Novel Neuronal Signalling Mechanisms (A*)

Question 1

What is the relative abundance of glia with respect to neurones?

Answer 1

Glia are 10-50x more abundant than neurones.

Question 2

List 5 functions of glia.

Answer 2

Functions of glia include:

1 - Guiding connecting.

2 - Physical support.

3 - Metabolic support.

4 - Myelination.

5 - Signalling.

Question 3

List the types of glia.

Give the relative abundance of each.

Answer 3

Glia are divisible into two categories:

1 - Microglia (10%).

• This category has no subtypes.

2 - Macroglia (90%).

• Ependymal cells (5%).
• Oligodendrocytes (5%).
• Astrocytes (80%).

Question 4

What is the function of microglia?

Answer 4

Microglia are the resident CNS macrophage-like cells.

Question 5

What is the function of ependymal cells?

Answer 5

Ependymal cells produce CSF.

Question 6

What is the function of oligodendrocytes?

What is the function of Schwann cells?

Answer 6

• Oligodendrocytes myelinate CNS neurones.

- Schwann cells myelinate PNS neurones.

Question 7

What are the functions of astrocytes?

Answer 7

Functions of astrocytes include:

1 - Metabolic support by coupling neuronal activity with blood flow.

2 - Extracellular ion homeostasis by K+ buffering.

3 - Neurotransmitter uptake.

4 - Gliotransmitter release.

Question 8

What is a tripartite synapse?

Answer 8

A tripartite synapse is the notion of the functional interplay and close proximity of a presynaptic neurone, postsynaptic neurone and a glial cell.

Question 9

Which cellular response occurs in glial cells when neurotransmitters bind to glial neurotransmitter receptors?

Why is this important?

Answer 9

• When neurotransmitters bind to glial receptors, Ca2+ channels open, causing Ca2+ influx into the glial cell. This results in:

1 - Gliotransmitter release

2 - The induction of a Ca2+ wave which propagates between neighbouring glial cells through gap junctions.

• This Ca2+ excitability is important for mediating the astrocyte response to neuronal activity, enabling neurone-glial and glial-neurone signalling.
• Despite this, glia are not considered ‘excitable’ cells.

Question 10

What is the importance of glial ATP receptors?

What is the function of these receptors in glia?

Answer 10

• Glial P2X receptors (an LGIC that responds to ATP) are important because, in the extracellular space, ATP is a damage signal (since it is released by perforated cells).
• In glia, ATP is detected by P2X receptors, and this triggers two defensive signalling mechanisms:

1 - Induces a Ca2+ wave from intracellular stores by triggering a downstream signalling pathway mediated by inositol (1,4,5) triphosphate.

• Both the Ca2+ and inositol (1,4,5) triphosphate can enter adjacent glia through gap junctions, spreading the signal.

2 - Stimulates the cell to release more ATP into the extracellular space in order to spread the signal.

Question 11

List 5 identified gliotransmitters.

What is known of the process of gliotransmitter release?

Answer 11

Identified gliotransmitters include:

1 - ATP.

2 - GABA.

3 - D-serine.

4 - Glutamate.

5 - TNF-alpha.

• All that is known of the process of gliotransmitter release is that it is Ca2+ dependent.

Question 12

Describe the role of glutamate as a gliotransmitter.

Answer 12

• Excessive glutamate release caused by high frequency neuronal firing can result in synaptic spillover when uptake mechanisms become overwhelmed.
• In response to synaptic spillover of glutamate, extracellular glutamate receptors are stimulated on surrounding glial cells.
• This stimulates glia to regulate synaptic strength by releasing glutamate:
• Negative feedback occurs when this glutamate binds to presynaptic mGluRs.
• Positive feedback can also occur when this glutamate binds to extrasynaptic NMDARs.

Question 13

How might astrocytes be involved in seizure activity?

Answer 13

• Seizure activity is characterised by synchronous neuronal firing.
• An initial excitation at one synapse in the brain could be picked up by an astrocyte, and spread to other synapses through the astrocyte’s other processes.
• Since an astrocyte makes many synapses with many different neurones, this could result in the spread of synchronous firing.

Question 14

Which coagonists must also bind to glutamate receptors in order to induce a receptor potential at NMDA receptors?

Answer 14

In order to induce a receptor potential, glutamate must bind to NMDA receptors with either:

1 - Glycine.

2 - D-serine.

