Systems Neuroscience Notes

8/6/2019 Systems Neuroscience Notes

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Chemical anatomy of pain pathways

Primary afferent nociceptors are specialised free nerve endings of primary

afferents ² A& C fibres. They detect noxious stimuli ² anything that has

produced tissue damage or threatens to do so.

y A fibres

o M yelinated ² fast conduction velocity -> sharp, pricking, fast

(epicritic) pain ² informative (precise location of insult) --> reflex

withdrawal

o Activated by mechanical/thermal stimuli (high threshold)

Not all A fibres are noxious ² some are Down Hair

receptors

o 2 main classes of A fibres -> can be distinguished by their

differential responses to heat

o A fibres also exist which are propr ioceptors in muscle spindles &

A fibres which are low threshold mechanoceptors but 20% are

high threshold so can serve as nociceptors

Both types innervate specific peripheral receptors

y C fibres

o Unmyelinated ² slow conduction velocity -> slow, burning

(protopathic) pain --> changes behaviour of person & emotional

reactiono Polymodal (activated by mechanical, thermal & chemical stimuli)

o Not all C-fibres are nociceptors ² there are also some unmyelinated

low threshold mechanoreceptors

Cell bodies of the nociceptors are located in the dorsal root ganglion. They are

small. Small DRG cells can be divided as:

M yelinated (A) ² contain neurofilament NF200

Unmyelinated (C) ² petidergic; contain substance P, CGRP &somatostatin

o Start in stratum spinosum& ends in lamina I and lamina II outer

o Express TrkA -> depend on NGF for survival

Unmyelinated (C) ² non peptidergic; contain FRAP & cell surface

glycoproteins which bind the plant lectin IB4

o Start in stratum granulosum& ends in lamina II inner



o Mass related gene coupled protein receptors distinguishes

subtypes

o Express GFR1/2 & receptor tyrosine kinase Ref -> depend on

GDNF for survival

o Also express P2X3 receptors

Cell marker correlates with peripheral target tissue innervated too:

Visceral ->peptidergic

Skin -> mostly peptidergic

Muscle -> small % non peptidergic

Sensory transduction: noxious stimuli detected -> transduced into inward

currents that if large enough generates APs along axon to CNS

Ion channels expressed in axon terminals and ce ll soma when activated lead to

inward currents that depolarise the axon (generator potential) -> if large enough

APs fires. Lots of different types:

TRP ² opens in respond to heat, H +&capsacin

o TRPV1 ² expressed by 60% of peptidergic& 75% of non -peptidergic

neurones

P2X3

Na+ channels i.e. 1.8, 1.9

Target tissue --> peripheral nerve --> DRG (soma) --> dorsal root --> spinal cord

Cell bodies with the largest diameters give rise to myelinated, rapidly

conducting A fibres ->most detect innocuous stimuli but 20% are HT Ms. B y

contrast, small & medium diameter cell bodies give rise to most of the

nociceptors.

Dorsal horn can be divided into 6 parallel laminae ² patterns of termination of

primary afferents within the spinal cord related to axonal diameter andreceptive field modality.

Superficial dorsal horn (I-III)

Main target for nociceptive primary afferents

Lamina II can be divided into IIi&IIo



Remaining 3 laminae for innocuous stimuli but nociceptive afferents also

terminate here

Lamina I (marginal layer) ² projection neurones & interneurons

y Projection neurones larger than interneurons (esp. marginal cells ofWaldeyer)

Lamina II (substantiagelatinosa ² lack of myelinated fibres) ² interneurons

y Most of the interneurons packed into IIo

o Can differentiate between IIo& Iii due to lack of myelin in Iii as

there are no interneurons

Lamina III ² high density of interneurons (but bigger than those in lamina II)

Lamina IV-VI ² heterogenous with neurones of different sizes including

projection neurones (esp. V)

Afferent fibres entering through dorsal root divide into ascending and

descending branches; mostly terminate near point of entry but can travel

several segments up/down in Lissauer·s tract.

Primary afferents release primarily glutamate onto neurones within the dorsal

horn including:

Projection axons ² convey info to the brain

Interneurones ² remain in spinal cord and serve 3 major functions:

o Relay sensory input from the periphery that may modulate motor

output i.e. reflexes

o Relay as well as modulate descending pathways

Excitatory: glutamate (stalked ² VGlut2/PKC)

High threshold ² noxious stimuli only

Low threshold ² innocuous stimuli only

Wide dynamic range ² noxious & innocuous stimuli

Inhibitory: GABA, glycine (islet ² GABA)

A fibres activate the dorsal column medial lemniscus



DRG -> dorsal column ->cuneate/gracile nucleus -> internal arcuate

(decussating) fibres -> medial lemniscus -> VPL thalamus -> somatosensory

cortex

A fibres activate the spinothalamic tract -> fast pain

Ventral white commissure (decussate straight away) -> spinal lemniscus ->

VPL thalamus -> somatosensory cortex

C fibres activate the spinoreticular tract -> slow pain

Neurochemistry of dorsal horn interneurons:

Inhibitory

o GABAergic but not glycinergic

Neuropeptides: NPY, galanin, encephalin Miscellaneous: NOS

o GABAergic but glycinergic

Miscellaneous: parvalbumin, NOS

Excitatory (VGlut2)

Neuropeptides: somatostatin, neurotensin

Miscellaneous: calbindin, PKC

Receptors: µ, NK1

Projection neurones: express NK1 receptor which binds substance P

Descending monoaminergic axons:

Serotonin from medullary Raphe nuclei

Noradrenaline from locus coerulus

Modulation by interneurons: intrathecal administration of GABA A and glycine

receptor antagonist can cause allodynia (innocuous stimuli causes nociception i.e.

brushing of skin) -> suggests inhibitory interneurons supress activity evoked by

tactile afferents so not perceived as painful.

Counter irritation: rub injury site to soothe pain

T ENS: transcutaneous electrical nerve stimulation -> passage of small current

with + A fibres



Transmission between nociceptive afferents (A& C) and dorsal horn

interneurons is mediated by:

Glutamate ² acts on AMPA receptors -> fast EPSP

o Metabotrophic: mGluRs

o Ionotrophic: AMPA, NMDA (Mg2+ block)

Neuropeptides i.e. substance P ² acts via NK1 receptor to elicit slow EPSP

allowing for temporal summation & glutamate response

Intrathecal injection of substance P conjugated to saporin destroys NK1

expressing neurones in the dorsal horn

Rats treated with this signs of hyperalgesia in inflammatory &

neuropathic pain

But substance P KO & NK1 KO mice show little reduction in hyperalgesia

and NK1 antagonists were disappointing

Concluded that NK1 is important for hyperalgesia but not mediated by SP

on NK1 but glutamate acting on cells (substance P just heightens it)

Two types of pain:

y Nociceptive pain

y Clinical pain

o Inflammatory

o Neuropathic



Pain is useful because it warns us of actual or potential tissue damage but if pain

systems become too sensitive it will cause us pain that provides us of NO

benefit.

HOW PAIN SYST EMS CHANGE:

Allodynia: when thresholds are lowered so innocuous stimuli produce pain

Hyperalgesia: noxious stimuli produce exaggerated/prolonged pain

Caused by peripheral and central sensitisation

Peripheral sensitisation = reduction in the threshold and increase in

responsiveness of peripheral ends of nociceptors due to actions of

inflammatory chemicals

o Primary hyperalgesia ² region of damage hypersensitive to heat and

thermal stimuli

Central sensitisation = increase in excitability of neurones within the CNS

caused by a burst of activity in the nociceptors which alters strength of

synaptic connections between nociceptors and spinal cord neurones

o Allodynia

Secondary hyperalgesia ² region outside injury site becomes

hypersensitive to mechanical stimuli

o Spontaneous pain

Inflammatory pain

NEUROTROPHIC FACT ORS ² regulate the long-term survival, growth or

differentiated function of discrete populations of nerve cells i.e. NGF (nerve

growth factor)

NGF- highly basic protein made of 120 amino acids ² exist as homodimers (2

identical polypeptide chains) ² important in the development of the nervous

system

Binds low affinity p75 receptors and high affinity tyrosine kinase receptors

(trk).

p75 ² preferred receptor for proNGF and modulates the activity of the

trk receptors.



