Sensory Mechanisms of the Spinal Cord: Volume 1 Primary Afferent Neurons and the Spinal Dorsal Horn

ISBN-13: 9780306480331 / Angielski / Twarda / 2004 / 581 str.

Jr. William D. Willis; Richard E. Coggeshall

Opis

The third edition of this monograph continues to have the goal of providing an overview of current thought about the spinal cord mechanisms that are responsible for sensory processing. We hope that the book is of value to both basic and clinical neuroscientists. Several changes have been made in the presentation, as well as additions because of the research advances that have been made during the past decade. Chapters 3 and 4 in the previous edition have been subdivided, and now the morphology of primary afferent neu rons of the dorsal root ganglia is described in Chapter 3 and the chemical neuroanatomy 4. The description of the dorsal hom in the previous Chapter 4 of these neurons in Chapter is now included in Chapter 5, and the chemical neuroanatomy of the dorsal hom in Chapter 6. Furthermore, discussions of the descending control systems have now been of Chapter 12. consolidated at the end The authors would like to express their appreciation for the help provided by several individuals. R.E.C. wishes to acknowledge the many things he learned about primary afferent neurons from conversations with Dr S. N. Lawson. He also thanks Lyn Shilling for her assistance with the typing. WDW thanks Dr Nada Lawand for her critical reading of parts of the manuscript, Rosaline Leigh for help with the manuscript, and Griselda Gonzales for preparing the illustrations."

Spis treści

1: Introduction.- Sensory Modalities and Channels.- Specificity versus Pattern Theories of Sensation.- Historical Perspective.- Specificity Theory.- Pattern Theory.- Gate Theory of Pain.- Supporting Evidence.- Molecular Basis of Specificity of Primary Afferent Neurons.- Sinclair’s Challenge and Evidence for Specificity from Microneurography.- Sensory Role of Single Tactile Afferents in Human Subjects.- Criticism of Microneurography Results.- Rebuttal of this Criticism.- Microneurography and Pain.- Somatosensory System Dualities.- Epicritic versus Protopathic Sensations.- Lemniscal versus Non-lemniscal Systems.- Large- versus Small-Fiber Systems.- Conclusions.- 2: Sensory Receptors and Peripheral Nerves.- Composition of Peripheral Nerves.- Sensory Receptors.- Cutaneous Receptors.- Cutaneous Mechanoreceptors.- Mechanoreceptors in the Glabrous Skin of the Primate Hand.- Cutaneous Displacement and Velocity Detectors.- SA I Receptors.- SA II Receptors.- Cutaneous Velocity Detectors.- FA I receptors.- Hair Follicle Receptors.- Field Receptors.- C Mechanoreceptors.- Cutaneous Transient Detectors.- FA II Receptors.- G1 Hair Follicle Receptors.- Sinus Hairs.- The Role of Growth Factors in the Regulation of Mechanoreceptors.- SA I (Merkel Cell) Endings.- FA I (Meissner Corpuscle) Receptors.- FA II (Pacinian Corpuscle) Receptors.- Hair Follicle Receptors.- Transduction in Mechanoreceptors.- Cutaneous Nociceptors.- A? Nociceptors.- C Nociceptors.- Transduction in Nociceptors.- Sensitization of Nociceptors.- Neurogenic Inflammation.- Peptidergic and Non-Peptidergic Nociceptors.- Nociceptors and Neurogenic Inflammation.- Interaction of Growth Factors with Nociceptors.- Inhibitory Interactions with Peripheral Cutaneous Nociceptor Endings.- Cutaneous Chemoreceptors Possibly Responsible for Itch.- Cutaneous Thermoreceptors.- Cold Receptors.