Question 15

List the receptors expressed by microglia.

Answer 15

Microglia express both immunological and neurotransmitter receptors:

Immunological:

1 - LPS.

2 - gp120HIV coat protein.

Neurotransmitter:

1 - ATP.

2 - Fractalkine.

Question 16

List 5 consequences of microglia receptor activation.

Answer 16

Microglia receptor activation results in:

1 - Microglia proliferation.

2 - Phagocytosis.

3 - Upregulation of NOS.

4 - Upregulation of cytokines.

5 - Upregulation of chemokines.

Question 17

List 3 roles of microglia in pathology.

Answer 17

Roles of microglia in pathology include:

1 - Chronic pain:

• Activated microglia release signalling molecules that result in astrocyte activation.
• Activated astrocytes amplify spinal pain signalling, causing chronic pain (see A* card 38).

2 - Neurodegenerative disorders (e.g. Alzheimer’s and Parkinson’s).

• Due to the neurotoxic nature of activated microglia.

3 - Ischaemic brain damage.

• Due to the neurotoxic nature of activated microglia.

Question 18

List 2 neuroprotective functions of microglia.

Answer 18

Neuroprotective functions of microglia include:

1 - Glutamate uptake, preventing excitotoxicity.

2 - Phagocytosis of :

• Dead neurones.
• Amyloid plaques.
• Pathogens.

Question 19

List 5 possible areas for pharmacological control of glial activation.

Give examples for each.

Answer 19

1 - Blockade of glial activation.

• E.g. fluorocitrate or minocycline.

2 - Inhibiting proinflammatory cytokine synthesis.

• E.g. propentofylline or thalidomide.

3 - Cytokine receptor antagonists.

• E.g. anakinra or etanercept.

4 - Disrupting proinflammatory cytokine signalling cascades.

• E.g. MAPK inhibitors.

5 - Gap junction modulators.

• No drugs yet.

Question 20

What is the psychoactive component of cannabis that is responsible for the euphoria / high?

Which component of cannabis is thought to have therapeutic potential?

Answer 20

• Tetrahydrocannabinol (THC) is responsible for the euphoria / high produced by cannabis.
• Cannabidiol (CBD) is the component of cannabis that is thought to have therapeutic potential.

Question 21

Describe the distribution of cannabinoid (CB) receptors in the body.

Answer 21

Cannabinoid (CB) receptors are found in:

1 - Presynaptic CNS neurones:

• Cortex.
• Basal ganglia.
• Cerebellum.
• Hippocampus.

2 - Presynaptic PNS neurones.

3 - The immune system.

Question 22

Where on the synapse are cannabinoid receptors usually located?

Answer 22

Cannabinoid receptors are usually located presynaptically.

Question 23

List 2 endogenous cannabinoid receptor ligands.

From which molecule are they synthesised?

Answer 23

Endogenous cannabinoid receptor ligands include:

1 - Anandamide (AEA).

2 - 2-Arachidonoylglycerol (2-AG).

• These cannabinoid receptor ligands are synthesised from membrane lipids, and contain arachidonic acid.

Question 24

Describe the process of cannabinoid synthesis.

Answer 24

• Cannabinoids are synthesised on demand rather than being stored.
• Cannabinoid synthesis is triggered by Ca2+ influx.
• Ca2+ influx activates PLD, which converts NAPE into anandamide.
• Ca2+ influx activates PLC and DAGL, which together convert DAG into 2-AG.

Question 25

Describe the process of cannabinoid release, reuptake and enzymatic breakdown.

Answer 25

• Cannabinoid release is triggered by Ca2+ influx, and occurs at the point of synthesis (they are not stored because they are lipophilic).
• Release occurs by diffusion across the synaptic membrane.
• Active reuptake transporters are responsible for reuptake.
• Enzymatic breakdown of anandamide occurs by FAAH.
• Enzymatic breakdown of 2-AG occurs by MAGL.

Question 26

List the subtypes of cannabinoid receptors.

What type of receptors are cannabinoid receptors?

Answer 26

• Subtypes of cannabinoid receptors include CB1 and CB2.

- Cannabinoid receptors are GPCRs.

Question 27

Where are CB1 and CB2 receptors found?

Answer 27

• CB1 receptors are found on presynaptic neurones in the CNS and PNS.
• CB2 receptors are found in the immune system.

Question 28

Give an example of an agonist and antagonist for CB1 and CB2 receptors.