TrkA ² NGF (trkB ² BDNF/neurotrophin 4 & 5; trkC ² neurotrophin 3 [but

in vitro neurotrophin 3 can activate all of them)

NGF binds -> receptor dimerization ->autophosphorylation of receptor ->

tyrosine kinase domain activated -> phosphorylates SH2 receptors on other

proteins inc:

PI3-K

MAP-K (via ras)

trkA principally expressed on peptidergic, small diameter DRG cells

ASSOCIATION BETWEEN NGF AND INFLAMMAT ORY PAIN:

Systemic NGF administration induces thermal hyperalgesia in rodents

within 30 mins and thermal & mechanical hype ralgesia after a few hourso Subcutaneous NGF injections produce thermal and mechanical

hyperalgesia at injection site

IV injections of NGF in humans produce widespread aching pains in deep

tissues and hyperalgesia at injection site

Elevated NGF in human inflammatory states i.e. bladder of cystitis

patients, synovial fluid of arthritis patients

Intraplantar injection of carrageenan produces acute inflammatory

reaction and thermal hyperalgesia is prevented if patient given trkA -IgG

too

Anti-NGF antibodies & NGF antagonists also reduce hyperalgesia

NGF produces sensitization of nociceptors:

Short term changes:

DIRECTLY = binds to trkA receptors on nociceptors -> phosphorylates PLC-->

PLC- converts PIP2 to DAG -> PIP2 mobilises Ca2+ stores from endoplasmic

reticulum via IP3

-> DAG activates PKC -> PKC phosphorylates receptors and ionchannels inc. TRPV1, Nav 1.8 -> increase Ca and Na -> depolarisation

INDIRECTLY = mast cell proliferation & degranulation via trkA -> histamine,

serotonin & numerous cytokines



Limiting amounts in skin -> cytokines produced during inflammation cause

proliferation of fibroblasts and keratinocytes -> release more NGF -> activate

nociceptors

May also target eosinophils, B- and T- proliferation

Activate sympathetic efferents -> release eicosanoids -> activate nociceptors

(but COX inhibitors don·t affect NGF induced hyperalgesia)

May activate 5-lipoxygenase which converts arachidonic acid into

leukotrienes ->chemoattract neutrophils (neutrophils may be important

for sensitisation coz animals without them don·t show thermal

hyperalgesia to NGF)

Long term changes:

Alters gene expression too via Ras-MAPK pathway which promotes CREB which

induces gene expression inc. TRPV1, ASICs, P2X3 and Nav1.8 -> retrograde

transport to DRG (cell body)

MAPK (and other erk fragments) translocate to nucleus where they

phosphorylate and activate various transcription factors which lead to gene

expression -> increased numbers of

Inflammation can also create a humoral cytokine sig nal which produces IL-1B

which recruits and activates microglia (neuropathic pain)

Can lead to central sensitisation -> activation and sensitisation of primary

afferents lead to enhanced spinal cord input which may trigger central

changes/increased NT or neuromodulator release from nociceptive terminals in

DH -> wind up etc.

Spinal cord processing of pain

Causes of central sensitisation:

1) Wind up

a. Homosynaptic (only activated synapse shows change)

b. Increase in DH neurone output over the course of a train of inputs

c. Corelease of neuromodulators i.e. substance P (acts via NK1

receptor) and CGRP which activates metabotrophic receptors -



>temporal summation -> Mg2+ block removed from NMDA receptors

-> voltage gated Ca2+ currents

d. Activity dependent; stops after stimulus stops

2) Classical heterosynaptic central sensitisation

a. Heterosynapticb. Activity dependent; outlasts stimulus by 10s of minutes

c. Reduction of threshold due to AB recruitment/DH neurones

increase number of Aps to suprathreshold stimuli/expand receptive

fields (secondary hyperalgesia)

d. Nociceptors release glutamate which bind AMPA, NMDA and

mGlus& SP which binds NK1 and BDNF which binds trkB -> increase

in Ca2+ -> activates serine/threonine protein kinases i.e. PKA, PKC,

CAMKII &Src -> phosphorylate NMDA (NR1, NR2A & NR2B

subunits) & AMPA receptors increasing sensitivity (increase open

time, remove Mg2+) -> PKC & PKA + ERK which phosphorylates

potassium channels -> PKC also inserts AMPA receptors into

membrane

i. MK-801 , NMDA receptor antagonist, reduces pain

hypersensitivity but not always i.e. don·t contribute to

mechanical hypersensitivity following bee venom

inflammation, Sorkin et al.

ii. Not sure phosphorylation of which subunit is important ->studies have shown that NR2B is most important & blocking

its phosphorylation correlates with reduced hyperalgesia

BUT contradictory findings

e. Noxious stimuli and inflammation + expression of c-Fos& COX-2

(early genes) and prodynorphin, NK1 and TrkB (late genes) via ERK

dependent activation of CREB in spinal cord neurones

f. Primary afferents are also affected -> increased levels of

substance P and BDNF/changes in expression of hundreds of genes

that alter their excitability & transmission properties/phenotypic

switch in some DRG neurones so some begin to express substance P

and BDNF so some non-nociceptor afferents can induce central

sensitisation =>allodynia

3) Long term potentiation

a. Homosynaptic enhancement of EPSPs



b. Brief duration, persistent, high-frequency nociceptor stimulation

i. Nociceptors do not normally fire at high frequencies so LTP

probably constrained to intense stimuli

c. NMDA-receptor induced Ca2+ influx -> activation of CAMKII ->

phosphorylation of AMPA receptors & trafficking of AMPAreceptors to synapse

Opioids and pain control

Opioid ² any substance whose actions are reversed by naloxone

y Includes a number of opiates derived from opium (dried sap from seed

capsule of poppy)

o Morphine (potent)

o Codeine (weak)o Thebaine (opioid precursor)

Act on (at least) 3 subtypes of opioid receptor:

Mu (µ) ² brain (cortex, thalamus, PAG), spinal cord (substantiagelatinosa)

o µ 1 - supraspinal analgesia/µ 2 ² respiratory depression, miosis,

euphoria, gastrointestinal motility, physical dependence

Delta () ² brain (hypothalamus, PAG), spinal cord (substantiagelatinosa)

o Spinal analgesia, sedation, miosis, inhibits ADH release

Kappa () ² brain (pons, amygdala, olfactory bulbs)

Have been cloned and their cDNA sequences deduced -> allowed us to determine

their structure and function.

The opioid peptides ² 3 families derived from 3 pro-hormones:

-endrophins from POMC - µ & receptors

Enkephalins from pro-enkephalin ² receptor

Dynorphins from pro-dynorphin ² receptor

All share the same N-terminal sequence: Try-Gly-Gly-Phe-Met/Leu

All belong to G protein-coupled receptor superfamily -> 7 hydrophobic

transmembrane domains interconnected by short loops & have an extracellular

N-terminus & intracellular C-terminus.



Mu, delta & kappa = highly homologous (tran smembrane domains &

intracellular loops best conversed)

o T ransmembrane domain form an opioid binding pocket that is

similar across all receptors

E xtracellular loops, N- or C- termini differ greatly o D educed which loops were important for binding of ligand s using

loss -of -function & binding rescue e xperiments

E 1 & E3 = mu selective compounds (DAMGO)

E3 = delta selective compounds (DPDPE )

E2 = kappa selective compounds

All coupled to G i /G0 p roteins (con fi rmed usin g Pe rtussis to xin )

(-) AC -> cytoplasmic events

o Confirmed using forskolin-stimulated cAMP accumulation

(+) MAPK -> transcriptional activity of cells

(-) insulin receptor signalling cascades

Activation of all 3 types inhibits Ca2+ entry via voltage gated Ca2+ channels

Activation of µ/ receptors activate inwardly rectifying K + channels

Predominantly found on presynaptic nerve terminals & inhibit NT release but

some found postsynaptically& hyperpolarise cell neuronal excitability.