- Warm Receptors.- Transduction in Thermoreceptors.- Muscle Receptors.- Stretch Receptors.- Muscle Spindles.- Golgi Tendon Organs.- Pressure-Pain Endings and Muscle Nociception.- Group III Muscle Afferents.- Group IV Muscle Afferents.- Sensitization of High Threshold Muscle Afferents.- Joint Receptors.- Joint Mechanoreceptors.- Slowly Adapting Joint Receptors.- Rapidly Adapting Joint Receptors.- Joint Nociceptors.- Sensitization of Joint Nociceptors.- Peptide Content of Fine Joint Afferents.- Joint Inflammation.- Role of Dorsal Root Reflexes in the Neurogenic Inflammation of Arthritis.- Visceral Receptors.- Visceral Mechanoreceptors.- Visceral Nociceptors.- Peptides in Visceral Afferents.- Growth Factors and Visceral Afferents.- Sensitization of Visceral Afferents.- Visceral Thermoreceptors.- Conclusions.- 3: Dorsal Root Ganglion Cells and their Processes.- Dorsal Root Ganglion Cell Bodies.- DRG Cell Classifications.- Cytologic and Size Classifications.- Myelination.- Axon Conduction Velocity.- Action Potentials and After Polarizations.- Classifications by Chemical Neuroanatomical Criteria.- Topographic Organization.- Innervation of Ganglion Cell Bodies, Pericellular Baskets.- Dorsal Root Ganglion Cell Processes.- Dermatomes.- Branching.- Ventral Root Afferent Fibers.- Spinal Cord.- Blind Fibers.- Looping Fibers.- Pial Fibers.- Function.- Segregation of Dorsal Root Fibers.- Conclusions.- 4: Chemical Anatomy of Dorsal Root Ganglion Cells.- Amines.- Catecholamines (CAs).- Histamine.- Serotonin.- Amino Acids.- Glutamate.- Aspartate.- GABA.- Glycine.- Channels.- Calcium Channels.- Sodium Channels.- Voltage-Gated Sodium Channels.- Cell Size.- Co-localizations.- ? Subunits.- Development.- Inflammation.- Peripheral Axotomy.- Nerve Growth Factor (NGF).- Peripheral Sensory Axons.- Non-voltage Gated Sodium Channels.- Enzymes.- Alkaline Phosphatase.- Carbonic Anhydrase (CA).- Cholinergic Enzymes.- ChAT.- ACHE.- Cytochrome Oxidase.- Fluoride-resistant Acid Phosphatase (FRAP).- Monoamine Oxidase.- Nitric Oxide (NO), Nitric Oxide Synthase (NOS) and NADPHd.- Ca++ Calmodulin Kinase II.- Other Kinases.- Hormones.- Corticotrophin Releasing Factor (CRF).- Insulin.- Peptides.- Angiotensin.- Atrial and Brain Natriuretic Peptide.- Bombesin.- Cholycystokinin (CCK).- Calcitonin Gene-related Peptide (CGRP).- Classification.- Age.- Development.- Co-localizations.- SP.- GAL.- SOM.- CCK.- VIP.- Bombesin, ENK, DYN, VIP, NT, and NPY.- FRAP.- Cysteine Proteases.- Amino Acids.- IB4.- Peripherin.- BDNF.- GDNF.- TrkA.- P75.- Experimental Manipulations or Disease.- Dorsal Rhizotomy.- Peripheral Nerve Lesions.- Inflammation.- Diabetes.- Hypoxia.- Hereditary Sensory Neuropathy.- Capsaicin.- Axonal Transport Blockers.- NGF Treatment.- Female Steroid Hormones.- Sympathectomy.- Dorsal Roots.- Periphery.- Somatic Innervation.- Visceral Innervation.- Endothelin (ET).- Galanin.- Nerve Lesions.- Inflammation.- Resiniferatoxin.- Axonal Transport Blockers.- Co-localizations.- CGRP.- SP.- VIP.- NPY.- 5-HT.- Glucocorticoid Receptors.- Dorsal Roots.- Neuropeptide FF (NPFF).- Neuropeptide Y (NPY), Polypeptide Y (PPY), and Peptide YY.- Peripheral Axotomy or Compression.- Trophic Factors.- Development.- Co-localizations.- Peripheral Innervation.- Neurotensin (NT).- Opioids.- Dynorphin (DYN).- Endomorphin.- Endorphin.- Enkephalin.- Nociceptin.- Oxytocin and Vasopressin.- Secretoneurin and the Chromagranins.- Somatostatin.- Co-localizations.