Answer 28

CB1:

• Agonist: ACEA.
• Antagonist: Rimonabant (more on the last card).

CB2:

• Agonist: JWH-133.
• Antagonist: AM630.

Question 29

What is the primary effect of cannabinoid receptor activation on neuronal function?

List 2 mechanisms of cannabinoid receptors.

Answer 29

• Cannabinoid receptors produce retrograde inhibitory receptor potentials to influence synaptic plasticity.
• CB1 and CB2 receptors cause inhibition because they are Gi/o-coupled GPCRs.

Mechanisms include:

1 - Increasing G-protein coupled inward rectifier K+ channels, resulting in an increase in K+ influx.

2 - Inhibiting voltage-gated Ca2+ channels.

Question 30

List 2 functions of cannabinoids other than retrograde signalling.

Answer 30

1 - Non-retrograde signalling.

• Cannabinoids released at postsynaptic neurones can activate CB1 receptors and TRPV1 channels on the same postsynaptic neurone (see somatosensory system lecture for TRPV1 channels and A* cards 39 and 40 below).

2 - Neurone-astrocyte signalling.

• Cannabinoids released at postsynaptic neurones can activate CB1 receptors on astrocytes.

Question 31

Which cannabinoid ligand contributes mostly to neuroplasticity?

Describe the mechanism by which this ligand brings about neuroplastic changes.

Answer 31

• 2-AG contributes mostly to neuroplasticity.
• It mediates a negative feedback mechanism used to offset excessive excitation OR inhibition (in this example, excitation):

1 - Ca2+ influx on postsynaptic neurones triggers a downstream pathway involving PLC and DGL-alpha.

2 - This signalling pathway results in 2-AG synthesis and release into the synapse

3 - The 2-AG binds to presynaptic CB1 receptors.

4 - Activation of presynaptic CB1 receptors results in the inhibition of presynaptic voltage-gated Ca2+ channels, which are required to induce neurotransmitter release.

5 - Inhibition of presynaptic Ca2+ channels results in a decrease in neurotransmitter release, weakening the synapse in response to overstimulation (a form of negative feedback).

• Since cannabinoids are lipophilic, this retrograde signalling mechanism can also affect neighbouring neurones, resulting in ‘heterosynaptic’ long-term potentiation or long-term depression.

Question 32

List 5 clinical effects of marijuana smoking.

Answer 32

Clinical effects of marijuana smoking include:

1 - An increase in appetite.

2 - Anxiolysis.

3 - Analgesia.

4 - Antiemetic effects.

5 - Anticonvulsant effects - particularly useful for paediatric epilepsy.

Question 33

Briefly describe the mechanism for cannabis oil for the treatment of epilepsy.

What is Epidiolex?

Answer 33

• Cannabis oil contains both THC and CBD.
• THC acts via the the CB1 receptor.
• CBD has a low affinity for both CB1 and CB2 receptors.
• There is a synergistic effect between CBD and THC that underlies their anticonvulsant effect.
• Epidiolex is used to treat the same condition, but it contains no THC.

Question 34

What is Sativex?

What is it used for?

Answer 34

• Sativex is a drug containing CBD and THC.

- It is used for its antispasmodic effect to treat multiple sclerosis.

Question 35

What is Rimonabant?

What is it used for?

Answer 35

• Rimonabant is a CB1 receptor antagonist.
• It is used as an anorectic (anti-obesity drug - CB1 antagonist reduces appetite).
• It was withdrawn in 2008 because it increases the risk of psychiatric disorders, but it’s back now!

Question 36

Huge boi A*:

List 6 arguments against the notion of gliotransmission in physiological conditions.

List 5 points opposing these arguments.

Answer 36

Arguments against the notion of gliotransmission in physiological conditions (from Fiacco et al., 2018):

1 - Most methods used to demonstrate gliotransmission induce gliotransmitter release by physiologically irrelevant stimuli, e.g. optogenetics and mechanical stimulation using pipettes.

2 - Ligands used in studies to demonstrate gliotransmission, such as DHPG (an mGluR agonist) also have receptors on neurones. Therefore, the observed effect on neuronal transmission might not be a result of gliotransmission.

3 - Evidence for the existence of Ca2+ waves is confined to studies of the hindbrain. Studies that have found evidence for Ca2+ waves using forebrain neurones have only achieved this in vitro, and without using intact slices.