Spinal analgesia: act to inhibit release of glu tamate & substance P from

primary afferents/hyperpolarise & decrease excitability of projection

neurones

Supraspinal analgesia: noxious stimuli (+) amygdala, parabrachial area &

central grey -> emotional aspects of pain/(+) thalamus -> sensory aspects

o Exogenous injection of morphine/glutamate/electrical stimulation

into PAG & RVM ->antinociception

Two types of cell in RVM:

y ON cells ² pronociceptive ( firing with nociceptive

spinal reflexes i.e. tail flick/paw withdrawal)

o Express opioid receptors . .. when opioids from

PAG present

y OFF cells ² antinociceptive



o Don·t express opioid receptors; opioids bind

GABAergic neurones hyperpolarising them dis -

inhibiting these so 5-HT can be released

y Neutral cells ² don·t know what these do

opioids (+)periaqueductal grey & nucleus reticularisparagiganticellularis ->(+) nucleus raphe magnus-> (+) serotonin/encephalin pathways (+

noradrenaline from LC) -> decrease transmission of nociceptive

information through DH

ANTIOPIOID SYST EM ² up-regulation of CCK mRNA following nerve injury in

DRG -> mobilises Ca2+ from intracellular stores/decreases the availability of

enkephalins/activates ON cells to induce thermal hyperalgesia

Weak CCK antagonist (proglumide) ² acts on CCK receptors & enhances

opioid analgesia

o Unfortunately, CCK2 selective antagonist didn·t augment analgesic

effect of morphine in patients with chronic neuropathic pain

Neuropathic pain: smaller & spatially restricted loss of opioid receptors/up -

regulated Ca2+ channel activity/increased excitation of spinal neurones due to

activation of NMDA receptors i.e. windup



Other subtypes have been postulated i.e. µ 1& µ 2

Recently, an opioid-receptor like orphan receptor has been cloned ² ORL1

o Believed to bind orphanin FQ (nociceptin) [17 aa peptide] but

physiology of system still unknown

Possibly involved in central modulation of pain (NOT

associated with respiratory depression)

Opioids used clinically ² primarily activate µ receptors

Morphine

Diamorphine (heroin) ² more lipophilic and more potent than morphine

Codeine ² weak opioid (mild pain)

Pethidine ² labour & minor surgery

o Plasma half-life of morphine is 3-6 hrs& longer in neonates since

they lack appropriate liver enzymes/also risk of neonatal

respiratory depression

Pethidine doesn·t rely on liver metabolism so safer

Methadone ² long acting, once daily dosing, used to treat addicts

Buprenorphine ² very lipophilic, partial agonist, used to treat addicts

Fentanyl ² very lipophilic, short-acting, used as part of pre -medication for

surgery (patches used in cancer patients)

Analgesia ² used to treat mild-severe pain; less effective against neuropathic

pain

Euphoria ² state of optimism, cheerfulness and well -being

Respiratory depression ² medulla becomes less sensitive to change in pCO2 ->

cause death in overdose (naloxone used to treat overdose)

Nausea and vomiting ² administered along with an anti-emetic which blocks

effect of chemical trigger zone in area postrema

Miosis ² pin point pupils in overdose

Inhibition of cough reflex ² NOT opioid effect; (+) dextromethorpham

receptor? Used in cough remedies



Severe constipation due to high level of receptors on enteric nerves ² kaolin &

morphine used to treat diarrhoea

Contract gall bladder & constrict bilia ry sphincter ² use with care in pain of

gallstones

Dependence ² psychological (take drug for its pleasurable effects)/physical

(stop unpleasant side effects) -> opioids produce both

Related to development of tolerance ² caused by adaptive changes i.e.

AC levels

Mo st opioid s admini st er ed ora lly or by inj ect ed/bupr enorphin e gi ven

sub lingua lly/cod ein e &diamorphin e ar e m etabo li sed to morphin e in th e body ->

morphin e m etabo li sed in li ver & m etabo lit es excr et ed in urin e

Purin es and pain contro l

P2 purinoc eptor s:

y Ionotrophic (P2X)

y Metabotrophic (P2Y)

Bind ATP

P2X receptors ² ligand gated ion channels ² cation selective channels (Ca 2+

mostly but also Na+& K+)

y Molecular cloning identified 7 genes encoding P2X subunits

o All possess 2 transmembrane regions & have intracellular N and C

termini & a long extracellular loop between the transmembrane

regions

1/3rd of aa in extracellular loop are conserved in ~ 6 subunits

-> involved in ATP binding

y Functional P2X receptor channel formed by a number of P2X subunits(like most other ionotrophic receptors) -> possibly 3 or 4

o Can be homomers/heteromers

P2X4

1. Activated microglia* after nerve injury increase expression of P2X

receptors



a. Activation of microglia is TLR2/4 dependent (LPS from

pathogens/endogenous ligands bind & activate

b. CCL2 from damaged nerves can also stimulate CCR2 receptors on

microglia

2. ATP released from primary afferents/astrocytes bind to P2X4 receptors3. intracellular Ca2+ & activation of p38MAPK (affects transcriptional activity)

4. Release of bioactive diffusible factors i.e. BDNF

&proinflammatorychemokines (CatS) and cytokines (IL-1) which acts directly

on neurones

a. Release of bioactive diffusible factors also caspase 1 and its

accessory protein ASC dependent

b. CatS cleaves soluble transmembranefractalkine -> this interacts with

CX3CR1 receptors on microglia & (+) further release of diffusible

factors

i. Cytokines &chemokines attract more spinal microglia &

astrocytes

5. BDNF down-regulates KCC2 channels, which renders GABAergic currents

from inhibitory interneurons depolarising

6. May interact with excitatory synapses of neighbouring DH neurones &

excitability

*Activated microglia show amoeboid morphology, more phagocytic ac tivity &

increased immune molecule expression (when quiescent exhibit ramified

processes which are highly motile & express low levels of immune molecules)

P2X3 ² can form homo-oligomeric channels or assemble with P2X2 subunits into

hetero-oligomers

ATP causes pain ² applied ATP by ionophoresis into the skin of human fore -arms

-> elicited mild sensation of pain but pain was enhanced by other inflammatory

mediators i.e. capsaicin/UV light/nocifensive behaviour such as hindpaw licking &

lifting following subplantar administration of ATP ² like in humans, increasednocifensive behaviour when coapplied with prostaglandin

E2/formalin/carrageenan

P2X3 receptors are found on non-peptidergic sensory nerurones² clear

excitatory effects of ATP & meATP from in vitro recordings of skin nerves



from rats -> increased in preparations when skin had previously been inflamed

with carrageenan

y In P2X3 KOs, non-peptidergic C-fibres are intact

Two kinds of response to ATP & meATP:

Rapidly rising inward current that desensitises during maintained

applications -> homo-oligomeric

Slowly rising inward currents that declines little if at all during repeated

applications -> hetero-oligomeric(likely to play a physiological role)

Where does endogenous ATP come from?

DAMAGED TISSUE

y Primary afferent nerves

o Sympathetic nerves ² copackaged with noradrenaline i.e. in vas

deferens/arteriolar smooth muscle

y Endothelial/epithelial cells

o Release of ATP from bladder epithelia in response to distention ->

acts on P2X3 receptors on primary afferents beneath urothelium ->

initiates voiding (start urination)

P2X3 KO mice must be distended to a larger volume before

voiding reflex initiatedy Lysed cells ² rheumatoid joints have more ATP than normal

Chronic inflammation (complete Freund·s adjuvant administered to hindpaw) ->

expression of P2X3 protein expression in dorsal root ganglia & larger than

normal ATP-induced currents.

Evidence that it plays a role in pain:

y Antagonists

o Sumarin reduces pain in man but it has actions at so many different

receptors so findings cannot be attributed to P2X

o T NP-ATP reduces nocifensive behaviour elicited by intraplantar

injection of formalin/intra-abdominal injection of acetic acid ²

almost as potent as morphine



Blocks P2X1 & P2X3 receptors but using P2X1 receptor

blocker determined antagonism of P2X3 caused effects

o A-317491: P2X3 antagonist ² produced analgesia following complete

Freund·s adjuvant (inflammatory)/chronic constriction injury

(neuropathic) in rat specieso PPADS

y P2X3 subunit knock down with antisense oligonucleotides - expression of

P2X3 in DRG -> marked reduction in chronic (but not acute) inflammation -

induced thermal & mechanical hyperalgesia& spinal nerve ligation induced

mechanical allodynia

y P2X3 gene KOs

o Homozygotes ² no detectable P2X3 protein in afferents, dorsal

root &nodose ganglia -> no currents elicited by ATP analogues

Modest reduction in hindpaw licking & lifting after

intraplantar formalin

Paradoxical potentiation of thermal hyperalgesia following

complete Freund·s adjuvant

Impairment in ability to sense bladder filling

Mechanisms of neuropathic pain

Neuropathic pain: pain initiated or caused by a primary lesion or dysfunction in

the nervous system; can be:

y Peripheral

y Central

y Mixed

Causes of neuropathic pain:

1) Infection i.e. HIV, herpes simplex virus

2) Metabolic i.e. diabetic neuropathy

3) Neurotoxicity i.e. chemotherapy (vincristine)4) Traumatic i.e. surgical damage

5) Central i.e. SCI

Symptoms: paradoxic because cutting axons should dea den sensation but there

is also evidence of:



Abnormal sensations which occur spontaneously

o Burning/coldness

o Cramping

o ´Pins and needles

sensation

o Itching Pain hypersensitivity

o Allodynia ² pain towards innocuous stimuli

Mechanical (tactile)

Static ² pain in response to light touch/pressure

Dynamic ² pain in response to brushing

o (Hyperalgesia ² stimuli produce exaggerated/prolonged response)

