Download Dorsal Horn Structure/Function

Dorsal Horn Structure/Function
Spinal Cord Organization
SG-Substantia Gelatinosa
NP-Nucleus Proprius
DC-Dorsal Columns
mouse
Spinal Dorsal Horn
Medullary Dorsal Horn
rat
Spinal Cord Organization
SG-Substantia Gelatinosa
NP-Nucleus Proprius
DC-Dorsal Columns
mouse
Rexed’s Laminar Scheme
(applied to rat spinal cord)
Rexed based his based on
cytoarchitecture
In the original study (Cat) Rexed
equated lamina II with the SG.
Differences in staining patterns of these two markers have lead to the assumption
that IB4 positive non-peptidergic C-fibers project to the inner part of lamina II and
peptidergic fibers project to laminae I and II outer. This has lead to the belief that
IB4 binding is a marker for the inner part of lamina II.
Cutaneous CPM nociceptor – IB4 positive – projects to lamina I
Examples of primary afferent terminations and “laminar boundaries” in the adult mouse.
Projections of IB4+ C-fibers
IB4-HRP labeling on left side
(A,C,E)
Projections of A-fibers
B-HRP labeling on right side
(B,D,F)
SG-Substantia Gelatinosa
NP-Nucleus Proprius
DC-Dorsal Columns
mouse
Primary Afferent Projections to the Spinal Cord
Cutaneous Afferent Fibers
Innocuous
Aβ
Noxious
>>
Hair Follicle
SA1
Aδ
HTMR/
heat
<
D-Hair
HTMR/
heat
C
CLTMR
<<
HTMR/
heat/cold
I
IIo
IIi
III
IV
V
Deep Afferent Fibers
Innocuous
Noxious
Muscle Group II/III
Muscle C-fiber
Visceral C-fiber
III
IV
V
X
VI
I
IIo
IIi
Sagittal view of Aβ
hair follicle afferent
in cat
Saggittal view of Aβ
slowly adapting type
1 mechanoreceptor
Sagittal view of a
myelinated Aβ-HTMR
(Cat)
Same fiber - low
power reconstruction
Major point of emphasis
It is clear that both myelinated and unmyelinated afferent fibres that
respond to noxious stimulation in the periphery project predominantly
to the superficial laminae of the dorsal horn. However, it is
also clear that myelinated and unmyelinated fibres that signal the
presence of innocuous mechanical and thermal stimuli also project
to these same laminae. Therefore, the anatomical substrate of pain
information processing cannot be easily distinguished from substrates
involved in other functions such as homeostasis.
Interestingly, recent reports suggest that while there is significant overlap
in the projections of fibers signaling different stimulus intensities, at least
some degree of functional segregation exists at the postsynaptic level in
the superficial laminae.
Development of Superficial Dorsal Horn
Pediatric Pain – premature human infants = neonatal rodents
Plasticity – does response to injury recapitulate development?
In rodents the superficial dorsal horn is still in a developmental state
at birth.
Early postnatal development P0-P14 or P21
ƒ Neurons are hyperexcitable.
ƒ They have large cutaneous receptive fields.
ƒ Excitatory inputs predominately from A-fiber inputs.
ƒ Weak inhibitory inputs both from spinal origins and from
descending inhibitory (e.g. 5-HT).
Beta-Cholera Toxin- HRP labeling
rat
Fitzgerald, et al., J.C.N. 348:225-233, 1994.
mouse
Beggs et al., Eur. J. Neurosci. 16:1249-1258, 2002
EPSCs evoked in lamina II cells in response to dorsal root stimulation.
Immature rat
(P21)
Mature rat
(P56)
Nakatsuka et al., Neurosci. 99:549-556, 2000
.
What are some of the possible mechanisms for this hyperexcitability?
The nervous system contains 3 basic types of neurons
1) Sensory neurons
2) Motor neurons
3) Interneurons
In the spinal dorsal horn the cells (interneurons) are
divided into two main groups based on the projections of
their axons.
Projection (Tract) neurons - axons project over many segments some terminate
outside the spinal cord.