- GDNF and Peripheral Axotomy.- Development.- Periphery.- Substance P (SP).- Segmental Levels.- Age and Development.- Co-localizations.- Tachykinins.- CGRP.- GAL.- SOM.- CCK.- ENKandDYN.- Bombesin, VIP, NT, and NPY.- FRAP.- NOS.- Glutamate.- IB-4.- 200 KD Neurofilament Protein.- Peripherin.- GDNF.- Opioid Receptors.- Experimental Manipulations.- Dorsal Rhizotomy.- Peripheral Nerve Lesions.- Inflammation.- Trophic Factors.- Adrenalectomy.- Resiniferatoxin.- Periphery.- Somatic Innervation.- Visceral Innervation.- VIP.- Proteins.- Calcium Binding Proteins.- Glutamate Transporters.- Reg-2.- Bone Matrix Proteins.- ?-arrestin.- Receptors.- Aminergic Receptors.- Adrenergic Receptors.- Dopamine Receptors.- Histamine Receptors.- 5-HT Receptors.- 5-HT1A Receptors.- 5-HT1B Receptors.- 5-HTlC Receptors.- 5-HT1D Receptors.- 5-HT1E Receptors.- 5-HT1F Receptors.- 5-HT2 Receptors.- 5-HT3 Receptors.- 5-HT4, 5-HT5, and 5-HT7 Receptors.- Amino Acid Receptors.- Glutamate Receptors.- Ionotropic Glutamate Receptors.- Metabotropic Glutamate Receptors.- GABA Receptors.- Glycine Receptors.- Cholinergic Receptors.- Nicotinic Receptors.- Muscarinic Receptors.- Hormone Receptors.- Androgen Receptors.- Estrogen Receptors.- Gender Differences.- Development.- Castration.- Glucocorticoid Receptors.- Insulin Receptors.- TRH Receptors.- Vitamin D Receptors.- Peptide Receptors.- Angiotensin Receptors.- Bombesin Receptors.- Bradykinin Receptors.- Cannabinoid Receptors.- CCK Receptors.- CGRP Receptors.- Endothelin (ET) Receptors.- Galanin Receptors (GALR).- Neuropeptide Y Receptors (Y-Rs).- Neurotensin Receptors (NT-R).- Neurokinin-1 Receptors (NKR1s).- NPFF Receptors.- Opioid Receptors.- MORs, DORs, and KORs.- Co-localizations.- Peripheral Axotomy.- Inflammation.- Development.- Periphery.- Endomorphin Receptors.- Endorphin Receptors.- Opioid-like Receptor 1 (ORL1).- Sigma Receptors.- Oxytocin and Vasopressin Receptors.- Somatostatin Receptors.- Vasoactive Intestinal Polypeptide (VIP) Receptors.- Purinergic Receptors or Purinoceptors.- Adenosine Receptors.- ATP (P) Receptors.- P2X Purinoceptors.- P2Y Purinoceptors.- Vanilloid Receptors (VRs).- Conclusions.- 5: Structure of the Dorsal Horn.- Lamina I.- Cell Types.- Classic Types: Marginal and Smaller Cells.- Modern Types.- Primary Afferent Input into Lamina I.- The Marginal Plexus.- Large versus Small Fibers.- A? versus C Fibers.- Propriospinal versus Primary Afferent Fibers.- Cutaneous, Muscular, and Visceral Afferent Input.- Neuropil Organization.- Lamina II (The Substantia Gelatinosa).- Cell Types.- Classic Cell Types.- Limiting (Limitrophe, Border) Cells.- Central Cells.- Funicular Cells.- Short-Axoned Cells.- The Gelatinosa as a Closed System?.- Modern Cell Types.- Stalked Cells.- Islet Cells.- Other Cell Types.- Arboreal Cells.- II-III Border Cells.- Spiny Cells.- Filamentous Cells.- Curly Cells.- Stellate Cells.- Adequacy of Dendritic Classifications for Lamina II Cells.- Axonal Projections.- Primary Afferent Input.- Coarse Primary Afferents.- Fine Primary Afferents.- Fine Myelinated (A?) Primary Afferents.- Neuropil Organization.- Glomeruli.- Central Terminals.- Dense or Dark Terminals.- Light (Electron-lucent or Regular Synaptic Vesicle) Terminals.- Large Dense-Core Vesicle Terminals.- Peripheral Dendrites.- Peripheral Terminals.- Chemical Markers.- Nonglomerular Synapses.- Lamina III.- Cell Types.- Axonal Projections.- Primary Afferent Input.- Neuropil Organization.