Primary (damaged tissues) -> heat

Secondary (surrounding undamaged tissues) -> mechanical

Can b e continuous/ ep isodic (p aroxysmal)*

*Lik ened to electrical shock

Animal models of neuropathic pain: one a p ortion of the afferents going to the

foot arelesioned

Spinal nerve ligation ² one or more spinal nerves going to foot are ligated

and cut

Partial sciatic ligation ² portion of sciatic nerve is tightly ligated Chronic constriction injury ² placement of 4 loos chromic-gut ligatures on

sciatic nerve -> immune response leads to nerve swelling and constriction

Spared nerve injury ² common peroneal&tibial nerves cut but sural nerve

left intact

Streptozotocin ² model of diabetes associated with development of

neuropathy

Pain hypersensitivity easy to measure in animals (i.e. Von Frey Test of

Mechanical Threshold, Radiant Heat Test of Thermal Sensitivity) bu t abnormalpain sensations difficult [autotomy (self -amputating limbs), cellular markers of

increased neuronal activity i.e. c -Fos, fMRI/PET imaging)

Animal models in some cases have been inconsistent with human models ->

diabetic neuropathy infrequent in humans but robust in rats; NK1 works in

animals not humans



Reflex measures of pain i.e. paw-withdrawal threshold method tests

efferents NOT afferents

Possible mechanisms

Spontaneous neural activity & ectopic sensitivity to mechanical stimuli atthe site of nerve injury

Expression of different molecules in the DRG of injured nerve is up/down

regulated reflecting loss of trophic support from periphery &

spontaneous neural activity in the DRG

Distal part of injured nerve undergoes Wallerian degeneration exposing

surviving nerve fibres to cytokines & growth factors

Partial denervation of peripheral tissues leads to an excess of trophic

factors tissue leading to peripheral sensitisation

Expression of different molecules in the DRG of uninjured nerves

up/down regulated reflecting enhanced trophic support

Sensitisation of postsynaptic DH neurones

Activated microglial cells contribute to development of central

sensitisation

Changes in descending modulation of DH neurones

Injured afferent hypothesis

y Spontaneous firing in some afferents & abnormal responsiveness tothermal, mechanical & chemical stimuli in others

o Neuroma endbulbs ² proximal end of axon seals off and forms

terminal swelling

o Regenerating/collateral sprouts ² fine processes that grow from

endbulb and endeavour to reform connections with target tissues

Without growth guidance from Schwann cells form entangled

mess which is painful (don·t know why ² genetics? Location?)

o Cell soma in DRG

o Patches of demyelination

y However, studies found that only A-fibres show ectopia not C-fibres

o Problem because C-fibres were thought to induce central

sensitisation but A-fibres undergo phenotypic switch & express

substance P after nerve injury



y Evidence suggests that local anaesthetic blockade of injured ne rves

relieves pain

y Reversed neuropathic behaviours in animal models treated with

anaesthetics/TT X

Intact nociceptor hypothesis

y Average discharge frequency low but incidence of spontaneously firing

nociceptors is high

y Become sensitised particularly to n oradrenaline & T NF

y Increased responsiveness to heat -> TRPV1

Ectopic neurones -> distort & amplify other Aps with afterdischarges (EADs)

and extra-spike formation

Growth factors

y Nerve injury induces changes in trophic factors in the tissue of

deprivation , Schwann cells without an axon anymore, DRG & dorsal horn

y Increase in NGF -> binds TrkA -> transport to DRG -> affects factors

such as BDNF

o Regulates TRPV1 expression

o Abnormal expression of transduction molecules

o Threshold for activation of transduction decreases i.e. sodium

channels -> Nav1.3 is unregulated in DRG of injured axons

Sodium channels accumulate in membrane at sites of nerve

injury

y Undertrophed neurones which have lost connectivity -> without GDNF

become ectopic (treatment with GDNF reverses neuropathic pain

behaviour)

y Overtrophed neurones -> too much NGF?

Central sensitisation

y Homosynaptic (windup)/heterosynaptic mechanisms -> increased release

of excitatory NTs/enhanced synaptic efficacy

o Presynaptic changes



Released of glutamate inhibited by µ-opiod receptors, GABAB

and adenosine receptors -> down-regulated -> glutamate

release

Up-regulation of voltage-gated Ca2+ channels -> glutamate

release A fibres undergo phenotypic switch & express substance P

Synapse in substantiagelatinosa& form synapses with

projection neurones

o Postsynaptic changes

AMPA receptors on cell surface (and NMDA in diabetic

neuropathy)

substance P and other peptide release

o Interneurone changes

Necrosis of inhibitory interneurons

LTD evoked by activation of NMDA receptors on GABAergic

neurones

Down-regulation of KCC2 symporter so opening Cl- by GABA

induces depolarisation -> KCC2 KOs show hyperalgesia

o Changes in descending modulation

RVM modulates inputs from cortical, thalamic &

periaqueductal areas -> decides whether to excite or inhibit

projection neurones Ablation of µ-opiod receptor expressing cells -> decreased

inhibition

o Immune/microglial mechanisms

Nerve damage ->denervated Schwann cells screte LIP &

MCP-1 -> macrophage infiltration, T-cell activation

&proinflammatory cytokines

MCP-1 -> receptor for CCR2 (KOs show no

hyperalgesia)

IL-1 -> NGF, T NF

Central microglia

Eph B receptors and pain modulation at spinal cord level

Understanding the tyrosine kinase family of Eph receptors and their ligand,

the ephrins



y Largest ¡

amil y ¢

¡

t yrosine £ inase receptors ¤

¥ 4 members ¦ ivided into

§ ̈

and B ̈ subclasses

y Bind transmembrane proteins called ephrins

Unde ©

nding their role in development

Guide migrating cells and neuronal growth cones towards their targets, modif y

cytoskeleton organisation and cell adhesion.

Knowing something about their mechanism of action

EphB receptor auto-inhibited via interaction with its own

uxtamembrane

domain. On activation phosphor ylation of

uxtamembrane t yrosine residues

removes inhibition.

Why EphB and ephrins B are related to sensory systems

mmunohistochemistr y has shown the presence of EphB receptors in the

superficial laminae of the dorsal horn and ephrin B2 in the dorsal root ganglion.

EphB-ephrin B2 forms a

ring-like tetrameric

structure with each ligand

interacting with 2 receptors

and each receptor

interacting with 2 ligands.



y Ephrin B2 agonists injected into the lumbar spinal cord resulted in a

short latency & prolonged behavioural thermal hyperalgesia, indicated by

a 50% reduction in withdrawal latencies to noxious heating of the

hindpaws in the plantar test

o Role in inflammatory pain?y This response was blocked by the NMDA receptor antagonist MK801

y Treatment with EphB1 receptor antibodies reduced thermal hyperalgesia

and mechanical allodynia following carrageenan intraplantar injection in

adult rats & c-Fos expression was reduced in DH

o Injecting EphB1 antibodies before formalin reduced pain related

behaviour

y Reduction in c-Fos expression in EphB1 KOs after carrageenan injection

Understanding the role of EphB in modulating pain processing at spinal cordle el interacting with NMDA glutamate receptors

Ephrin B2 -> Activated EphB1 receptor -> phosphorylation of NR2 subunit of

NMDA receptor by src -> amplification of NMDA activity

Evidence:

y Administering Ephrin B2 increases src

y EphB receptor contains YEPD motif which binds to src

y Phosphorylation of NMDA-R by EphB2-Fc prevented by prior injection ofPP2 (src blocker)

GLIAL NEUROBIOLOGY

Two types of brain cell

y Neurones (excitable)

y Glia (non-excitable) ² most populous; ~9

0%

Types of glia:

y CNS

o Microglia (non-neuronal -> originate from macrophages)

Ameboid ² found in fountains of microglia (corpus callosum)

so free movement -> prevalent in perinatal brain



(development when there are lots of extracellular debris &

apoptotic cells)

Ramified ² mature, resting -> found in strategic locations

throughout brain (regular mosaic pattern)

o Macroglia Astrocytes

Ependymal cells

Oligodendrocytes

y PNS

o Schwann cells

?Synantocytes

y Express the NG2 chondroitin sulphate proteoglycan (CSPG) -> considered

to be oligodendrocyte precursor cells that persist in the adult CNS to

generate oligodendrocytes throughout life

o Not in same place as o ligodendrocytes or express markers

y Stellate cells with large processes that form contacts with neurones at

synapses and nodes of Ranvier

y Neuronal activity (glutamate & ATP) act on receptors & evoke

intracellular Ca -> proliferate & change phenotype (become

oligodendrocyte/neurone) -> physiological function? Maybe specialised to

monitor signals from neurones and glia

Glial cell development:

