A.R. Flashcards
Definition of pain
“unpleasant sensory & emotional experience with actual or potential tissue damage or described in terms of such damage” - IASP
Nociceptive endings in the skin mostly have what type of fibres?
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
What are dorsal horn neurons?
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
How can dorsal horn neurons be classified?
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
How can different fibres in sensory neurons be classified?
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)
How have APs in nociceptive neurons been recorded?
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)
Why are APs in nociceptive neurons different shapes?
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
What receptors exist in the synapse between nociceptive neuron and dorsal horn?
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)
How is pain signal transmitted in the nociceptive neuron –> dorsal horn?
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
What are possible methods for stopping nociceptors sending signals to CNS?
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..
What are the different sensory receptors found on primary afferent nociceptive neurons?
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)
What is the role of TRPV1 in pain?
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.
What is the role of TRPV3 in pain?
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?
What is the role of TRPM8 in pain?
Cold / menthol activity
Relief of cold hypersensitivity in KO animals?
What’s the role of TRPA1 in pain?
Possible role in chemotherapy induced peripheral neuropathy (cold allodynia common symptom)
Cisplatin increases trpa1 expression?
Difference between acute and chronic noxious stimuli?
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
How does peripheral sensitisation occur?
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
What is peripheral sensitisation in terms of electrophysiology?
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)
How can you make cell more likely to fire without changing the stimulus?
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
What is the problem with recording from nociceptor To study how action potentials are initiated
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
Describe the VGSCs that make the upstroke of the AP
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
Which sodium channel subtypes are found in the DRG?
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)
Gating properties of VGSCs?
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)
What is steady state inactivation?
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
How do recovery times differ in VGSCs?
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
What are inactivation / activation curves
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)
What is the significance of TTX?
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
What is the possible role of sodium channel beta subunits in pain?
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
NaV1.3 - role in pain?
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
Nav1.7 - role in pain? lab experiments etc.
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
NaV1.8 - role in pain? lab experiments etc.
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)
Na1.7 - role in pain? (clinical conditions / clinical trial potential)
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)
Na1.8 - role in pain? (clinical conditions / clinical trial potential)
- Gain of function mutation = painful neuropathies
Selective blockers reverses NP and inflammatory pain in rats - but none through clinical trials (bad pharmacokinetics)
Na1.9 - role in pain?
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
Extra reading - Dib Hajj 2002? Others?
need to add..
Park & Luo (2010)
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
