Handbook of Innovations in Central Nervous System Regenerative Medicine

Handbook of Innovations in CNS Regenerative Medicine provides a comprehensive overview of the CNS regenerative medicine field. The book describes the basic biology and anatomy of the CNS and how injury and disease affect its balance and the limitations of the present therapies used in the clinics. It also introduces recent trends in different fields of CNS regenerative medicine, including cell transplantation, bio and neuro-engineering, molecular/pharmacotherapy therapies and enabling technologies. Finally, the book presents successful cases of translation of basic research to first-in-human trials and the steps needed to follow this path.

Areas such as cell transplantation approaches, bio and neuro-engineering, molecular/pharmacotherapy therapies and enabling technologies are key in regenerative medicine are covered in the book, along with regulatory and ethical issues.

• Describes the basic biology and anatomy of the CNS and how injury and disease affect its balance
• Discusses the limitations of present therapies used in the clinics
• Introduces the recent trends in different fields of CNS regenerative medicine, including cell transplantation, bio and neuro-engineering, molecular/pharmacotherapy therapies, and enabling technologies
• Presents successful cases of translation of basic research to first-in-human trials, along with the steps needed to follow this path

• Language: English
• Publisher: Elsevier Science
• Release date: Jun 10, 2020
• ISBN: 9780128180853

Book preview

Handbook of Innovations in Central Nervous System Regenerative Medicine

Edited by

Antonio J. Salgado

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Preface

Chapter 1. Insights on nervous system biology and anatomy

Abstract

1.1 Introduction

1.2 Development of the vertebrate nervous system

1.3 General organization of the nervous system

1.4 Cells of the nervous system

1.5 Technical approaches to study the nervous system

1.6 Conclusions

References

Chapter 2. Overview of Alzheimer's and Parkinson's diseases and the role of protein aggregation in these neurodegenerative diseases

Abstract

2.1 Alzheimer’s disease

2.2 Prevalence of Alzheimer’s disease

2.3 Diagnosis of Alzheimer’s disease

2.4 Neurodegeneration and neurobiology of Alzheimer’s disease

2.5 Progression of amyloid deposition throughout the brain

2.6 Genetic influences

2.7 The amyloid cascade hypothesis

2.8 Parkinson’s disease

2.9 Prevalence of Parkinson’s disease

2.10 Diagnosis of Parkinson’s disease

2.11 Neurodegeneration and neurobiology of Parkinson’s disease

2.12 Progression of α-synuclein deposition throughout the brain

2.13 Genetic and environmental causes

2.14 Common cellular mechanisms in neurodegenerative diseases

2.15 Conclusions

2.16 Acknowledgments

References

Chapter 3. Introduction to trauma in the central nervous system

Abstract

3.1 Introduction

3.2 The current landscape of central nervous system trauma

3.3 Stages of central nervous system injury

3.4 Traumatic spinal cord injury pathophysiology

3.5 Traumatic brain injury

3.6 Guidelines for the management of neurotrauma

3.7 Conclusion

Acknowledgments

References

Chapter 4. Current clinical approaches in neurodegenerative diseases

Abstract

4.1 Alzheimer’s disease and Parkinson’s disease in a clinical context

4.2 Current pharmacotherapies used in Alzheimer’s and Parkinson’s diseases

4.3 Pitfalls of the clinical trials

4.4 New drugs currently being developed

4.5 Conclusion and future challenges

References

Chapter 5. Neuroprotection in the injured spinal cord

Abstract

5.1 Spinal cord injury in a clinical context

5.2 Behind spinal cord injury

5.3 Current neuroprotective therapies in spinal cord injury

5.6 Final remarks

References

Chapter 6. The therapeutic potential of exogenous adult stem cells for the injured central nervous system

Abstract

6.1 Introduction

6.2 Adult stem cells and their sources

6.3 Differentiation along neural lineages

6.4 Challenges in expansion and transplantation

6.5 Adult stem cells in preclinical models of central nervous system diseases

6.6 Clinical trials of adult stem cells in the central nervous system

6.7 Conclusions

6.8 Acknowledgements

References

Chapter 7. Biomaterial-based systems as biomimetic agents in the repair of the central nervous system

Summary

7.1 Introduction

7.2 Considerations on the pathology of spinal cord trauma

7.3 Positioning biomaterials for central nervous system regenerative medicine

7.4 Biofunctionalized electroconducting microfibers as biomimetic agents in central nervous system repair

7.5 Central nervous system regeneration: decomposing the needs to recompose the strategy

7.6 Translational research on biomaterials for central nervous system repair

7.7 Acknowledgments

References

Chapter 8. Tissue engineering and regenerative medicine in spinal cord injury repair