- Fine Structure of Terminals of Identified Axons.- Lamina IV.- Cell Types.- Axonal Projections.- Primary Afferent Input.- Neuropil Organization.- Lamina V.- Cell Types.- Axonal Projections.- Primary Afferent Input.- Lamina VI.- Cell Types.- Axonal Projections.- Primary Afferent Input.- Conclusions.- 6: Chemical Anatomy of the Dorsal Horn.- Amines.- Catecholamines (CA).- Dopa.- Dopamine (DA).- Noradrenaline (NA).- Adrenaline.- Histamine.- Serotonin (5-HT).- Laminar Localizations and Intrinsic Cells.- Synaptic Organization.- Co-localizations.- Experimental Manipulations.- Development.- Amino Acids.- Glutamate.- Laminar Localizations.- Synaptic Organization.- Aspartate.- Gamma-Aminobutyric Acid (GABA).- Laminar Localizations.- Intrinsic Cells.- Synaptic Organization.- Axoaxonic Synapses.- Dendrodendritic, Dendrosomatic, and Dendroaxonic Synapses.- Postsynaptic Targets.- Co-localizations.- Glutamate.- Glycine.- ChAT.- NOS and NADPH Diaphorase.- CGRP.- Enkephalin.- Galanin.- Neuropeptide Y.- Neurotensin.- Somatostatin.- SP.- Thyrotropin-releasing Hormone.- Parvalbumin.- Glycine Receptor.- MORs.- Experimental Manipulations and Development.- Glycine.- Laminar Localizations.- Synaptic Organization.- Co-localizations.- Gephryin.- GABA.- Glycine Transporter GLYT2.- NADPH Diaphorase.- ChAT.- Somatostatin.- Taurine.- Channels.- Calcium Channels.- Sodium Channels.- Enzymes.- Cholinergic Enzymes (ChAT and ACHE).- ChAT.- AChE.- Cytochrome Oxidase.- Fluoride Resistant Acid Phosphatase (FRAP).- Monoamine Oxidase.- Nitric Oxide (NO).- Laminar Localizations.- Co-localizations.- Experimental Manipulations.- Dorsal Rhizotomy.- Spinal Damage.- Inflammation.- Development.- Protein Kinase C.- Hormones.- Corticotrophin Releasing Factor (CRF).- Thyroid-releasing Hormone (TRH).- Peptides.- Angiotensin.- Atrial and Brain Natriuretic Peptide.- Bombesin.- Cholycystokinin (CCK).- Calcitonin Gene-related Peptide (CGRP).- Laminar Localizations.- Intrinsic Neurons.- Synaptic Organization.- Dorsal Column Organization.- Descending Fibers.- Experimental Manipulations.- Deafferentation.- Peripheral Nerve Lesions.- Spinal Hemisection.- Inflammation.- Morphine and Tolerance.- Electrical Stimulation.- MK-801.- Dietary Calcium.- Development and Age.- Sprouting.- Endothelin (ET).- Galanin.- Laminar Localizations.- Synaptic Organization.- Experimental Manipulations.- Hypocretin.- Neuropeptide FF (NPFF).- Neuropeptide Y (NPY), Pancreatic Polypeptide (PPY) and Peptide YY.- Laminar Localizations.- Synaptic Organization.- Co-localizations.- Experimental Manipulations.- Dorsal Rhizotomy or Spinal Transection.- Peripheral Axotomy.- Inflammation.- Neurotensin (NT).- Opioids.- Dynorphin (DYN).- Laminar Localizations.- Intrinsic Cell Types.- Co-localizations.- Synaptic Organization.- Experimental Manipulations.- Dorsal Rhizotomy and Peripheral Axotomy.- Spinal Injury.- Inflammation.- Endomorphins.- Endorphin.- Enkephalin (ENK).- Laminar Localizations.- Segmental Levels.- Cell Types.- Synaptic Organization.- Development.- Experimental Manipulations.- Deafferentation.- Spinal Injury.- Nerve Transection.- Inflammation.- Ovariectomy and Noxious Stimuli.- Co-localizations.- Nociceptin (Orphanin FQ).- Oxytocin (OXY) and Vasopressin (VP).- Secretoneurin.- Somatostatin.- Laminar Localizations and Intrinsic Cells.- Deafferentation and Peripheral Axotomy.- Synaptic Organization.- Development.- Substance P (SP).- Laminar Localizations.- Intrinsic Cells.- Synaptic Organization.