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NS of Pain > T.P. - NA > Flashcards

T.P. - NA Flashcards

1
Q

What is the role of endogenous analgesia?

A

Endogenous analgesic circuits built into CNS, can regulate pain perception via sets of neurons that release substances that either suppress nociception or alter pain perception

Conditionally regulates gain of pain system, depending upon behavioural context (switch off pain, or pay more attention to it)- useful survival role. Brain has working model of how much pain to should expect in response to any given circumstance - therefore acts on spinal cord to control gain of information transfer (not just about nociceptive input generating pain)

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2
Q

Which part of the pain pathway does descending control particularly regulate?

A

Gain at the DH synapse

Either increasing nociceptive barrage, or suppression

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3
Q

Which brainstem locations are important in descending control?

A

Peri-aqueductal grey (PAG)

Locus coereleus (pons) - NA neurons
Rostral ventromedial medulla (RVM) - 5HT?
*both neuron classes project down to cord
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4
Q

What is ‘opiate burn’?

A

Endorphin burn - post exercise/stress - due to release of opiate like peptides:

Endorphins / Enkephalins
Noradrenaline
Serotonin

Opiates are gold standard / most potent pain relief - therefore built in system is capable of fairly profoundly regulating pain

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5
Q

How do chronic pain treatments affect the endogenous analgesic system?

A

Chronic pain >3 months (outlasts normal biological healing time). Many therapies interact with endogenous NA analgesic system e.g.

TCAs: block NA/5HT reuptake e.g. duloxetine

Manipulate NA levels: tramadol, tapentadol, clonidine

Deep brain stimulation (DBS), transcutaneous electrical nerve stimulation TENS + spinal cord stimulation

  • all seem to have an effect on release of monoamines (amongst other things)
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6
Q

What is the role of endogenous analgesia in chronic pain?

A

Edward (2005) - Deficiency of endogenous analgesia in chronic pain - evidence across variety of chronic pain conditions
(Measure endogenous analgesia in humans - quantify pain suppression in particular circumstances - CP patients engage endogenous analgesic circuits less)

Endogenous analgesia strength predicts risk of post-surgical chronic pain (patients before surgery who poorly recruit endogenous analgesia - higher risk of post-surgical chronic pain - CAUSAL not just association)

Endogenous analgesia strength predicts efficacy of reuptake inhibitors! esp. duloxetine - (Yarnitsky et al 2008, 2012) - if poor endogenous analgesia, more likely to respond to duloxetine than those with functional endogenous analgesic system

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7
Q

How are monamines synthesised?

A

From aromatic amino acids (tyrosine = essential AA) and all have a single amine group

Tyrosine → (tyrosine hydroxylase) L-DOPA → (DOPA decarboxylase) dopamine → (dopamine B-hydroxylase) NA → (PNMT) adrenaline

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8
Q

What are examples of monamines in brain?

A 
Serotonin
DA*
NA*
Adrenaline*
Histamine
  • = catecholamines
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9
Q

How are monaminergic neurons arranged?

A

Relatively small number of neurons (few 1000) that release monoamines (relative to vast number of neurons in brain - 100s of billions)

Organised in small clusters (cell bodies in brainstem / midbrain) with very large, extensive axonal projections.

Therefore ‘global’ neuromodulators i.e. widespread actions despite only few neurons -MODULATORS, fundamental to brain function. Dysfunction = profound disease states e.g. Parkinson’s

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10
Q

What sort of functions to monoamines subserve?

A

Global state changes:

  • sleep - wake cycles
  • mood/affect
  • alerting and arousal
  • reward and addiction

Some specific functions

  • movement control
  • sensory processing

Similarities between them but each subserve different roles / very distinct brain functions

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11
Q

How did Dahlstrom & Fuxe (1965) classify monoaminergic neruons?

A

Histochemically labelled NA neuron groups A1-A7 in brainstem, as they extend from caudal ventrolateral medulla → rostral lateral pons.