Neuroepithelium (neural progenitors) forming neural tube -> radial glial

cells -> generate majority of neurones &macroglia in CNS (neurogenesis)

from precursors (i.e. SC precursors)

o RGCs also form scaffold along which they can migrate from

ventricular zone to their ¶home·

o RGCs eventually become astrocytes

Small proportion become stem astrocytes for adult

neurogenesis

Astrocytes (star-like)

Most populous glial cells in CNS



ALL express intermediate filament protein: glial fibrillary acidic protein

(GFAP) or vimentin which forms cytoskeleton [levels vary btw cells]

o High tensile strength -> gives CNS its structure

Types of astrocyte:

y True astrocytes (with classical stellate appearance)

o Fibrous

White matter

Elaborate and complex processes

o Protoplasmic

Grey matter

Less elaborate but longer processes

y Radial glia (disappear following maturation & turn into true atsr ocytes;

some persist i.e. Muller cells in retina, Bergmann cells which support

Purkinje cells in cerebellum)

y Specialised astrocytes

Functions of astrocytes

1) Structure

a. Each protoplasmic astrocyte occupies a well -defined territory

i. Overlap between astrocyte territories doesn·t exceed 5%

b. Using these territories, astrocytes divide grey matter into domains i. Neurones, synaptic terminals & blood vessels are integrated

by distal processes of astrocytes

2) Blood brain barrier

a. Astroglialendfeet enwrap capillary wall & release regulatory

factors i.e. GDNF & TGF which induce formation of tight junctions

between endothelial cells

i. Endothelial cells communicate reciprocally with astroglia via

LIF

b. Endfeet endowed with glucose transporters, potassium channels

and water channels -> absorbs these and sends it to neurones in its

domain

c. Regulate cerebral blood flow



i. Increased blood flow: Glutamate release at synapses ->

activates Ca2+ influx -> propagates to end-foot ->arachidonic

acid release -> converted to PGs by COX -> vasodilation

ii. Decreased blood flow: Less glutamate release at synapses ->

less Ca2+

-> propagates to end-foot -> AA release ->converted to 2-HET E by P450 -> vasoconstriction

3) Neurotransmitter uptake

a. Remove glutamate released by neurones thru EAAT1 & EAAT 2 ->

convert to glutamine -> glutamine taken up by presynaptic terminal

-> recycled to glutamate

i. Terminates signal, recycles, protects against neurotoxicity

4) Metabolism

a. Astrocytes taken up glucose via GLUT1 -> convert into pyruvate ->

convert into lactate (using lactate dehydrogenase) -> glutamate

released during neuronal activity + release of lactate -> lactate

taken up by neurone (via monocarboxylase transporters)

[ACSTROCYTE-NEURONE LACT ATE SHUTT LE]

5) Potassium homeostasis

a. Potassium released into extracellular space during neuronal activity

(repolarisation) -> astrocytes taken up excess K+ thru K irchannels

and redistribute the K+ thru the astroglial syncytium via gap

junctions (Cx 43, 30 & 26) ² SPAT IAL POST ASSIUM BUFFERING i. Possible because astrocytes express high density of voltage-

gated potassium channels & have strongly ²ve RMPs

6) Neuron-glial signalling

a. Astrocytes express NT receptors (glutamate, ATP) -> when

activated produces IP3 which + ER to release Ca2+ -> Ca2+

transmitted via diffusion thru gap junctions (INTERCELLULAR

PROPAGAT ION) -> release of gliotransmitters which affect

neurone activity

i. Can occur at neurone-glial/tripartite synapses (pre-,

postsynaptic neurone & astrocyte; gliotransmitters can

affect metabotrophic receptors on pre- and ionotrophic on

post)

Oligodendrocytes, Schwann cells and myelination



Oligodendrocytes

y 4 types:

o Type 1/2 ² myelinate small diameter axons; 1:30

o Type 3 ² myelinate large diameter axons; 1:3-5

o Type 4 ² myelinate large diameter axons; 1:1

Schwann cells

y 3 types:

o M yelinating ² ensheath axons > 1 µm diameter; 1:1

o Non-myelinating ² ensheath multiple axons < 1 µm diameter; physical

support & separation, ion homeostasis, express cell adhesion

molecules; immature -> can adopt myelinating phenoty pe

o Perisynaptic ² ensheath terminal axon branches & synaptic boutons;covered with basal lamina which fuses with muscle fibre; essential

for synapse function

y Unlike oligodendrocytes, SCs form basal lamina which is continuous tube

along axon and bridges the nodes

o SC microvilli fill the nodal gap & are important for potassium

regulation at nodes

In PNS microvilli are formed by astrocytes & NG2-glia

Myelin sheath

Made of:

y 70% lipid ² insulating properties

y 30% proteins ² fuse & stabilise lamellae & mediate membrane-membrane

interactions (MAG mediates interactions in both)

o Major proteins in CNS:

MBP ² fuse cytoplasmic interface forming major dense line

PLP ² fuse extracellular interface forming intraperiod line

o Major proteins in PNS: Charcot-Marie-Tooth neuropathy

(dysfunctional proteins)

P0 ² mediates adhesion of lamellae

PMP22 ² myelin synthesis & assembly; also joins Schwann cell

to basal lamina

Cx32 ² gap junctions for ion homeostasis



De elopment

y Schwann cells: neural progenitors (if exposed to delta/notch, NRG -1 &

transcription factor SOX10) -> SC precursor -> immature SC (if exposed

to NRG-1 & transcription factor Krox-20) ->myelinating/non-myelinating

SC

y Oligodendrocytes: develop in ventricular zones under influence of

transcription factors and signalling molecules -> migrate to final sites in

CNS & undergo local proliferation & differentiation in response to growth

factors from astrocytes & interactions with axons

Axon-glial interactions in the control of myelination

1) M yelinating cells recognise which axons to myelinate ² when SC

integrins/periaxin binds L1 & when OL neurofascin binds L1a. NCAM also plays a role

b. Jagged/Notch prevents myelination by OLs

2) SCs & OLs organise sodium channel clustering at nodes of Ranvier

Multiple sclerosis

BBB normally prevents most lymphocytes & phagocytes entering CNS. This

coupled with a poor lymphatic drainage system & low expression of MHC means

antigen presentation is poor.

In inflamed state, BBB break dow n -> induces MHC expression on microglia &

endothelial cells -> interact with T-helper cells

y Episodes of neurological dysfunction secondary to inflammatory lesions

within CNS confined to small areas of demyelination

o Lesions often resolve with remyelination& clinical recovery but with

time, permanent loss of myelin occurs with secondary axonal loss &

fixed disabilities

Oligodendrocyte precursor cells proliferate &remyelinatethe axons but ultimately fail due to lack of growth factors &

too much inhibition

Events that occur:

1) T-cells activated against myelin components



2) T-cells attack myelin & activate microglia

3) Activated microglia release cytokines, activate astrocytes & attack

oligodendrocytes& neurones

Alzheimer·s disease

Dementia progressive decline of cognitive function usually affecting

cortex as a whole but sometimes patchily; impairment of

intellect, memory & personality without disturbance of

consciousness

Alzheimer·s disease

CLINICAL FEATURES AND DIAGNOSIS

y Commonest dementia (accounts for ~65% of dementia in any age group)

y Cardinal clinical features:

o Amnesia (memory loss) ² inability to learn, retain & process new

information

o Aphasia (decline in language) ² difficulty in naming and in

understanding what is being said, simplification of language,

descriptive power declines -> complete loss of communication

o Apraxia ² impaired ability to carry out skilled motor activities i.e.eating, dressing, drawing, waving goodbye despite intact sensory &

motor systems

o Agnosia ² failure to recognise object i.e. prosopagnosia (faces),

places, own body parts

o Mood disorders, delusions, hallucinations, misidentifications,

behavioural changes (agitation, aggression, wandering)

y Differential diagnosis: delirium, pseudodementia, vascular dementia,

dementia with Lewy bodies, fronto-temporal demntia (difficult to

recognise each type of dementia)

o Also difficult to define landmarks that separate dementia from

mild decline in cognitive function associated with ageing

y Diagnosis based on:

o History (particularly collateral history from informant i.e. carer)

o Mental State Examination



o Physical examination esp. cardiovascular risk factors & neurological

problems

o Investigations i.e. blood, urine, imaging

AET IOLOGY

y Early-onset AD (familial ² emerges before 65 yo) ² AUT OSOMAL

DOMINANT (1/2 the offspring of affected parents will get disease)

o Amyloid precursor protein (chr. 21)