Abstract

8.1 Introduction

8.2 Experimental models of spinal cord injury: methodology, advantages, disadvantages, and behavioral testing

8.3 Treatment strategies

8.4 Cell therapy: overview, comparison of various types of stem cells, methods of application

8.5 Antioxidant treatment

8.6 Biomaterials in spinal cord injury

8.7 Low-level laser therapy

8.8 Future perspectives

8.9 Acknowledgements

8.10 Contribution

References

Chapter 9. Toward the therapeutic application of small interfering RNA bioconjugates in the central nervous system

Abstract

9.1 Considerations on therapeutic drug delivery for neurological disorders

9.2 Small interfering RNA

9.3 Barriers for siRNA delivery

9.4 Chemical modifications

9.5 Ribose modifications

9.6 Structural modifications

9.7 Bioconjugates

9.8 Dynamic polyconjugates

9.9 Other delivery systems: nanocarriers

9.10 Future perspectives

Acknowledgements

References

Chapter 10. Gene therapy approaches in central nervous system regenerative medicine

Abstract

10.1 Gene therapy

10.2 Gene therapy vectors

10.3 Gene therapy for nervous system

References

Chapter 11. Gene editing and central nervous system regeneration

Abstract

11.1 Introduction

11.2 Targeted nucleases for efficient genome editing

11.3 Nuclease-mediated alterations: resolving double-strand breaks

11.4 CRISPR-Cas9 technology

Acknowledgment

References

Chapter 12. Molecular therapeutic strategies in neurodegenerative diseases and injury

Abstract

12.1 Introduction

12.2 Spinal cord injury

12.3 Traumatic brain injury

12.4 Amyotrophic lateral sclerosis

12.5 Multiple sclerosis

12.6 Alzheimer’s disease

12.7 Parkinson’s disease

References

Chapter 13. Spinal cord stimulation for the recovery of function following spinal cord injury

Abstract

13.1 Introduction

13.2 A brief history into electricity induced neuromodulation

13.3 Modulation of spinal circuits

13.4 Neuromodulation of motor circuits

13.5 Conclusion

Acknowledgements

References

Chapter 14. Electroceutical therapies for injuries of the nervous system

Abstract

14.1 Introduction

14.2 Effects of electrical fields on neural growth in vitro

14.3 Electrical stimulation for peripheral nerve injuries and regeneration

14.4 Electrical stimulation in spinal cord injuries

14.5 Electrical stimulation in brain injuries

References

Chapter 15. Role of mesenchymal stem cells in central nervous system regenerative medicine: past, present, and future

Abstract

15.1 Mesenchymal stem cells: origins

15.2 The paradigm shift: from differentiation to secretome

15.3 In vivo veritas

15.4 What lies ahead

15.5 Conclusion

Acknowledgments

References

Chapter 16. Three-dimensional culture systems in central nervous system research

Abstract

16.1 Introduction

16.2 Organoids

16.3 Spheroid systems

16.4 Scaffold-based models

16.5 Challenges and future directions

16.6 Concluding remarks

Acknowledgments

References

Chapter 17. Scaffolds for spinal cord injury repair: from proof of concept to first in-human studies and clinical trials

Abstract

17.1 Scaffold-based strategies to facilitate spinal cord injury repair

17.2 The mechanisms of motor function recovery in complete transected spinal cord injury animals

17.3 Clinical study of stem cells and scaffold transplantation for spinal cord injury repair

17.4 Perspectives and challenges

Acknowledgments

References

Chapter 18. Animal models of central nervous system disorders

Abstract

18.1 Introduction

18.2 Naturally regenerating animal models

18.3 Rodents as a model of central nervous system disorders

18.4 Rodents as a model for spinal cord injury

18.5 Rodents as a model for Parkinson’s disease

Acknowledgments

References

Chapter 19. Bioethics in translation research and clinical trials

Abstract

19.1 Introduction

19.2 The role of research ethics committees

19.3 Conclusion

References

Index

Copyright

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List of Contributors

Armando Almeida

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Guimarães, Portugal

Rita C. Assunção-Silva

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Sandra Barata-Antunes

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Assumpcio Bosch

Institut de Neurociènces (INc), Department of Biochemistry and Molecular Biology, Universitat Autònoma Barcelona, Bellaterra, Spain

Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Centro de Biología Molecular SEVERO OCHOA, Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain

Vall d’Hebron Institut de Recerca (VHIR), Research Group on Gene Therapy at Nervous System, Barcelona, Spain

Nicholas M. Boulis,     Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA, United States

Alice Braga,     Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

Bing Chen,     State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

Miguel Chillon

Institut de Neurociènces (INc), Department of Biochemistry and Molecular Biology, Universitat Autònoma Barcelona, Bellaterra, Spain

Vall d’Hebron Institut de Recerca (VHIR), Research Group on Gene Therapy at Nervous System, Barcelona, Spain

Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Jorge E. Collazos-Castro,     Neural Repair and Biomaterials, National Hospital for Paraplegics, Toledo, Spain

João Cortinhas

i3S—Institute for Research and Innovation in Health, University of Porto, Porto, Portugal

INEB—Biomedical Engineering Institute, University of Porto, Porto, Portugal

Faculty of Engineering of the University of Porto (FEUP), Porto, Portugal

Abel Salazar Institute of Biomedical Sciences (ICBAS), University of Porto, Porto, Portugal

Jianwu Dai,     State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

Ignacio Delgado-Martínez,     Institute of Neurosciences and Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, and Network Center of Biomedical Research on Neurodegenerative Diseases (CIBERNED), Bellaterra, Spain

Jaume del Valle,     Institute of Neurosciences and Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, and Network Center of Biomedical Research on Neurodegenerative Diseases (CIBERNED), Bellaterra, Spain

Madalena Esteves

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Guimarães, Portugal

Razan Faraj,     Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA, United States

Thais Federici,     Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA, United States

Michael G. Fehlings

Division of Neurosurgery, Department of Surgery, University of Toronto, Toronto, ON, Canada

Division of Neurosurgery, Krembil Neuroscience Centre, Toronto Western Hospital, University Health Network, Toronto, ON, Canada

Aline M. Fernandes

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Miguel Gago,     Neurology Department, Hospital da Senhora da Oliveira, Guimarães, Portugal

Guillermo García-Alías

Institute of Neurosciences and Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, and Network Center of Biomedical Research on Neurodegenerative Diseases (CIBERNED), Bellaterra, Spain

Institut Guttmann of Neurorehabilitation, Badalona, Spain

Julian L. Gendreau,     Department of Neurosurgery, Mercer University School of Medicine, Macon, GA, United States

Eduardo D. Gomes

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Alex Greven,     Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA, United States

Laureen D. Hachem,     Division of Neurosurgery, Department of Surgery, University of Toronto, Toronto, ON, Canada

Regan Hamel,     Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

R.M. Ichiyama,     School of Biomedical Sciences, University of Leeds, Leeds,United Kingdom

Pavla Jendelová

Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

Second Faculty of Medicine, Charles University, Prague, Czech Republic

Kristýna Kárová,     Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

Kristýna Kekulová,     Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

R.W.P. Kissane

Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom

School of Biomedical Sciences, University of Leeds, Leeds,United Kingdom

Zuzana Kočí,     Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

Šárka Kubinová,     Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

Hugo Leite-Almeida

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Guimarães, Portugal

Mariah Lelos,     Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, Wales, United Kingdom

Zhijia Liang,     Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA, United States

Rui Lima

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Guimarães, Portugal

Sara Monteiro Lopes

Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

Institute for Interdisciplinary Research, University of Coimbra, Coimbra, Portugal

Alvaro Machado,     Neurology Department, Hospital de Braga, Braga, Portugal

Cláudia R. Marques

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Susana Monteiro

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Pedro M.D. Moreno

i3S—Institute for Research and Innovation in Health, University of Porto, Porto, Portugal

INEB—Biomedical Engineering Institute, University of Porto, Porto, Portugal

Xavier Navarro

Institute of Neurosciences and Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, and Network Center of Biomedical Research on Neurodegenerative Diseases (CIBERNED), Bellaterra, Spain

Institut Guttmann of Neurorehabilitation, Badalona, Spain

Itse Onuwaje

Department of Pharmacology, UCL School of Pharmacy, University College London, London, United Kingdom

UCL Centre for Nerve Engineering, University College London, London, United Kingdom

Ana P. Pêgo

i3S—Institute for Research and Innovation in Health, University of Porto, Porto, Portugal

INEB—Biomedical Engineering Institute, University of Porto, Porto, Portugal

Faculty of Engineering of the University of Porto (FEUP), Porto, Portugal

Abel Salazar Institute of Biomedical Sciences (ICBAS), University of Porto, Porto, Portugal