Rat brain: Major cluster (A6) in locus coeruleus - project to forebrain and also some to cord/cerebellum (similar principle in humans)

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12
Q

What is the locus coeruleus? How does it function in the brain?

A

Homogenous noradrenergic nucleus - only contains NA neurons

  1. Coupled, synchronous activity
  2. Global effector (projects widely)
  3. Volume transmission
    (like sprinkler system, spouts NA and cells nearby with appropriate receptors respond accordingly)
  4. Few synaptic specialisations (unlike GABA/glutamate which has precise neuron-neuron areas of action, micron precision)
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13
Q

What does activity in the locus coeruleus affect?

A

Activity reflects global brain state (silent when sleep, but fires relatively slowly/tonically when awake)

Signals salience & arousal
(most salient stimulus = noxious stimulus, therefore LC fires lots when noxious stimulus applied)

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14
Q

What is the key debate about the general influence of the LC?

A

Responds to salient stimuli + part of global arousal system

On the other hand - NA system involved in control and suppression of pain

Essentially opposite functions - alerting cortex but also suppress spinal cord input?

Ongoing active debate for noradrenaline - high profile papers currently supporting both perspectives
* Uematsu et al (2015)

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15
Q

What paper supports the LC as having global activity?

A

Chandler et al (2014) - brainwide actions

  • Widespread projections
  • Volume transmission
  • Homogenous cells
  • Any specificity determined by post synaptic cells
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16
Q

What paper supports the LC as having regional activity?

A

Schwartz et al (2014) - modality specificity

  • Targeted projections
  • Synaptic specialisations
  • Modular organisation
  • Differential co-release of NTs (i.e. some other NTs released)
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17
Q

What did Reynolds (1969) show?

A

Deep brain stimulating electrode into rat midbrain + pontine sites evoked analgesia

  • Found to be modality selective
    via inhibition of spinal sensory neurons
  • Laparotomy without anaesthetic when stimulating midbrain in region of PAG
  • led to idea that midbrain releasing opiates to cause analgesia
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18
Q

What did Bandler and Shipley (1994) show?

A

Chemical activation (or disinhibition) of specific areas revealed regional specificity

Shows chemical stimulation (glutamate injection) of PAG produces same effects as electrical stimulation - therefore not just an artefact of electrical stimulation (defined columns in PAG that evoked particular types of analgesic effect)

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19
Q

What is deep brain stimulation used for?

A

Patients can show selective analgesia without motor/autonomic side effects with deep brain stimulation (aim for PAG/PVG/thalamus)

Occasionally done for specific refractory pains

Pickering et al (2009)

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20
Q

What are the nodes and monoamines identified in descending control systems?

A

Nodes:

  • hypothalamus
  • peri-aqueductal grey (PAG)
  • dorsolateral pons
  • locus coeruleus, A7 (A5?)
  • rostral ventromedial medulla

Monoamines (in addition to role of opiates):

* NA - LC, A7, A5
* Serotonin - RVM
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21
Q

What did Eipperts et al (2009) show?

A

Placebo paradigm: fake LA gel

Heat stimulus to arm (fMRI of DH - activity at spinal level)

Explained that putting on LA cream + turned down the heat to mimic the effect of LA

In scanner, then applied same stimulus as first - subjects believed it hurt less - activity in spinal cord was extinguished

Convincing evidence: beliefs believe can influence transmission of nociceptive information at the SPINAL LEVEL + suggests descending control circuits active in every day life?

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22
Q

What is the RVM?

A

Rostral Ventromedial Medulla

Relay site (medulla → to spinal cord) interfacing between the PAG & higher centres

On cells: activated when apply a noxious stimulus (pronociceptive, therefore inhibitied by opioids)

Off cells: inhibited when apply noxious stimulus (anti-nociceptive, therefore activated by opioids)

Both cell types project to the DH + have wide receptive fields - often affect whole body

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23
Q

How does the RVM affect the withdrawal response?