Increased incidence in Down·s syndrome sufferers by 40 yo

o Presenilin 1 (chr. 14)

o Presenilin 2 (chr. 1)

y Late-onset AD (sporadic)o ApoE4 (chr. 19 -> encodes glycoprotein, apolipoprotein E; involved in

cholesterol transport & metabolism; 3 alleles ² ApoE4: high risk)

No copies ² lower risk than population

1 copy ² 4 fold increase in risk

2 copies ² 8 fold increase in risk

o Epidemiology risk factors: age, head trauma, cholesterol, CVD

PATHOPH YSIOLOGY

1) Gross cerebral atrophy

2) Senile plaques



y Spherical lesions measuring up to 100 µm

o Fully developed stage (neuritic plaque) ² central core of amyloid

surrounded by dystrophic neuronal processes, reactive astrocytes

& microglia

Diffuse plaques also presenty Amyloid precursor protein ² transmembrane protein that projects into

ECF from all nerve cells

y Can be hydrolysed at 3 different sites by -, - or -secretase

o When hydrolysed by -secretase, nontoxic peptide products

formed (A40 ² not prone to aggregation)

o When hydrolysed by - or -secretase, polypeptides with 40-42

aas are produced that are toxic

Most toxic = A42

Form extracellular aggregates (together with altered nerve

fibres and reactive glial cells) -> stick to AMPA receptors &

Ca2+ channels -> Ca2+ influx -> apoptosis

y They can bind to surface receptors i.e. prion proteins

(also linked with CJD) on neurones and change synaptic

communication

Initiate inflammatory response & inhibit neurotrophic factor

release -> intracellular tangles

Also builds up in the mitochondria o f cells and inhibit certainenzymes disrupting glucose metabolism

N-APP (fragment of APP) may trigger cells to self -destruct

by binding onto death receptor 6

Astrocytes detect -amyloid released by affected neurones and withdrawtheir

process from affected & neighbouring neurones -> these neurones die without

support -> the -amyloid released accumulates in astrocyte -> astrocyte death ->

debris attracts microglia -> plaque formation

3) Neurofibrillary tangles

y Found in neurones, senile plaques & dendrites

y Hyperphosphorylation of tau protein (microtubule -associated protein) by

active GSK-3 which pairs with other threads to form paired helical

filaments



y Microtubules disintegrate -> transport system collapses

y Interfere with cellular functions by displacing organelles

4) Neurone and synapse loss

y Starts in the transentorhinal cortex (hippocampus &neocortex)

5) Neurotransmitter changes

y Acetylcholine

o Loss of cholinergic neurones in the basal forebrain, decreased

acetylcholine levels and a decrease in choline acetyltransferase

(CHAT) which synthesises ACh

o Animal models show that ACh plays a crucial role in information

processing & memory

o Senile plaques may affect cholinergic synapses --> in vitro studies

show ACh release

Reductions in APP in depressed patients· CSF who are taking

drugs with anti-cholinergic side effects

o ACh -> (-) PKC -> increased activity of GSK-3 -

>hyperphosphorylation of tau

y Glutamate

o Inactivation of EAAT1, EAAT 2 & glutamine synthetase ->

glutamate in synaptic clefto VGlut1 which pumps glutamate into vesicles

o Vesicular glutamate released into cleft by Ca 2+ dependent stimulus

coupled exocytosis (synaptophysin?)

Interesting to note that tetrahydro-9 aminoacridine

improves cognitive performance in some AD patients &

reduces synaptic Ca2+ coupling & glutamate release

o glutamate -->excitotoxicity which kills cells by necrosis &

apoptosis; activation of mGlu2 can exacerbate problem

Activation of NMDA receptors causes dissociation of protein

phosphatase-2A --> tau phosphorylation

o Not beneficial for LTP because tonic rather than phasic stimulation

of NMDARs --> background ¶noise· which impedes detection of

relevant signals (partial depolarisation -> background Ca 2+ influx)



Decreased numbers of pyramidal and stellate cells that stain

for glutamate and glutaminasein hippocampus -> neurones

that are present have disorganised and shortened dendrites

AMPAkines ² positive modulators of glutamate action at

AMPA receptors/increase production of neurotrophins i.e.BDNF & NGF in brain areas involved in memory using slices

of brain tissue from aged rats & mice

Memantine ² moderate affinity, non-competitive NMDA

receptor antagonist

MK801 ² high affinity, competitive NMDA receptor

antagonist

y Serotonin

o Reduction of 5-HT in post mortem AD brains -> 5-HT reuptake

sites in temporal cortex /Raphe nucleus ² preferential site for NF

formation/ 5-HT 2A receptor

o Frequent co-existence of depression?

Spinal cord injury and repair

Describe the gross anatomy of the spinal cord

EX T E RN AL FEATURE S

y Provides sensory, motor & autonomic innervation for the trunk and limbsy Occupies the spinal canal within the vertebral column

y ~ cylindrical but diameter varies at different levels

o Bears 2 enlargements:

Cervical ² provides innervation for the upper limb via the

brachial plexus

Lumbar ² innervates the lower limb via the lumbar & sacral

plexi

y Terminates at vertebral level L1-2 in the adult (conusmedullaris)

o Filumterminale [connective tissue] attaches conusmedullaris to

first coccygeal vertebra

y 31 pairs of spinal nerves attach to the spinal cord through dorsal &

ventral roots, carrying afferent & efferent fibres respectively

o Exit the vertebral canal via intervertebral foramina

o Below termination of cord, spinal nerves descend as caudaequina



y Cord is covered b y 3 meninges pia, arachnoid dura mater

o Subarachnoid space between pia& arachnoid mater) contains CSF

N ! ERN " L FEATURES

y Central canal continuous with cerebral ventricular s ystem # contains CSF)

y Gre y matter $ nerve cell bodies, dendrites & s ynapses

o Dorsal horn % main site of termination of afferents

o Lateral horn &

preganglionic s ympathetic fibres o Ventral horn ' lower motor neurones

y White matter ( nerve fibres

o Ascending tracts ) dorsal columns, spinothalamuc tract,

spinoreticulothalamic s ystem, spinocerebellar tract

o Descending tracts 0 corticospinal tracts, rubrospinal tracts,

tectospinal tracts, vestibulospinal tracts, reticulospinal tracts

Describe the neuropathology of spinal cord injury

y Spinal cord injur y refers to an y injur y that is caused b y trauma instead

of disease.

y Depending on where the spinal cord is damaged, the s ymptoms can var y

widel y 1 generall y, weakness/paral ysis/spasticit y and sensor y loss at and

below the point of injur y:

Dorsal funiculus

Ventral funiculus

Lateral funiculus

Central canal

Lateral horn

Dorsal horn

Ventral horn

Dorsal medial sulcus

Ventral median fissure Ventral white commissure



o Cervical ² quadriplegia (+ blood pressure problems, breathing

difficulties, abnormal sweating, breathing difficulties, problems

maintaining steady body temperature)

o Thoracic ² paraplegia

o Lumbarsacral ² bladder, bowel & sexual dysfunctiony All can be described as:

o Incomplete ² partially injured

o Complete ² severely injured

Estimated 2.5 million people live with SCI & 130,000 new injuries reported each

year ² significant impact on QoL, life expectancy and economic burden (due to

primary care & loss of income).

Causes:

Motor vehicle accidents

Falls

Violence

Sports injuries

Minor injuries inc. whiplash

Pathophysiology of SCI

1. Acute

a. Haemorrhage occurs -> localised oedema -> loss of microcirculation

by thrombosis & vasoconstriction of blood vessels

i. Vertebral disruption/oedema compresses spinal cord &

exacerbates problem

b. Mechanical (injury) & ischaemic insults -> necrosis (particularly in

grey matter)

c. Injured nerves respond with an injury -induced barrage of APs

i. Electrolytic shift -> spinal shock (generalised failure of

spinal cord)2. Secondary

a. extracellular glutamate (and other excitatory aas& free -radicals)

-> apoptosis forming cyst/cavity which interrupts cord

i. Wallerian degeneration (degeneration of axons distal to an

injury ² when it has become separated from cell body)



ii. M yelin disintegrates & fragments but oligodendrocytes

survive

iii. Reactive gliosis (microglia activated) ² P2X & glutamate

receptors so detect apoptosis

Activated astrocytes grow & reproduce -> GFAP,growth factors, inflammatory mediators i.e. IL -1

o Neutrophils which clear debris invade followed

by lymphocytes

iv. Astrocytes also form glial scar which protects other

neurones from damage but blocks regenerati on & reforms

BBB (angiogenesis)

Synantocytes also help form glial scar & enable

oligodendrocytes to survive

3. Chronic

a. Cysts/cavities can continue to enlarge (syringomyelia) - FATAL

b. Compensatory sprouting (limited)

c. Compensatory cortical, brainstem & spinal plasticity (limited) ² new

spinal circuits can bypass the lesion i.e. rubrospinal tract can take

over job of corticospinal tract/cortical sensorimotor areas can

functionally rearrange

d. Little/no neurogenesis but proliferation of ependymal cells can give

glial precursors mainly

Describe animal models of spinal cord injury

Vast majority of research conducted using adult mice (transgenic) & rats

(larger) because they are cheap, although some has been done using cats and

dogs and increasing work is being done using nonhuman primates.