Inês M. Pereira

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Guimarães, Portugal

Luís Pereira de Almeida

Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

Luca Peruzzotti-Jametti,     Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

James B. Phillips

Department of Pharmacology, UCL School of Pharmacy, University College London, London, United Kingdom

UCL Centre for Nerve Engineering, University College London, London, United Kingdom

Stefano Pluchino,     Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

Sofia Rocha,     Neurology Department, Hospital de Braga, Braga, Portugal

Nataliya Romanyuk,     Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

Carola Rutigliani,     Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

António J. Salgado

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

ICVS/3B’s Associate Lab, PT Government Associated Laboratory, Braga/Guimarães, Portugal

Nadine Correia Santos

Life and Health Sciences Research Institute (ICVS), School of Medicine—University of Minho, Braga, Portugal

Center for Digital Medicine P5, School of Medicine, University of Minho, Braga, Portugal

Nuno A. Silva

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal

ICVS/3B’s—PT Government Associate Laboratory, Guimarães, Portugal

Jayden A. Smith,     CITC Ltd, St. John’s Innovation Centre, Cambridge, United Kingdom

Barbora Svobodová

Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

Second Faculty of Medicine, Charles University, Prague, Czech Republic

Fábio G. Teixeira

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Andreia Teixeira-Castro

Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Campus de Gualtar, Braga, Portugal

ICVS/3B’s PT Government Associate Laboratory, Braga/Guimarães, Portugal

Pavlos Texakalidis,     Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA, United States

Muhibullah S. Tora,     Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA, United States

Lucia Machova Urdzíková

Institute of Experimental Medicine, Czech Academy of Science, Prague, Czech Republic

Second Faculty of Medicine, Charles University, Prague, Czech Republic

Zhifeng Xiao,     State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

Bryan Yu,     Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom

Yannan Zhao,     State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

Preface

The central nervous system (CNS) is still considered to be the holy grail of regenerative medicine. Its intrinsic low regenerative properties, as well as its highly functional specialization, pose challenges that no other tissue or organ present. Even with the tremendous advances achieved in the last century in medicine, most of the severe conditions that affect the CNS are considered untreatable. Indeed, the complexity of CNS disorders demands the development of innovative therapies. Areas such as cell transplantation approaches, bio- and neuroengineering, molecular/pharmacotherapy therapies, and enabling technologies are key in regenerative medicine approaches that target injury and disease within the CNS. In the last decade, we have witnessed the emergence of different approaches within these fields that will probably pave the way for the emergence of innovative therapies that can target CNS injury and disease in the future. However, when developing such complex and combined strategies for therapeutic use, one has to be aware of both regulatory and ethical issues. Particularly, those in academia need to know the procedures and appropriate paths to follow to adequately take a concept from the bench, to a clinical product or methodology, and to the bedside. Therefore, it is thus essential to provide information that covers both aspects of translational research, but above all immerses them in a unique multidisciplinary approach that ultimately can foster new ideas and strategies, and with it pave their way within the future of the CNS regenerative medicine field. Having this in consideration the present book is focused on topics that go from the basic biology of injury and disease in the CNS, to upcoming fields in CNS regenerative medicine including cell transplantation, bio- and neuroengineering, molecular/pharmacotherapy therapies, and enabling technologies. By presenting these topics in a sequential and integrated manner it is expected that the reader acquires a multidisciplinary vision of CNS regenerative medicine, and with it, pave the way to new regenerative routes that will overcome the limitations of the present ones.

Chapter 1

Insights on nervous system biology and anatomy

Madalena Esteves¹, ², Armando Almeida¹, ² and Hugo Leite-Almeida¹, ²,    ¹Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal,    ²ICVS/3B’s—PT Government Associate Laboratory, Guimarães, Portugal

Abstract

The mammalian nervous system is possibly the most fascinating product of evolution. It is inherently complex, but our knowledge on its intricacies has been increasing over decades. In this chapter an overview of the mammalian nervous system development from the neural plate and of the resulting anatomical organization, is provided. Functional aspects of each region will also be presented. Finally, a brief description of the main technical approaches used in neuroscience will be discussed.