- Experimental Manipulations.- Deafferentation.- Peripheral Nerve Lesions.- Spinal Lesions.- Inflammation.- Nerve Growth Factor.- Local Anesthesia.- Capsaicin.- MK-801.- Thiamine Deficiency.- Electrical Stimulation.- Prostate Stimulation.- SP Antisera.- Hereditary Sensory Neuropathy.- Age and Development.- Vasointestinal Polypeptide (VIP).- Laminar Localizations.- Intrinsic Cells.- Synaptic Organization.- Experimental Manipulations.- Dorsal Rhizotomy.- Peripheral Nerve Section or Crush.- Spinal Transection.- Proteins.- Calcium Binding Proteins.- Glutamate Transporters.- Receptors.- Aminergic Receptors.- Adrenergic Receptors.- ? Adrenergic Receptors.- ?1 Receptors.- ?2 Receptors.- Dopamine Receptors.- 5-HT Receptors.- 5-HT1 and 5-HT1A Receptors.- 5-HT1B Receptors.- 5-HT1C Receptors.- 5-HT1D Receptors.- 5-HT1s Receptors.- 5-HT2 Receptors.- 5-HT3 Receptors.- 5-HT7 Receptors.- Amino Acid Receptors.- Glutamate Receptors.- Ionotropic Glutamate Receptors.- Metabotropic Glutamate Receptors.- GABA and Benzodiazepine Receptors.- GABAA Receptors.- GABAB Receptors.- Glycine Receptors.- Cholinergic Receptors.- Nicotinic Receptors.- Muscarinic Receptors.- Hormone Receptors.- Androgen Receptors.- Corticosteroid Receptors.- Corticotrophin Releasing Factor (CRF) Receptors.- Estrogen Receptors.- Insulin Receptors.- TRH Receptors.- Peptide Receptors.- Angiotensin Receptors.- Bombesin Receptors.- Bradykinin Receptors.- Cannabinoid Receptors.- CCK Receptors.- CGRP Receptors.- Endothelin Receptors.- Galanin Receptors.- Neurokinin Receptors.- Laminar Localizations.- Segmental Localizations.- Intrinsic Neurons.- Synaptic Organization.- Experimental Manipulations.- Neurotoxicity and Therapy.- Neural Injuries.- Development.- Other Neurokinin Receptors.- Neuropeptide Y Receptors (Y-Rs).- Neurotensin Receptors.- NeuropeptideFF (NPFF) Receptors.- Opioid Receptors.- MORs, DORs, and KORs.- Laminar Localizations.- Autoradiographic Binding.- Immunocytochemical Labeling.- Segmental Variability.- Proportions of Opioid Receptor Subtypes.- Primary Afferent Input.- Intrinsic Neurons.- Synaptic Organization.- Co-localizations.- Experimental Manipulations.- Development.- Estrus.- “Other” Opioid Receptors.- Endomorphin Receptors.- Endorphin Receptors.- ORL1 Receptors.- Sigma Receptors.- Oxytocin and Vasopressin Receptors.- Somatostatin (SRIF) Receptors.- Vasoactive Intestinal Polypeptide (VIP) Receptors.- Purinoceptors.- Vanilloid Receptors.- Conclusions.- 7: Functional Organization of Dorsal Horn Interneurons.- Electrophysiological Recordings from Interneurons.- Recording Techniques.- In Vivo Preparations.- Extracellular Recordings from Interneurons.- Intracellular Recordings from Interneurons.- In Vitro Preparations.- Responses of Interneurons to Peripheral Nerve Volleys Evoked by Electrical Stimulation.- Volleys in A Fibers.- Volleys in A and C Fibers.- Population Responses.- Cord Dorsum Potentials and Intraspinal Field Potentials.- Cord Dorsum Potentials.- Negative Field Potentials.- Population Responses to Muscle Afferent Volleys.- Population Responses to Joint Afferent Volleys.- Population Responses to Visceral Afferent Volleys.- Positive Cord Dorsum Potential and Primary Afferent Depolarization.- Primary Afferent Depolarization.- Changes in Metabolic Activity and Intracellular Signaling.- 2-Deoxyglucose.- Expression of Immediate, Early Genes.- Summary.- Afferent Projections to the Dorsal Horn.- Laminae I-IV.- Laminae V, VI, and X.