4 - Methods used to stimulate gliotransmission are likely to interfere with other Ca2+ dependent functions of astrocytes. Namely:

• Extracellular ion homeostasis, e.g. of K+.
• Glutamate uptake.
• Synthesis and release of neurotrophic factors.

5 - Although there is evidence for the presence of machinery for glutamate exocytosis (e.g. SNARE proteins - see A* card 38), a recent study (Chai et al., 2017) found that neurones derived from the hippocampus and corpus striatum did not express synaptotagmin or vGLUTs.

• This would imply that the astrocyte would be unable to sense Ca2+ influx for exocytosis, and glutamate would not be able to be loaded into vesicles for exocytosis.

6 - Many methods have only demonstrated gliotransmission in animal experiments, and this might not reflect signalling in the human brain. For example, early research into gliotransmission identified mGluR5 receptors on astrocytes derived from mice. This was thought to play a major role in gliotransmission, but later studies revealed that mGluR5 receptors are not expressed in adult human astrocytes.

7 - Various functions that could be attributed to astrocytic gliotransmission are able to occur in IP3R knockout mice. Since IP3 generated from GPCR signalling is thought to be the primary source of Ca2+ in astrocytes, removal of the IP3R should attenuate gliotransmission, and hence block functions such as cerebral autoregulation and synaptic plasticity.

8 - Studies that have induced Ca2+ activity in astrocytes have not observed concomitant effects on synaptic plasticity.

Arguments for the notion of gliotransmission in physiological conditions (from Savtchouk and Volterra, 2018):

1 - (versus point 6 above) Although mGluR5 receptors have been shown to be absent in adult human astrocytes, gliotransmission has also been shown to occur in response to activation of other astrocytic receptors such as CB1 receptors, cholinergic receptors and P2Y1 receptors.

2 - (versus point 7 above) Studies using IP3R knockouts rely on the assumption that IP3 signalling is the only mechanism by which intracellular Ca2+ can be raised in astrocytes, when in fact there may be other mechanisms involved.

3 - (versus point 8 above) Studies should not expect changes to synaptic plasticity simply by inducing single elevations of Ca2+ in astrocytes, because the astrocytic response to raised intracellular Ca2+ depends on the spatiotemporal nature of the Ca2+ activity.

• E.g. Jouaville et al. (1995) demonstrated a differential effect on oocyte ‘excitability’ response to varying velocity, frequency and amplitude of an inositol (1,4,5) triphosphate-induced Ca2+ wave. Considering the ubiquity of this signalling mechanism, the quality of Ca2+ signalling should not be ruled out as an insignificant determinant the astrocyte response.

4 - (versus point 8 above) Studies investigating effects of astrocytic Ca2+ activity on synaptic plasticity generally fail to consider the broader scope of mechanisms underlying long-term potentiation, which is not itself a single process.

5 - (versus point 5 above) The argument ‘gliotransmission does not occur due to the absence of astrocytic vGLUTs and synaptotagmin’ relies on the assumption that the mechanism by which astrocytic glutamate release occurs is identical to the process in neurones.

• In fact, the mechanisms are likely to be different due to the difference in the spatiotemporal nature of Ca2+ events in astrocytes vs neurones. Even within astrocytes, heterogeneity of the spatiotemporal aspects of Ca2+ signalling is a major determinant of the cellular response (Gomes et al., 2018).

Question 37

A*:

What is metaplasticity?

How might astrocytes contribute to metaplasticity?

Answer 37

• Metaplasticity is the modulation of factors that induce plasticity.
• It is a means of priming, or preventing the onset of, plasticity.
• One example of astrocytic control of metaplasticity is the changing of the physical arrangement of the tripartite synapse.

Example:

• The supraoptic nucleus synthesises oxytocin, which together with prolactin, promotes lactation.
• It was found that in lactating rats, the degree of astrocytic coverage of synapses was decreased as a space formed between the astrocytes and the synapse (Panatier et al., 2006).
• This meant that gliotransmission between the astrocytes and the neurones of the tripartite synapse was reduced, as the concentration of gliotransmitter in the synapse decreased.
• Specifically, a reduction in D-serine gliotransmission (an agonist for the glycine binding site of NMDA receptors) led to changes in neuronal NMDA receptor activity, which in turn modulates LTP.
• These metaplastic changes were not seen in non-lactating rats, implying that the process is important for the timely onset and degree of lactation.