A

Around time that animal withdraws: on cells start firing and off cells switch off
(balance between 2 classes determines when this decision to withdraw occurs)

If give opiates - increase activity in off cells and decrease activity in on cells - part of analgesic effect

*lots of evidence for this + for dysfunction of this system in the setting of chronic pain

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24
Q

How does serotonin affect the RVM?

A

Original idea: 5HT in off cells + released at spinal level to inhibit nociception (now less accepted)

Current idea: Serotonergic cells are NEUTRAL cells making up 20% of the RVM (less clear role in nociception).

Serotonin facilitates or inhibits experimental nociception, depending on receptor expression on spinal neurons e.g. 5HT1A inhibitory, 5HT2/3 - excitatory

*RVM definitely involved in pain perception, but probably more by GABA/opiates rather than serotonin?

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25
Q

Evidence to support serotonin in control of pain?

A

Role for 5-HT1A agonists in treatment of migraine (possibly by peripheral action on blood vessels - stopping vasospasm rather than neural effects)

Little other effect of 5-HT agonists / antagonists or uptake blockers in human acute or chronic pain

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26
Q

What noradrenergic neurons are involved in central control of pain?

A

Rat brain: spinal cord projections mostly from LC, but some also come from A7 and A5. LC (A6) also projects to other brain areas - but predominantly cord

Westlund et al (1983): Anatomical studies- spinal innervation from pontine NA neurons A5, LC (A6) + A7

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27
Q

What adrenoceptors are present at spinal level? What are their signalling pathways and which one is important?

A

α₁: Gq: excitatory via PLC (↑ P1 & DAG) -

α₂: Gi/o: inhibition of AC: ↓ cAMP: opens K+ channels, inhibits calcium channels (like MORs)

β₁,₂: Gs: excitation via ↑ cAMP

*NA will act on all classes of adrenoceptor, and all are present in spinal cord, but α₂ seems to be particularly important in analgesia

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28
Q

Why is it difficult to study role of NA neurons in descending control?

A

Cell bodies of NA neurons located deep in brainstem (CV control, breathing, arousal/consciousness) - inaccessible as damage would be serious/fatal

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29
Q

How do we know that NA neurons may have an anti-nociceptive role?

A

Direct activation of LC + A7 = anti-nociceptive

  1. Jones & Gebhart 1986:
    Stimulating electrode in LC = suppression of evoked nociceptive withdrawal response
  2. Hentall et al 2003:
    Stimulation of pons causes release of NA in spinal cord
    (cyclic voltammetry to detect oxidisable chemicals)
  3. Hirata + Ashton Jones 1994:
    LC neurons fire AP bursts in response to noxious stimulus (very salient stimulus)

Therefore: feedback inhibitory circuit (LC neurons are excited by nociceptive stimulus , release NA at spinal level +
a2 receptors exist on spinal cord - demonstrated in anaesthetised animal)

30
Q

How did Jones (1991) demonstrate role of spinal adrenoceptors in pain suppression?

A

Jones 1991: intrathecal α₂ antagonists block LC-evoked analgesia + attenuate the analgesia from PAG + RVM stimulation (upstream of LC)

31
Q

What other pharmacological evidence has demonstrated the role of spinal adrenoceptors in pain suppression?

A

NA: potent analgesic actions (similar to opioids) - both systemically + intrathecally
(Rat models: Reddy & Yaksh 1980 - humans: Eisenanch et al 1995).

Systemic α₂ agonists exert analgesic effects via spinal site as greatly attenuated by intrathecal α₂ antagonist

Epidural and intrathecal α₂ agonists used in humans. Systemic α₂ agonists only used peri-operatively (as cause sedation)

32
Q

What sites does NA act on a2 receptors to suppress pain?