4 types of SCI

Cord maceration (morphology seriously distorted) ² subdural insertion and

inflation of a balloon/clip compression

Cord laceration (gun shot/knife wounds) ² surgical incision

Contusion injury (bruising injury) ² weight drop

Solid core injury ² none available



Animal model depends on hypothesis ² neuroprotection (contusion/maceration);

pro-regeneration (laceration ² can distinguish between regenerating axons from

spared/collaterals)

Problems:

No studies showing any improvements after intervening > 1 month post-

injury (long-standing injuries in humans)

Only assess outcome over period of weeks/months -> do these remain

stable beyond 2/3 months

Bipedal locomotion differs radicall y from quadrupedal

locomotion/partially upright locomotion ² posture, kinematics, physiology,

anatomy

Differences in responses to SCI between species & members of the same

species

o Cysts/cavities form in humans but not in mice

Animal models indicate that axon regeneration by a small % is sufficient to

restore partial function

Describe possible mechanisms contributing to loss of function

Critically e2 aluate current treatments (pharmacological, rehabilitati2 e)

REHABILITATIVE

1. Patients are immobilised at scene of injury until they deduce there is no

damage to highest portions of the spine

2. Brought to hospital ² evaluated for SCI using X-ray/CT scan

3. Complications ² spinal shock -> autonomic dysreflexia/respiratory failure,

pulmonary oedema, pneumonia/DVT ( can be recognised early and avoided ²

kept in ICU)

4. Surgery to remove any bone fragments and stabilise the spine

(decompress)5. Treated with corticosteroids (methylprednisolone) to reduce

inflammation

6. Rehabilitation

a. Support and prevention ² gives individual sense of control over

situation/prevention of pressure sores in bed & wheelchairs



b. Support and education for individual & caregivers ² evaluation of

limb function to determine what patient can do/selfcare

skills/eating, bowel & bladder management/mobili ty/sexual health

& function

c. Assistive devices i.e. wheelchairs/sliding transfer boards/grabbars

d. Patient·s living environment needs to be modified i.e. ramps/lifts

Improved locomotor function seen in mammals with complete/incomplete SCI

following exercise. Even enhances ability to walk on a treadmill when body -

weight is supported ² spinal circuitry does not become silent but maintains

active and functional neuronal properties & can respond to peripheral input from

below lesion site ² can generated oscillating, coordinated motor patterns &

considerable plasticity. Functional electrical stimulation (electrophysiologicalstimulation of spinal cord/peripheral nerves/muscle) can also induce step -like

movements in patients with complete SCI.

Helps chronic (faster overground walking speeds but patients only used

wheelchairs) & recent patients.

Combination of treadmill training and spinal cord epidural FES improved quality

& quantity of stepping during the training session and resulted in the im mediate

improvement in quality of overground walking

Epidural + locomotor training = BEST

Improved cardiovascular performance, reduced spasticity, bone loss &

bladder/bowel dysfunction

However, can pose risks i.e. autonomic dysreflexia, fracture, muscula r injury,

hyperthermia (atypical responses to exercise)

Ichiyama et al. given enough physiological activation (ES + quipazine) afferent

input can drive spinal circuits to enable stepping and that repetitive practiceallows the isolated lumbosacral circuits to find new solutions in executing motor

& postural problems imposed by SCI ² stepping more consistent and fewer

neurons activated in trained (body weight support system for bipedal stepping)

vsnontrained spinal rats



Fong et al . robotic step tra ining a nd a serotonin a gonist, quipa zine genera ted

significa nt recovery of l ocomotor function in compl ete spinal cord tra nsected

mice tha t otherwise coul d not step ² extent of recovery a chieved when these

trea tments were combined exceeded tha t obta ined when either trea tment wa s

a ppl ied independentl y

Prostheses: a rtificial device extension tha t repla ces a missing body pa rts ² ma y

suppl ement/repla ce biol ogical thera pies

PHARMACOLOGICAL

PNS neurones regenera te l ong dista nces by Wall eria n dege nera tion

y M yelinating Schwann cells dedifferentiate and myelin fragments leaving

basal lamina intact

y Macrophages remove debris

y Schwann cells line up and release growth factors which cause axon to

sprout collaterals and regenerate

y Perisynaptic Schwann cell s reform connections

y Schwann cells remyelinate axons

CNS neurones grow poorly because:

Limited by inhibitory influences of glia & the extracellular environment

Glia ² produce factors that inhibit remyelination& axon repair i.e. NOGO

o Orientated perpendicular to the neuraxis so physical barrier

Axons ² lose capacity to regenerate due to less GAP 43, RARb

Environment ² myelin-associated inhibitors, astrocytes, oligodendrocytes,

oligodendrocyte precursors, lack of laminins

Chondroitin sulphate proteoglycan

Astrocytes (+) CSPGs (requires RhoA pathway)

All CSPG inhibitory to neurite outgrowth (projection from axon) ²

chondroitin-4 proteoglycan was the most inhibitory

NG2 is a GSPG that is expressed by oligodendrocyte precursor cells in

glial scar -> following injury to CNS, NG2 expressing OPCs are seen

around the site of injury within 24 hours of the initial injury and remain



elevated for ~ 8 weeks. In vitro studies show neurite growth inhibition

(neurones didn·t bind NG2)

o Cultured with NG2 & adhesion molecules ² significantly reduced

neurite extension with NG2 than without

o Cultures created with striped surfaces ² NG2 lanes & adhesivemolecule lanes ² neurones stick to lane with no NG2

Shows CSPGs stop neuronal growth into glial scar

Delivery of chondroitinase ABC (bacterial enzyme) which degrades CSPGs

promotes regeneration of injured CNS axons

o Delivery of bacterial proteins carries a risk of nonspecific immune

responses & further doses may not be possible if body becomes

immune

o Human enzymes i.e. hyaluronidases/matrix metalloproteinases

might avoid these problems

Rolls et a l. used xyloside to inhibit CSPG forma tion a t different time

points a fter injury a nd a na lysed phenotype a cquired by

microglia /ma cropha ges in lesion site

o Immediate inhibition of CSPG IGF-1 & T NF--> impaired

functional motor recovery & tissue loss

o Delayed inhibition improved recovery

Protein tyrosine phosphatase sigma (PTP) deficient mice = don·t respond

to CSPGs ..

. axon regeneration

Semaphorin 3A ² present in glial scars & contributes to outgrowth inhibitory

properties of these scars

Ephrin B3 ² functions through EphA4 receptor and inhibits remyelination

NOGO

M yelin expresses growth inhibitory molecules including: Nogo -A, MAG

&OMgp

o IN-1 antibody binds Nogo-A & neutralises inhibitory effects ->

CNS axon growth & induced CNS axons to sprout collaterals

reported in animal models

Recently shown to promote growth of corticospinal axons in

marmoset monkeys



Lee et al . in 2009 cla im there is no corticospinal a xon

regenera tion however!