Question 38

A*:

Describe the role of astrocytes in normal pain modulation.

Answer 38

• Astrocytic ATP is converted by ATPases in the extracellular space into adenosine.
• Adenosine binds to A1 receptors in neurones in the ascending pain pathways.
• A1 receptors are Gi/o coupled, and therefore cause inhibition when activated.
• However, there is also evidence of the involvement of other gliotransmitters in pain modulation, as pain threshold was reduced in mice underexpressing glial SNARE proteins (Foley et al.,2011).
• This suggests involvement of other gliotransmitters requiring Ca2+dependent transport, such as glutamate, GABA or D-serine, in potentiating pain transmission.
• This study was met with controversy over the exclusivity of the targeted SNARE protein to astrocytes. However, the targeted SNARE protein was shown to colocalise with the astrocyte-specific protein, GFAP, but not the neuronal-specific protein, NeuN, or microglial-specific protein, Iba1. These are widely used and well-validated markers with high specificity to these cell types, hence the SNARE protein was likely astrocyte-specific.

Question 39

A*:

Describe the roles of cannabinoids in learning and memory.

How might this relate to the memory deficits in Alzheimer’s disease?

Answer 39

Two opposing roles of cannabinoids in learning and memory:

1 - In the hippocampus, CB1 receptors are expressed presynaptically on inhibitory GABAergic neurones.

• CB1 signalling onto presynaptic receptors can cause an inhibition of GABA release into the synapse.
• This disinhibition is a known as long-term depression of inhibition, and strengthens excitatory neural pathways in the hippocampus.
• It has been shown that application of amyloid beta to these synapses prevents this process of long-term depression of inhibition. This is a potential mechanism by which memory formation might be impaired in Alzheimer’s disease.

2 (repeated in card 40) - In the dentate gyrus, continued activation of postsynaptic mGluR5s upregulates expression of of anandamide.

• Anandamide binds to TRPV1 autoreceptors on the postsynaptic neurone, leading to long-term depression.
• This has the opposite effect to the mechanism in point 1, as it inhibits excitatory neural pathways in the dentate gyrus, preventing formation of memories.

Question 40

A*:

List 2 non-retrograde signalling mechanisms of cannabinoids.

Answer 40

Non-retrograde signalling mechanisms of cannabinoids:

1 - In the neocortex, it has been shown that continued activation of postsynaptic GABAergic neurone leads to Ca2+-mediated 2-AG synthesis.

• The 2-AG binds to CB1 autoreceptors on the postsynaptic neurone.
• Activation of these CB1 receptors activates inward-rectifier K+ channels, causing inhibition.
• This is another example of long-term depression of inhibition.

2 (repeated in card 39) - In the dentate gyrus, continued activation of postsynaptic mGluR5s upregulates expression of of anandamide.

• Anandamide binds to TRPV1 autoreceptors on the postsynaptic neurone, leading to long-term depression.

Question 41

A*:

Describe a possible mechanism of action for the antidepressant effects of cannabinoids.

Answer 41

• In depression, underexpression of BDNF caused by hyperactivity of the HPA axis is responsible for decreases in neurogenesis of hippocampal dopaminergic neurones. This contributes to pathogenesis of depression in 2 ways:

1 - Hippocampal dopaminergic neurones normally have a role in negative feedback of the HPA axis, however in depression this function is lost due to poor neurogenesis. The resulting elevated cortisol is thought to play a role in the pathogenesis of depression.

2 - Loss of hippocampal neurones leads to hypoactivity of the mesolimbic reward pathway.

• Cannabinoids are thought to exert an antidepressant effect through mTOR signalling in hippocampal neurones (Sartim et al., 2018).
• These findings are in agreement with a report by Sales et al. (2019), in which a similar mTOR-mediated antidepressant effect of cannabinoids was demonstrated in the prefrontal cortex.
• Activation of CB2 receptors activates downstream cascades involving PI3k and Akt.
• This signalling cascade activates mTOR, which is a kinase that leads to expressional changes, e.g. upregulation of BDNF, promoting neurogenesis, neuroplasticity and neuroprotection.
• Hence, mTOR signalling is an emerging target for antidepressant drugs, and can be targeted by cannabinoids and other drug classes (for more details on emerging drugs, see card 15 of depression deck).

Question 42

A*:

What is different about CB receptors in astrocytes?