A

Two sites:

Descending NA fibres in WM tract; project into DH and release NA → presynaptic a2 adrenoceptors on primary afferents → inhibition of VCCCs → inhibit Glu release from the primary afferents

*Also a2-receptors on DH: second order neurons also affected by the NA → open K+ channels → hyperpolarisation

Normal innocuous sensory pathway has no a2 receptors:
therefore stimulating a2 receptors does not cause numbness, just suppresses nociceptive transmission

33
Q

At what site does NA acts on a1 adrenoceptors to suppress pain? How do we know this?

A

Circuit controlled by local inhibitory neurons (tonically active in spinal cord)

Local inhibitory neurons have α1 receptors (excitatory): a1 activation ↑ activity of local inhibitory circuits (inhibitory amino acids released)

Shown through reduced preparations:

Histology (immunohistochemistry + in situ hydbridisation). In vitro slice: (intracellular, patch clamp). In vivo (anaesthetised animals -
extracellular, ionophoresis, patch clamp).

Limited ability to relate to nociceptive circuits in behaving animals, - functional significance
i.e. which info modalities and which circumstances are the circuits are engaged - why they might fail

34
Q

What methods are there to examine the functional role of descending NA control?

(in vivo animals)

A
  1. Electrical / chemical stimulation of pontine nuclei (Jones 1986, Yeomans et al 1992)
  2. Recording LC neurons: (Hirata + Aston-Jones et al 1994)
  3. Electrical lesion of LC (Tsuruoka & Willis 2003)
  4. Chemical lesion of LC e.g. anti-DβH-saporin (Jasmin et al 2003)
  5. Intrathecal α₂ or α₁-selective antagonists
  6. KO of α₂ receptors or DβH - (Stone 1997, Jasmin 2002)
    * phenotype unclear - less dramatic than may expect
35
Q

What methods are there to examine the functional role of descending NA control?

(Humans)

A

Intrathecal or epidural) α-agonists

Systemic NA re-uptake inhibition (TCAs, duloxetine)

Imaging studies (Jon Brooks lectures)

36
Q

Why is it so difficult to determine the functional role of descending NA control in humans?

A
  • Recording neurons in vivo is difficult therefore so only have partial view
  • Ablating neurons damages the circuit trying to look at, but also often damage things that are nearby - also often compensatory effect to the damage
  • KO experiments less dramatic than expected

Ongoing debate about role of system and how it is engaged

37
Q

What is basal tone theory?

A

NA neurons fire when awake, therefore continuous NA level in spinal cord -increasing / decreasing from this level will regulate nociception

We know there is ongoing NA activity, but contradictory

38
Q

What evidence is there for role of NA control in inflammatory pain?

A

Consistent evidence from
formalin test / carrageenan injections etc.

  1. LC ablation augments pain (Tsuruoka et all 1996)
  2. Intrathecal antagonists augment pain (Omote et al 1998)
  3. Animal increases levels of spinal NA in response to sensitisation as compensatory mechanism (Omote et al 1998)
  4. KO α₂ receptors, augment pain phenotype

Suggests system can be engaged when a PERSISTENT noxious stimulus is present -descending NA control opposes the spinal sensitisation seen in persistent inflammatory pains

39
Q

What evidence is there for role of NA control in inflammatory pain?

A

Consistent evidence from
formalin test / carrageenan injections etc.

  1. LC ablation augments pain (Tsuruoka et all 1996)
  2. Intrathecal antagonists augment pain (Omote et al 1998)
  3. Animal increases levels of spinal NA in response to sensitisation as compensatory mechanism (Omote et al 1998)
  4. KO α₂ receptors, augment pain phenotype

Suggests system can be engaged when a PERSISTENT noxious stimulus is present -descending NA control opposes the spinal sensitisation seen in persistent inflammatory pains

40
Q

What evidence is there to suggest that NP pain patients have deficits in NA control?