Local spinal interneurons – axons project over short distances usually remaining
within the same spinal segment or one or two segments rostral or caudal
Local spinal interneurons are divided into two groups: 1) excitatory; 2) inhibitory
polysynaptic
Presynaptic
inhibition
hyperpolarization
monosynaptic
Synaptic Ultrastructure
In the superficial dorsal horn
Type 1 glomerulus
C – central terminal from
primary afferent.
A – terminals from Gaba/Gly
inhibitory interneurons
Type 2 glomerulus
Excitatory synapses are
asymmetrical
Inhibitory synapses are
symmetrical
Peptides
Primary Sensory Neurons
Dorsal Horn Cells
X
Sub-P
CGRP
Under some circumstances primary afferent depolarization can be sufficient
to elicit action potentials in the terminals. These centrally generated action
potentials propagate back through the dorsal roots (dorsal root reflex) into
the periphery and can result in release of peptides.
Information processing in the dorsal horn allows us to discriminate
many qualities peripheral stimuli.
Modality - Thermal, Mechanical, Chemical – segregation and convergence
Innocuous (pleasant) “slow brush cells” or menthol;
Noxious (painful)
Localization
Depending on modality or tissue type this can be very precise or very imprecise.
How do we localize a stimulus?
A sample of receptive fields of afferent fibers in adjacent dorsal roots shows
significant overlap in the area of skin represented. In the dorsal horn this this
Discontinuous Dermatomal Representation converted to a Continuous
Somatotopic Representation (map) of the body surface.
Tactile acuity is directly proportional to area of neuropil devoted to body region
which is directly proportional to innervation density.
This principals are the same throughout the neuraxis.
“Referred” Pain
Dorsal Horn Cells Responsible for Signaling Pain
Perl and colleagues showed that there are cells in the superficial dorsal horn that
respond exclusively to noxious stimuli
Stroking with a glass rod
Squeezing with smooth forceps
Squeezing with serrated forceps
Christensen & Perl, 1970
Lamina V -
Willis 1981
Lamina V -
Willis 1981
There is a great deal of debate about
which types of dorsal horn cells adequately
encode noxious stimuli.
Some suggest that it is the nociceptive
specific (HT) cells, while others suggest
that WDR cells are equally efficient in
coding pain information leading to the
perception of pain.
Andrew and Greenspan ‘99
Andrew and Craig ‘02
Functional classification of dorsal horn
neurons
Type
Input
Location
Function
Nociceptive
Specific
C, Aδ
L I,V
Nociception
Wide Dynamic
Range
C, Aδ, Aβ
L I,II,V
Nociception?
Low Threshold
Aβ
L III,IV
Touch
Proprioceptive
Aα
L VI
Proprioception
Methods for examining the
processing of cutaneous afferent
inputs from single fibers.
(Cat)
Example of intracellular
recording from a lamina I HT neuron in the cat.
Single stimuli produce sub-threshold
excitatory post synaptic potentials
(epsps).
When pairs of stimuli are applied
the faster (sub-threshold - low
threshold) inputs show depression of
their evoked response while the
slower high threshold inputs exhibit
facilitation evoking action potentials.
Whole nerve or dorsal root stimulation has drawbacks
A.
20 μm
B.
C.
sub-threshold
0.7 mN
1.0 mN
threshold
suprathreshold
5.6 mN
-70 mV
28.4 mN
20 mV
100 ms
20mV
20 ms
Simultaneous recordings from pairs of neurons in lamina II
Lu and Perl ‘03
Lu and Perl ‘03
Summary of results from dual recordings depicting excitatory circuit
Lu and Perl ‘05
AXON
TERMINAL
AMPA
Glu
P
DORSAL HORN
NEURON
TrK B
BDNF
PKC
Mg2+
P
Src
Glu
NMDA
Ca2+
Glu
mGluR
SP
NK1
IP3
The majority of spinothalamic tract
neurons located in lamina I contain the
substance P receptor NK1.