What effect might this have on synaptic plasticity?

Answer 42

• CB1 and CB2 receptors are generally considered to be coupled to Gi/o. However, in astrocytes, the receptors are thought to be coupled to Gq.
• This would suggest that CB receptors are a possible mechanism by which neurones are able to induce Ca2+-dependent gliotransmitter release in astrocytes.
• CB-induced gliotransmitter release in hippocampal glial cells has been evidenced in mice by a study by Navarette and Araque (2008). Activation of glial CB receptors by anandamide led to PLC-dependent release of Ca2+ from internal stores, resulting in release of astrocytic glutamate. This, in turn, produced a slow excitatory potential in the postsynaptic neurone through action at NMDA receptors. This is a form of short-term plasticity.

Question 43

From neuropeptides A* cards (perhaps look at the neuropeptides lecture to see what NAAG is):

Describe the role of NAAG in development.

Answer 43

The role of NAAG in development involves neurone-glial and glial-glial signalling:

• Neurones release NAA and NAAG in the extracellular fluid.
• NAA signals to oligodendrocytes to promote myelination.
• NAAG signals to astrocytes to promote neuronal metabolic support.
• Oligodendrocytes also synthesise and release NAAG to signal to astrocytes, and conversely astrocytes hydrolyse NAAG into NAA, which is released to signal to oligodendrocytes.
• These signalling pathways are thought to be involved in:

1 - Controlling release of trophic factor.

2 - Transmitting information regarding the direction and distance between neurones and glia.

• Deficiency of this pathway is thought to result in poor formation of the brain in developmental abnormalities.

Question 44

From pain and analgesia A* cards:

Describe the role of cannabinoid receptors in pain signalling.

Answer 44

Background:

• CB receptors involved in the modulation of pain are found mostly on Adelta fibres, but also on some C fibres.
• Noxious stimuli increase endocannabinoid release.
• CB1 receptors are located both centrally and peripherally in immune cells.
• CB2 receptors are located peripherally in immune cells.
• Both CB receptors are Gi/o GPCRs.

Analgesia mechanism:

• CB1 receptors can attenuate synaptic transmission of ascending pain pathways (through the downstream effects of Gi/o).
• Both CB1 and CB2 receptors are expressed in various inflammatory cells, and have an analgesic effect in inflammatory hyperalgesia.
• This effect is through attenuating NGF-induced degranulation of mast cells, reducing inflammation-induced peripheral sensitisation.
• Therapeutically, CB1 agonists are not preferable because of the side effects associated with the modulation of central synaptic transmission.
• Selective CB2 agonists are a potential avenue for analgesic drugs.
• Tolerance and dependence are potential issues with these treatments.

Question 45

From pain and analgesia A* cards:

Describe the role of glia in chronic pain.

List 2 analgesic drugs that target glia.

Answer 45

• Glia do not directly mediate pain, but can enhance existing pain signals.
• Glia can be activated by a variety of neuronal factors, such as those released in the inflammatory soup (card 13) and paradoxically by opioids.

4 successive glial activation states have been reported in chronic pain (Ji et al., 2013):

1 - ‘Glial reaction’.

• Upregulation of mitogens.
• Hypertrophy.
• Proliferation.

2 - Activation of MAPK signalling, e.g. ERK, due to the activity of mitogens which are upregulated in the glial reaction.

• It has been shown that ERK signalling mediates central sensitisation in dorsal horn neurones, because ERK regulates glutamate and K+ transmission.

3 - Increased expression of ATP, intercellular connexin channels (AKA hemichannels) and chemokine receptors, and decreased expression of glutamate transporters.

• ATP influences pain signalling via P2Y receptors on neurones and glia.
• Hemichannels permit flow of small signalling molecules such as ATP, ions and microRNAs.
• Chemokines attract inflammatory cells, promoting an inflammatory response.
• Glutamate transporters are required for glutamate uptake from the synapse. This is an important process in the cessation of signal generation at glutamatergic synapses. Hence, reduced expression of glutamate transporters prolongs glutamate-induced pain signals.

4 - Increased expression of growth factors (e.g. NGF - card 26), chemokines, cytokines and other glial mediators.

• These changes (1-4) alter neurone-glial and glial-neurone communication, and hence contribute to chronic pain by causing both central and peripheral sensitisation.

Analgesic drugs that inhibit glial activation include:

1 - Propentofylline.

2 - Minocycline.