A
  1. NA reuptake inhibitors (tramadol, tapentadol) = a main treatment - RCTs modestly effective (Finnerup 2010)
  2. Viisanen & Pertovaara (2007)
    LC stimulation evoked less analgesia in NP patients compared to control patients

Spinal receptors functioning, fibres present in cord + LC still responds to noxious stimuli (even more strongly?) - BUT if stimulate electrically, no analgesic effect - break between LC + cord somewhere?

Why can’t descending control compensate for excessive sensitisation in NP pain?

Descending control hypoactive, with spinal hypersensitivity to NA (cord NA sensitive but lack of NA release)

41
Q

What evidence is there to suggest that NP pain patients have an intact NA control system?

A

Animal studies:

  1. Pertovaara 2006 / Eisenach 1995: Enhanced analgesic action of intrathecal a2 agonists (probably more potent than in a naive control animal, so receptors still intact
  2. Ma & Eisenanch 2003: Patients have upregulated spinal NA fibres (DBH containing fibres) - x Tony disagrees, he thinks there is plasticity but his data suggests in opposite direction (down-regulation of spinal fibres)
  3. LC neurons more strongly activated by noxious stimuli (not by touch) - so no failure in this part of system
  4. Malmberg 2001: KO a2 receptors shows no NP pain phenotype (so lack of a2 receptors unlikely to be causing NP pain)
  5. Ablation of NA neurons - contradictory findings (Jasmin et al 2003)
42
Q

Why do endogenous analgesic systems not compensate for / switch off NP pain? (theories)

A
  1. Loss of central drive to pontine NA neurons?
  2. Altered balance with overwhelming pro-nociceptive facilitation by (RVM)?
  3. Or LC tries to compensate, but overwhelmed by setting of NP pain (switches off inflammatory, but can only control NP to a smaller degree?

Is descending NA control regulating some sensory modalities?

43
Q

What evidence is there that descending NA control only regulates some sensory modalities?

i.e. cannot control NP pain, but better in inflammatory pain

A

NA blockade can uncover latent NP pain

  1. Xu et al 1999

Cut branch of sciatic nerve, 80% animals develop sensitisation (only proportion of patients get NP pain - rest just get numb patch).

Remaining 20% given intrathecal a2 antagonist: all developed hypersensitivity (normally animals discarded as failure of model, but actually modelling appropriate suppressive response from descending control system so do not exhibit symptoms!!)

  1. De Felice (2012): 90% Spring Dawley rats show sensitisation but just 50% of Holson rats

Holson seem to have upregulation of NA system when compared to SD (so NA system in 50% of those rats can suppress NP sensitisation)

44
Q

What is the next focus for correcting the imbalance of descending control in NP pain?

A

Need new strategies to define + correct imbalance

  1. Targeted, temporally restricted interventions aimed at NA system
  2. Allow activation / inhibition in conscious behaving animals
45
Q

What is the targeted noradrenergic vector strategy?

A

Replication deficient adenoviral vector (i.e. gene therapy)

Catecholaminergic specific promotor (PRSx8): selective for catecholaminergic neurons, has transcription factor binding domain upstream of dopamine beta hydroxylase (DBH) gene

PRSx8 = 8 repeats of binding motif over ~250 base pairs (small, efficient + selective)

  • promotor is selectively activated by transcription factor (Phox2) that is only switched on in NA + adrenergic neurons (not DA neurons). With Phox2 present, promotor will express the gene put downstream of it

Virus delivers chosen gene segment into neurons without specificity - but will only transcribed in neurons that have Phox2 - which creates specificity. Virus must be injected directly into spinal cord or brain tissue.

46
Q

What can the targeted noradrenergic vector strategy be used to do?

A

Can separate neuronal populations basis on anatomy

Express eGFP (green) or mRFP (red) in neurons by adding eGRP/mFRP sequence to the PRSx8 region. Inject virus that tends is taken up by axons + delivered to cell body by RETROGRADE transport

e.g. Injected into DH then stain for protein. 3 weeks later: many LC neurons filled with green protein (therefore LC neurons must project to cord)

Projections from cord: 80% = LC, 12% = A5, 8% = A7.