Lamina I cell retrogradly label from
thalamic injection of fluro-gold
Spinothalamic projection neurons that
contain NK1 receptors predominately
receive input from peptidergic fibers
regardless of their laminar location
Similar specificity can be seen for projections of cells
containing specific peptides
Examples of co-localization of markers found in excitatory and
inhibitory interneurons in the superficial dorsal horn
Receptors in spinal dorsal horn
•Adrenergic: α1, α2
•Hormones: androgen,
•Dopaminergic: D1, D2
corticosteroid, CRF,
•5-HT: 5-HT1A-D,2, 3,7
estrogen, insulin, TRH
•Glutamatergic:
•Peptide:
iGluR: AMPA, KA, NMDA
angiotensin, bombesin,
mGluR1-8
BK, cannabinoid, CCK, CGRP,
•GABAergic: GABAA,B
endothelin, galanin, NK1-3, NPY,
•Benzodiazepine
NT, Som, VIP
•Glycine receptors
•Opioidergic: mu, delta, kappa
•Cholinergic: Nicotinic, muscarinic
•Purinergic: P2X, P2Y
•Neurotrophins: TrkA-C
•Vanilloid receptors
A. NB-stained lamina I marginal layer cell in transverse spinal section also
immunostained for NK1 and PKCγ. B. It was found to be NK1 positive and
PKCγ negative. C. The cell responded to mechanical stimulation of the
skin and D. to both cooling and heating of the skin.
Lawson, McIlwrath, Koerber
QUESTIONS
Studies have documented both preand postsynaptic mechanisms for the
effects of μ-opioid receptors on laminae
I and II neurons. The postsynaptic
hyperpolarization is caused by the
activation of a G protein coupled,
inward-rectifying potassium channel.
The observed presynaptic effect is a
decrease in spontaneous excitatory
postsynaptic potentials (EPSPs),
presumably mediated by a decrease in
free intracellular calcium in presynaptic
terminals.
In addition, there are some cells in the
superficial dorsal horn that are
apparently excited by opioids. For
example at least one study has
suggested that lamina I cells
responding to innocuous cooling are
depolarized following activation of this
receptor.
Light and Willcockson, ‘99
Central projections of a myelinated
low-threshold rapidly adapting
mechanoreceptor (D-hair) from
a P6 mouse. Does not project into
lamina II
Central projections of (A) a low-threshold mechanoreceptor (P3)
and (B) a C-fiber nociceptor (P4)
Myelinated HTMR
projecting to laminae I-V
P6 mouse
Central projections of two
myelinated nociceptors from
a P3 and P6 mice
Myelinated HTMR
Projecting to laminae I-V
(P21 mouse)
Beta-Cholera Toxin – HRP Labeling
Adult Mouse
Dorsal Horn Structure/Function II
A.
B.
20 μm
10 mV
5s
Thermal Stimulator
Contact
52ºC
34ºC
10 mN 25 mN
50 mN 100 mN 200 mN
5ºC
A.
-39 mV
50 μm
20 mV
5 msec
B.
-52 mV
D.
C.
-48 mV
1 mV
10 msec
-48 mV
20 msec
Correlation between morphology and function of cells found in
the superficial dorsal horn
Over the past 25-30 years a
large number of studies have
documented cell types found in
the superficial dorsal horn.
Recently, in series of studies by
Ed Perl colleagues have
attempted to take these types of
studies a step further.
These are examples of lamina I
cells (A) projections neurons and
(B) local interneurons.
(Grudt and Perl, ‘02)
Examples of different cell types found in
lamina II. (A) islet cells, (B) radial cells, (C)
central cells, (D) medial-lateral cells and (E)
vertical cells.
(Grudt and Perl, ‘02)
Unfortunately not all cells fall neatly into
this or any other classification scheme.
Examples of different morphological types of superficial dorsal horn neurons
injected with NB. This figure also shows examples of double immunostaining for
NK1 and PKCγ.
Lawson, McIlwrath, Koerber
Synaptic Ultrastructure
In the superficial dorsal horn
Non-peptidergic C-fiber
Type 1 glomerulus
GABA
C – central terminal from
primary afferent.
A – terminals from Gaba/Gly
inhibitory interneurons
Type 2 glomerulus
Axoaxonic terminals frequently
contain GABA in Type 1 and
GABA/GLY in Type 2
GABA/GLY
D-hair
GABA/GLY