- Responses to Electrical Stimulation.- Lamina I.- Lamina II.- Laminae III-VI.- Responses to Natural Stimulation.- Lamina I.- Lamina II.- Laminae III-VI.- Lamina X.- Classification of Dorsal Horn Interneurons.- Monosynaptic Input.- PAD-related.- Laminar Position.- A or A Plus C Input.- Bandwidth.- Cluster Analysis.- Dorsal Horn Neurons in Unanesthetized, Behaving Animals.- Summary.- Pharmacology of Dorsal Horn Interneurons.- Pharmacological Techniques.- Systemic Drug Administration.- Superfusion of the Spinal Cord.- Intrathecal Administration.- Microdialysis.- Microiontophoresis.- Limitations of Electrophysiological Recordings in Drug Studies.- Excitatory Transmitters in the Dorsal Horn.- Excitatory Amino Acids: Glutamate and Aspartate.- Release of Excitatory Amino Acids in the Spinal Cord.- Excitatory Action of Glutamate and Aspartate.- Excitatory Amino Acid Receptors.- Ionotropic Glutamate Receptors.- Functions of Ionotropic Glutamate Receptors.- Metabotropic Glutamate Receptors.- Functions of Metabotropic Glutamate Receptors.- ATP.- Purinergic Receptors.- Functions of Purinergic Receptors.- Excitatory Neuropeptides.- Substance P (SP).- Other Neurokinins: Neurokinin A (NKA) and Neurokinin B (NKB).- Calcitonin Gene-related Peptide (CGRP).- Combined Action of Excitatory Amino Acids and Peptides.- Other Excitatory Peptides.- Vasoactive Intestinal Polypeptide (VIP).- Neurotensin.- Cholecystokinin (CCK).- Thyrotropin-releasing Hormone (TRH).- Corticotropin-releasing hormone (CRH).- Pituitary Adenylate Cyclase-activating Polypeptide (PACAP).- Islet Amyloid Polypeptide (IAPP or Amylin).- Inhibitory Transmitters in the Dorsal Horn.- Inhibitory Amino Acids.- Gamma-aminobutyric Acid (GABA).- Glycine.- Acetylcholine (ACh).- Inhibitory Peptides in the Dorsal Horn.- Opioid Peptides.- Other Inhibitory Peptides.- Nocistatin and Nociceptin.- Somatostatin.- Galanin.- Bombesin.- Neuropeptide Y (NPY).- Pharmacology of PAD.- Role of Excitatory Amino Acids.- Role of GABA.- Ionic Mechanism.- Other Transmitters.- Potassium Release.- Summary.- Modulation of the Responses of Dorsal Horn Neurons.- Receptive Fields and Alterations in their Sizes.- Excitatory Receptive Fields.- Cascade Theory.- Somatotopic Organization.- Changes in Receptive Fields.- Long-term Potentiation and Long-term Depression.- Inhibitory Receptive Fields.- Gate Theory and Diffuse Noxious Inhibitory Controls.- Gate Theory of Pain.- Diffuse Noxious Inhibitory Controls.- Summary.- Models of Human Disease.- Inflammatory Pain.- Experimental Arthritis.- Kaolin and Carrageenan Model.- Adjuvant Model.- Cutaneous Inflammation.- Mustard Oil.- Carrageenan.- Complete Freund’s Adjuvant.- Formalin Injection.- Bee Venom Injection.- Intradermal Injection of Capsaicin.- Neuropathic Pain.- Deafferentation.- Spared Root Paradigm.- Nerve Transection.- Autotomy.- Models of Peripheral Neuropathic Pain.- Bennett Model.- Seltzer Model.- Chung Model.- Models of Central Neuropathic Pain.- Cordotomy Model.- Contusion Injury Model.- Spinal Cord Hemisection Model.- Quisqualic Acid Model.- Ischemia Model.- Summary.- Genetic Influences on Spinal Cord Sensory Processing.- Genetic Basis of Spinal Cord Sensory Functions.- Mechanoreceptor-related Genes.- Cold-related Gene.- Nociception-related Genes.- TrkA and Congenital Insensitivity to Pain.- Deletion of Neurokinin-1 Receptors.- Deletion of CGRP.- Changes in Signal Transduction Molecules.- Opiate Receptors.- Summary.- Conclusions.- References.