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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 Neurons are hyperexcitable. They have large cutaneous receptive fields. Excitatory inputs predominately from A-fiber inputs. 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