47
Q

What did Howorth et al (2009) discover about the LC using targeted NA vector strategy?

A

Labelled neurons with GFP. Rat LC ~1600 neurons, ~150 labelled in experiment (<10% of LC is projecting to the spinal cord). Position of the 10% relatively consistent - suggests SUBGROUP of LC projects to cord.

Then gave animals noxious stimulus (formalin) - neurons projecting to LC were activated (cFos positive nuclei) - therefore confidence that this is the population of interest

48
Q

How can the FUNCTION of NA neurons be studied? (techniques)

A

Filling neurons with GFP

  • characterise anatomy of projection, and identify NA neurons in brain slices
  • associate with functional role

But to study function:

  • Alter behaviour of NA neurons (change their activity pattern)
  • Decrease excitability by increasing resting K+ channel expression (hyperpolarisation)
  • Assess consequences
  • in vitro: slices / slice cultures
  • in vivo: tests of nociception
49
Q

What did Howorth et al (2009) discover about the function of LC neurons in inflammatory sensitisation models?

A

Injected virus expressing Kir2.1 (K+ channel gene) + EGFP

In vitro: LC neurons transduced with virus: lower input resistance, more hyperpolarised + FIRED LESS (validading virus)

3-4 weeks later: variety of in vivo nociception tests

Naive animals (no formalin etc): little change in mechanical thresholds and small change in thermal thresholds

Sensitised with formalin / carrageenan test: virus animals had more flinches / paw lifts / licking & ↑ Cfos in spinal cord

  • Behavioural & anatomical evidence: inhibiting LC neurons a bit (not ablating) effects nociception in whole animal
  • Proves that NA system has a role, but not the negative direction (shows we can make inflammatory pain worse if system damaged)
50
Q

What did Howorth et al (2009) discover about the role of LC neurons in NP pain models?

A

NP model: tibial nerve transected, developed hind limb sensitisation over 2-3 weeks: (lower von Frey withdrawal threshold, more acetone withdrawals, mild heat hyperalgesia compared to sham).

Virus had NO EFFECT (system was already broken + not compensating for NP pain?)

51
Q

What did HUGHES et al (2013) discover about the role of LC neurons in NP pain models?

A

Same NP model as Howorth (2009) - but no viral vectors used.

Intrathecal catheter: delivered spinal Yohimbine (a2 antagonist) repeatedly over weeks: mechanical allodynia, + cold allodynia PRESENTED earlier but ended up the same level

As time increased - difference disappeared - therefore NA system fails over time, causing sensitisation to develop (lack of compensation)

*Reason untreated animals not sensitised early on because NA system is masking it, but eventually it fails - TEMPORAL LOSS OF DESCENDING CONTROL

52
Q

In addition to temporal loss of control, what else did HUGHES et al (2013) show?

A

Stimulated CL side (non-injured side) - for most animals, sensory inputs remained lateralised.

When given Yohimbine - UNMASKED CL/MIRROR SENSITISATION
- therefore, if block descending control system: CL side shows sensitisation

SPATIAL constraint being exerted by NA system on degree of sensitisation: without NA system - both limbs are sensitised (nerve injured side is worse, but CL limb also affected).

This also shows that the descending control must still be active in NP pain as normally remains lateralised?

53
Q

What did Hughes et al (2013) - find in NP pain models?

in addition to temporal loss of NA control + spatial constraints of NA system

A

Nerve injured animals has depletion of DBH containing fibres (i.e. NA neurons)

*Contrasts with Eisenanch (2003) findings (they found fibres upregulated in NP pain) - but correlates with idea that there is anatomical & functional deficit in this control system at the spinal level

54
Q

What did a patient case study of NP pain show?

A

After surgery for tibial plateau fracture, presented with ipsilateral pain (chronic regional pain syndrome - CRPS)

Months/years later, had pain in other leg, that was quite similar to the ipsilateral leg
(as if the sensitisation was spreading)

Was NA system failing to spatially constrain their sensitisation? Augmenting NA with reuptake inhibitors failed to cure pain :(

55
Q

Summary….