Past studies have produced mixed results regarding

generative phenotype of Nogo KO mice -> 2 lines didn·t

show any regeneration & 1 displayed modestregeneration; the 4th reported extensive regeneration

but confounded with an axon la belling artefact

o Peptide inhibitor (NEP1-40) also developed which targets Nogo-A

receptor & prevents neurones responding

Leucine rich repeat domains of the NgR necessary for interaction with

Nogo-A, MAG &OMgp -> GPI anchored protein so recruits co-receptor

molecules i.e. p75 (but most adult neurones don·t express p75!)

o Co-receptor activation (+) Rho & ROCK pathways -> modulates

cytoskeleton &neurite growth

o NgR-independent cAMP/PKA pathway also affects

Elevation of cAMP by direct injection in DRG overco mes

MAG & myelin induced inhibition ->upregulatesArginasel (+)

polyamines (+) axonal growth

Filbrin ² PirB may be another receptor that responds to myelin inhibitors

NgR1, LINGO-1 & TROY/p75 = shared receptor complex for myelin-

derived inhibitors (Mandemakers)

CELLULAR TRANSPLANTATION

Bridges cysts& cavities, replaces dead cells, provide new

neurones/myelinating cells, create favourable environment for

regeneration

Work through myelination, neuroprotection, plasticity

Neural progenitors from 8 week old human foetuses transplanted into

immunosuppressed nonhuman primates 9 days after cervical contusion -?

spontaneous locomotion within cage & forelimb grip power, cavitation &

no sign of tumour formation

Autologous remain to be tested (but working on how to stimulate patient·s

endogenous adult progenitors)

Purified Stem (can differentiate into any cell type ²

totipotent)/progenitor (can differentiate into many cell types) cells can

be collected at 3 different stages of development: inner cell mass of



blastocyst/ CNS, olfactory system or umbilical cord of fetus/CNS,

olfactory system, bone marrow or blood of adult

Each of these cell populations can be growin in vitro & engineered to

produce a molecule of in terest/restricted to a particular cell fate before

transplantation Some can be injected directly into the cord (fetus)

Some can be used for autologous transplantation (olfactory

system/umbilical cord blood cells ² which can be frozen at

birth/blood/bone marrow)

o Peripheral nerve

Autologous transplants in rats (+) growth of all axonal types

but not supraspinal axons/some spinal axons were found to

have regenerated 4 months after injury in nonhuman

primates

o Schwann cells

Bruce et al . huma n a xons remyel ina ted by Schwa nn cell s

Alternatively implant oligodendrocyte precursors

o Cells from embryonic & adult olfactory bulb/mucosa

Functional recovery and/or CNS axon regeneration when

transplanted immediately or up to 2 months after SCI in

adult rats

GROWTH FACT ORS

Following contusion/laceration injury growth factors become

undetectable esp. neurotrophins 3 & 4 or only slightly upregulated&

briefly i.e. NGF

Message levels for neurotrophin receptors i.e. trkA remain absent, trkB&

C remain unaltered, p75 upregu lated

o Upon ligand binding, receptors dimerise& p75 signals through Rho

but also MAPK pathways etc.

BDNF may not be available for signalling through trkB however since

there is a sustained upregulation of competitor binding sites

To promote CNS axon regeneration & functional recovery after SCI,

growth factors have been delivered to soma:

o B y direct injection

o B y osmotic mimpump



o B y fibrin glue

o B y gene transfer techniques

Delivery of these (+) recovery of CNS axons in vivo ² implantation of NT-

3 secreting fibroblasts into adult rats caused CST axons to grow up to 8

mm/BDNF-expressing fibroblasts (+) 7% rubrospinal axons regenerated Severely injured axons can be rescued by applying BDNF

Gave nonhuman primates NGF firoblasts but need to study functional

recovery in injured animals

Clinical trials using systemic NTs - side effects i.e. severe muscular

pain, depression & hallucinations (use viral vectors to target tissues)

Epilepsy and anticonvulsants

Epilepsy disorder of the CNS characterised by sudden, large increases in

electrical activity (seizures) that may be localised/generalised

*derives from G reek meaning ¶to seize· since ancients believed it was a

p ossession from G od

Normally sp read of electrical activity between neurones is restricted &

synchronous discharge is confined to small group s, p roducing normal EEG

rhythms. In seizures, large group s of neurones are activated rep etitively (but

low frequency), unrestrictedly and hyp ersynchronously and synap tic inhibition

fails p roducing high 3 oltage spike and wa 3 e acti 3 ity.

What causes this?

Pre 3 enting burst mode firing: It currents are low-threshold Ca2+ currents

which are activated ~RMP but are simultaneously inactivated to p revent burst

mode firing. Following an AP, to p revent summation of excita tion of synap tic

inp uts Ih currents which hyp erp olarise the cell are activated.

It makes cells more excitable

Ih causes greater summation of excitatory inputs Channelopathies: benign familial neonatal convulsions (non -functional

potassium channels)/generalised epilepsy with febrile seizures (beta

subunits alter inactivation kinetics of alpha subunit in Na + channel ->

slower inactivation)

Paroxysmal depolarising shift in a single cortical neurone



Sustained neuronal depolarisation resulting in a burst of action potentials

o Glutamate mediated (AMPA, NMDA)

Increased glutamate release and receptors in epileptics

Astrocytes (communicate with one another through Ca 2+

signalling) trigger synchronous release of glutamate whichacts on multiple neurones

o Ca2+ influx

Plateau-like depolarisation following completion of the action potential

burst

o Ca2+ opens voltage-gated Na+ channels

o Na+ influx

Rapid repolarisation followed by hyperpolarisation

o GABA mediated

Decreased GABA release in epileptics

o Glutamate depletion/receptor desensitisation

o Cl- influx/K+ efflux

This PDS is spread to neighbouring pyramidal neurones due a failure of

inhibitory feedback though local intern eurons (surround inhibition).

?Gain of inhibition

GABA excites: underexpression of KCC2 channels in epilepsy which

maintain low intracellular Cl - to ensure efflux upon GABA binding

o NKCC1 dominants which pump Cl - in resulting in depolarisation when

GABA opens chloride channels

GABA agonist drugs provoke seizures in animal models of absence epilepsy

GABA makes seizures persist: convulsant (kainite) infused into ipsilateral

hippocampus & evoked seizures in both ipsi - & contra-

o If contra- treated before it became epileptogenic with GABA

antagonist bicuculline it didn·t stop seizures but if treated after it

did

EEG (electroencephalography): recording of brain·s spontaneous electrical

activity over 20-40 minutes from multiple electrodes placed on the scalp.



y Electrical potentials generated by single neurones too small to be

detected so EEG reflects summation of synchronous activity of many

neurones with similar spatial orientation

o Have to have similar spatial orientations or volume conduction

cannot occur Volume conduction -> local currents generated (ion

movement) when synapses activate neighbouring neurones ->

attract/repel electrons in metal electrode generating

voltage

o Pyramidal neurones produce most of the EEG signal since they are

well-aligned and fire together

y Limitations: most sensitive to superficial layers of the cortex so can·t

measure activity of midline/deep structures i.e. hippocampus/meninges

and skull smear the EEG signal

o Alternatively can use MEG which measures magnetic fields

(electrical currents produce magnetic fields)

Types of epilepsy

y Generalised: affects the whole cortex (impaired consciousness)

o Primarily generalised: started at a focal point and spread out but

focal point unknown

Absence (petit-mal): sudden brief loss of consciousness;common in children

Tonic-clonic (grand-mal): sudden stiffening of muscles

following by jerking movements

Tonic phase caused by rapid discharge; frequency of

discharge slow in clonic

o Following seizure (post-ictal phase) discharges

still occur but no associated abnormalities

Status epilepticus: medical emergency when seizures

occur for >30 mins& consciousness not regained

(caused by grand-mal)

o Secondarily generalised: started from a specific focal point that

can be determined (mostly cortical -> temporal most prevalent esp.

hippocampus &entorhinal cortex)

y Partial (focal): affects one part of the cortex



o Simple: no loss of consciousness

Jacksonian: focal cortical seizure characterised by jerking

movements that begin in limbs and spread to rest of the

body

o Complex: impaired consciousnesso Aura: stereotyped perception before the seizure (impaired

perception after) i.e. smell (visual/gustatory), tingling in one limb or

strange inner feelings (déjà vu)

Treatments for epilepsy

1) Drugs which GABAergic activity

a. Benzodiazepines (diazepam) ² make GABAA receptors more

responsive to GABA

i. Only used in status epilepticus as it causes amnesia &

sedation and myoclonic seizures because they are muscle

relaxants

b. Barbiturates (phenobarbitone) ² make Cl - channels open longer

c. Vigabatrin ² x GABA transaminase

d. Tiagabine ² x GABA reuptake

2) Drugs for grand mal

a. Carbamapezine, lamogatrin, phenotyin ² bind inactivated Na+

channels & refractory periodi. Lamogatrin can be used for petit mal too

3) Drugs for petit mal

a. Sodium valproate ² x Na+ channels & GABA transaminase

i. Can also be used for grand mal

b. Ethosuxamide, gabapentin, pregabilin ² block T-type Ca2+ channels

i. Ethosuximide doesn·t bind Ca 2+ channels at synapses so can·t

block NT release

4) Atypical anticonvulsants

a. Retigabine ² activates KCNQ2/3 & KCNQ3/5 potassium channels

b. Felbamate ² blocks NMDA receptors

c. Topiramate ² blocks AMPA/kainite receptors

d. Levetiracetam ² blocks fusion of vesicles with membrane

e. Losigamone ² blocks Na+ currents on postsynaptic membrane

5) Ketogenic diet (high fat/low carbohydrate -> good for children)



6) Vagal stimulation

7) Surgery to remove the focal point

Sod ium channel b lock ers are commonly used in partial seizures

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Systems Neuroscience Notes

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