question 28

What is the possible role of sodium channel beta subunits in pain?

Answer 28

Can determine voltage-dependence

Nav1.8 (TTX resistant) has large currents when co-expressed with beta 1 subunit (changes activation curve)

If have more of certain beta subunits, cell would show greater excitability as AP threshold may be lower

In addition to alpha subunit, possible changes to levels/properties of beta subunits in various pain models (and human tissues) - mainly correlative

Question 29

NaV1.3 - role in pain?

Answer 29

Usually absent in adult DRG but re-expressed after AXOTOMY

May produce ECTOPIC APs in damaged nerves (spontaneous pain)

Streptozotocin-induced diabetes causes tactile allodynia and upregulation of 1.3/1.6/1.9

Carrageenan inflammation induced 1.3/1.7/1,8

Question 30

Nav1.7 - role in pain? lab experiments etc.

Answer 30

Functional expression controlled by NGF.

Major TTX-sensitive channel in DRG, but also in other cell types.

• Upregulated in 1.8 KO mice
• Contributes to enhanced TTX-sensitive current following carrageenan inflammation (current increases following)
• 1.7 KOs (specific KO - only in nociceptive neurons) = REDUCED MECHANICAL AND THERMAL RESPONSES + CHANGES TO INFLAMMATORY PAIN

Question 31

NaV1.8 - role in pain? lab experiments etc.

Answer 31

DRGs only and mainly nociceptors - expression regulated by inflammatory mediators (e.g. NGF, CCL2) . Current size/kinetics/activation voltage modulated by PGE2, adenosine, serotonin - probably via cAMP (inflammatory molecules?). Contributes to most of the TTX-insensitive current.

• KO contributes to inflammatory pain
• Knock down suggests role in neuropathic pain
• Upregulated in 1.7 KO mice
• Contributes to enhanced TTX insensitive current following carrageenan inflammation
• Current increases when treated with CCL2 (inflammatory chemokine)
• Key role in cold sensation (Zimmerman 2007)

Question 32

Na1.7 - role in pain? (clinical conditions / clinical trial potential)

Answer 32

Primary erythromyalgia = 1.7 gain of function
Paraoxysmal extreme pain disorder = 1.7 gain of function
Channelopathy-associated insensitivity to pain = loss of function SCN9A gene

Clinical trials: turning down 1.7 with dosing so not completely removed - still some pain (selective molecules in development, 2011 - phase I, on people with hyperactivity of 1.7)

Question 33

Na1.8 - role in pain? (clinical conditions / clinical trial potential)

Answer 33

• Gain of function mutation = painful neuropathies

Selective blockers reverses NP and inflammatory pain in rats - but none through clinical trials (bad pharmacokinetics)

Question 34

Na1.9 - role in pain?

Answer 34

Mainly expressed in nociceptors, sensory terminals

Down-regulated by axotomy, probably a persistent slow gating channel active at negative potentials (poorly understood, difficult to study)- may contribute to resting potential / AP thresholds

expression dependent on NGF/GDNF. regulated by intracellular GTP and probably Gq coupled GPCRs

KO: various inflammatory agents no longer sensitise nociceptors (Amaya et al 2006), Priest et al (2005)

Hard to study / no useful drugs
Gain of function mutation causes loss of pain
But another gain of function mutation causes familial episodic pain!! - conflicting

Question 35

Extra reading - Dib Hajj 2002? Others?

Answer 35

need to add..

Question 36

Park & Luo (2010)

Answer 36

Role of calcium channels

T type VGCC

Located in primary afferent terminals and DRG, 3.2 the most abundant. Electrophysiology suggest involvement lowering AP thresholds. Activation of T type VGCC close to resting potential allows Ca influx reducing threshold for high threshold spike generation.

Blocking T type can reduce excitability.

From animal studies -

• Small DRG neurons channel currents increased after chronic constriction of sciatic nerve, and in medium sized following chemical induced diabetic neuropathy. This may lead to increased excitability which is involved in mechanical allodynia and thermal hyperalgesia.
• 3.2 deficient mice fail to develop acid induced chronic mechanical hyperalgesia so involved in development chronic musculoskeletal pain syndromes.
• 3.2 KO mice show hypoalgesia to acute somatic visceral and tonic inflam - pronociceptive in processing noxious stimuli.
• 3.2 expression increased in DRG diabetic neuropathy and mechanical nerve injury.
• Knockdown 3.2 via siRNA or antisense oligonucleotides leads to anti nociceptive effects. 3.1 deficient mice show reduced nerve injury induced behavioural hypersensitivity.
• 3.1 KO increases visceral pain.
• Inhibiting T type VGCC with antagonists blocks and reverses tactile hypersensitivity and thermal hyperalgesia probably by reducing neurotransmitter exocystosis from primary afferents.
• L-cysteine enhances T type currents and promotes cutaneous thermal and mechanical hyperalgesia. Endogenous hydrogen sulfide may activate or sensitise elevated T type channels and contribute to neuropathic pain maintenance.

VGCC a2d1
α2δ-1 subunit of VGCC is binding site for gapapentin and pregabalin. α2δ-1 usually dysregulated in DRG in neuropathic pain models. Biochemical studies show increased expression of this subunit leading to increased calcium channel currents in DRG neurons, DH neuron hyperexcitability and behavioural hypersensitivity. Gabapentin can normalise these without affecting control groups. Elevated α2 δ-1 mediates behavioral hypersensitivity through enhanced excitatory pre-synaptic input that activates glutamate receptors at post-synaptic dorsal horn neurons