What we know about NA now, what we need to do for functional benefits..

A

Selective viral genetic manipulation of few 100 neurons can alter whole animal pain behaviours (profound effect)

  • NA system engaged by persistent inflammation
  • NA acts to spatially restrict & delay the onset of sensitisation in a neuropathic pain model

But to be useful we need: AUGMENTATION of NA system

Working on several strategies:

  • NA reuptake inhibitors
  • Channel rhodopsin
  • DREADDs
56
Q

What did Hughes et al (2015) show (using an intrathecal catheter)?

A

TNT with intrathecal catheter: REBOXETINE (NA reuptake inhibitor) - 2x daily.

Hour after reboxetine: sensitisation largely reversed - less mechanical/cold allodynia

When stopped 2x daily doses, sensitisation re-emerged (so masking not curing).

To ensure effect is on nociception / perceived pain rather than motor responses (withdrawal) - used place preference test. Injured rats chose reboxetine over saline, whereas control rats had no preference!

57
Q

What did Hughes et al (2015) show with systemic reboxetine?

A

Higher dose needed to get equivalent effect on withdrawal responses as the intrathecal dose (bigger volume of distribution)

Despite improved withdrawal, both nerve injured animals and naive animals AVOID reboxetine in place preference - adverse effects?

58
Q

What technique did Hickey et al (2014) use?

A

Optogenetics

Virally transduced LC neurons with channel rhodopsin (light sensitive ion channel - opens within µ-seconds of blue light) + mCherry to confirm that it worked

(Lenti-PRSx8-ChR2-mcherry)

Anaesthetised animal 3-4 weeks later + lowered optical fibre into pons (LC)

59
Q

What were Hickey et al’s (2014) findings?

A

Light: just over 50% of animals ↑ hind paw thermal withdrawal threshold (analgesia) - stimulating the WHOLE LC (not just spinally projecting) produced nociceptive suppression in anaesthetised animals.

BUT 7 animals had exact opposite (reduced threshold, hyperalgesia). Experiment much more specific (know only LC neurons targeted), so cannot say that another pathway is causing sensitisation in these animals.

Animals with analgesic effect had more neurons DEEPER in the nucleus that were transduced with the virus.

Then tried stimulating LC in same animal with optical fibre but at different depths
Dorsal stimulation = PRO-NOCICEPTIVE
Deeper/ventral stimulation = ANTI-NOCICEPTIVE

60
Q

How could the LC be modular? What is the significance of this?

A

Acting as separate modules instead of one unit - each releasing NA, but defined by different projection targets

Want to selectively target spinally projecting part (analgesic). Need a means to selectively target analgesic system

61
Q

How can retrograde optogenetics be used?

A

Canine adenoviral virus - does retrograde transduction well (inject in cord, labels LC neurons projecting to cord, allows expression of channel rhodopsin in them)

Functional effects in vivo for >6 months (to validate that virus working correctly, show over 6 months that can light activate the neurons and produce sleep-wake transitions)

Then inject virus at spinal level or at cortical level - looking for differences in the neurons labelled and the functional effects of activating them

62
Q

What did Li et al (2016) find?

*used retrograde optogenetics

A

mCherry (red) - injected in pre-frontal cortex
Green - virus injected in spinal cord

Then examined LC: shows DIVIDE between dorsal (red) + ventral (green)

At least 2 populations of LC neurons defined on basis of projection target + located in slightly different positions in the nucleus

*May explain previous ontogenetic results where pro-nociceptive effect occurred dorsally and anti-nociceptive effect ventrally (deep)

63
Q

How can a chemogenetic strategy be used to manipulate NA neurons?

A

Engineered neuronal nicotinic receptors (cation channel) so that no longer recognises ACh - recognises PSAM (pharmacologicaly selected activator molecule) instead.

Inject virus with engineered receptor + desired promotor (to ensure expression in NA neurons - only the NA neurons will be excited in response to drug).

Locally apply drug in vicinity of neurons - increase firing rate (excite the neurons)

*If give same drug intraperitoneally (systemic adminstration) also excites the neurons, discharge frequency - (rate count of firing) goes up about 8x then returns to baseline

64
Q

What did Hirschprung (2017) show?

heat withdrawal

A

Chemogenetics: showed selective pontospinal anti-nociception

Rats freely mobile, infrared heat source beneath paw, withdraw after certain latency (controls with CL paw, and with saline)

If spinally-projecting LC neurons transduced with chemogenetic receptor: withdrawal latency goes up with the drug (7.5s to ~12s) - comparable to morphine

If PFC-projecting neurons transduced: drug has no effect on nociception - therefore, different modules show specificity in their effects on nociception

65
Q

What did Hirschprung (2017) show?

1st experiment - place preference

A

Conditioned place preference test (heat maps to study movement)

Animals with control virus: no preference for drug/saline. Animals with chemogenetic virus into whole LC - preference for SALINE.

  • Remember NA reuptake inhibitors = aversive - likely a consequence of activating LC neurons that project to cortex.
  • Seems that activation of the LC is aversive (LC = alerting, arousing, stress-inducing nucleus)
66
Q

What did Hirschprung (2017) show?

2nd experiment - place preference

A

Targeting PFC projecting or spinal cord projecting with chemogenetic virus

If spinal cord projections transduced,
activation of neurons with drug = no preference.

PFC cortex transduced = some aversion to the activating drug.

67
Q

What did Hirschprung (2017) show about NP pain?

von Frey withdrawal

A

Activation of LC:SC is analgesic in neuropathic pain model

After nerve injury, lower von Frey withdrawal threshold (shows sensitisation)

Chemogenetic activation of ps:LC (spinally projecting neurons) SUPPRESSES NEUROPATHIC sensation in animals who have become sensitised by nerve injury
- when drug given in increasing doses, transiently reverse von Frey sensitisation (lasts few hours) then returns to sensitised state. Cold allodynia (acetone) also reversed at least in part.

*similar effect to intrathecal reboxetine

68
Q

What did Hirschprung (2017) show about NP pain?

conditioned place test

A

Nerve injured animal with chemogenetically activated ps-LC neurons

When animals naive - no preference drug/saline

After nerve injury: strong preference for drug side

Injured animals also weight bare more evenly on two hind paws after activating LC:SC neurons *not only suppressing evoked nociceptive response, also have effect on ongoing pain!

69
Q

Overall what did Hirschprung (2017) show about NP pain (summary)

A

Opposing effects on pain and effect - specificity in organisation of NA system (different modules)

In NP pain model; activating LC:PFC has negative affect and seems to increases spontaneous pain - no effect on evoked pain, but seem to increase amount animals attended to injured paw.

In contrast, activating LC:SC has positive affect, analgesia and less spontaneous pain

Is this translatable? how can we turn this into a useable therapy? number of avenues for benefitting humans;;;

70
Q

How can we make Hirschprungs findings about specific modules of the NA system translatable?

A

Currently able to deliver targeted, precise excitation (currently) - new experm. avenues:

  1. Examine mechanisms in vitro and in vivo (cell /molecular and behavioural level)
  2. Temporally controlled
  3. Vectors to selectively engage endogenous analgesic (EA) system over long term, conditionally
  4. Research expressing vectors in primary afferents, to see how the detector system is wired/behaves
  5. Test efficacy in acute + chronic pain models
  6. Define role of other NA modules in stress, anxiety, arousal

This has clarified some of the roles of NA system in acute & chronic pain - resolved some outstanding issues (not everyone agrees)

  • possible to augment to suppress chronic pain?
  • possible to prevent?

NS of Pain (7 decks)

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