Keywords

Central nervous system; peripheral nervous system; embryology; anatomy; histology

1.1 Introduction

The moment that humankind recognized the brain as the source of its intellect, sensation, and movement is lost in the midst of time. Skulls bearing signs of trepanation point to prehistory. Many of these present evident signs of healing (the subject outlived the surgery), but the motivation, ritual or therapeutical, remains controversial [1,2]. In ancient Egypt the stomach, liver, lungs, and intestines were deposited in canopical jars during the mummification process. The brain was discarded through the nostrils, meaning that it was not considered essential for the afterlife. Curiously, the oldest-known treatise on trauma and surgery, the Edwin Smith papyrus (circa 1600 BCE but probably based in older documents), has its origins in ancient Egypt. It contains what is considered the first inscription of the word brain as well as the earliest description of the meninges and cerebrospinal fluid (CSF). Also, it contains remarkable descriptions of several head and spinal injuries and associated complications—see for instance, [3–6], also [7]. In ancient Greece, Aristotle of Stagira (384–322 BCE) considered that the brain was a sort of radiator or cooling device for the heart, the latter being the organ of reason. Such view was later criticized by Galen of Pergamon (Aelius Galenus, CE 130–200), who considered the brain the center of mental activity based on observation brain injuries effects [8]. The Renaissance marks the beginning of systematic studies on the human body, including the central nervous system (CNS). Leonardo da Vinci (1472–1519) and Andreas Vesalius (1514–1564) were among these pioneers, the latter authoring the highly influential De humani corporis fabrica (1543). New views on the centrality of the brain emerged, markedly influenced by Rene Descartes (1596–1650) and his De Homine (1664). However, it was not until the 18th century that the experimental study of the nervous system started with the studies of Luigi Galvani, demonstrating the involvement of electricity in muscle contraction [9] (see also [10]) and the demonstration (traditionally attributed to Charles Bell [1783–1855] and François Magendie [1774–1842]) that anterior and posterior spinal nerve roots were respectively associated with motor and sensory functions [8] (see also [11]). The function of the different regions composing the nervous system became a point of interest for the following decades. The study of brains from aphasic patients by Marc Dax (1770–1837) and Paul Broca (1824–1880) marked the beginning of cortical function mapping, which would boost in the following century with the development of imaging techniques [12,13]. The foundation of modern neuroscience is however attributed to two histologists, Camillo Golgi (1843–1926) and Santiago Ramón y Cajal (1852–1934) both receiving the Nobel prize in physiology or medicine in 1906 [14,15].

The 20th century witnessed astounding scientific and technical developments that greatly expanded our knowledge on the CNS function and structure. Modern neuroscience includes areas as distinct as psychology, cellular and molecular biology, or anatomy and systems biology. The goal of this chapter is to provide a comprehensive overview of (1) CNS origin, (2) anatomy, and (3) function as well as on the techniques currently used in humans and experimental models to gain insights on its function.

1.2 Development of the vertebrate nervous system

1.2.1 Development of the trilaminar embryo

By the end of the second week after fertilization, the human embryo is fully implanted and has developed the bilaminar embryonic disk (bilaminar blastoderm), which is constituted by two cell layers: the epiblast (external) and the hypoblast (internal) corresponding to the primitive ectoderm and endoderm, respectively. At this stage, the precursors of the nervous system are not yet developed. During the third week, two main processes occur: primitive streak formation and cell migration through the primitive streak into the interior of the embryo (ingression), a phenomenon known as gastrulation. Gastrulation begins with the formation of a longitudinal midline structure located in the caudal region of the embryonic disk, the primitive streak, which defines the body axis (cranial-caudal, medial-lateral, and dorsal-ventral) and quickly elongates through the embryo. Epiblast cells then migrate and displace hypoblast cells, forming the definitive endoderm, which will later develop into gut, lungs, and liver. Other epiblast cells migrate to the space between the epiblast itself and the developing definitive endoderm, forming the intraembryonic mesoderm that later will give rise to connective tissues, muscle, and the vascular system. Also, a population of mesodermal cells form the notochordal process (the rudiment of the notochord) in the midline. The notochord is a critical source of signals that patterns the development of the surrounding tissues [16] (see Sections 1.2.2 and 1.3.1). The remaining epiblast cells constitute the ectoderm, the precursor of the epidermis and nervous system [17,18]. Thus, by the end of third week of gestation, the embryonic disk is trilaminar. The primitive streak gradually regresses and around day 20, some of its caudal remnants swell, producing the tail bud (or caudal eminence). This structure will be of importance for spinal cord development (see Section 1.2.3). Meanwhile, the remaining primitive streak gradually decreases in size, and by day 26 it disappears.

1.2.2 Neural induction

Neural induction occurs during gastrulation starting on day 18. In this process, the ectoderm cranial to the primitive node thickens to form the neural plate—neuroectoderm, a pseudostratified, single-layered columnar neuroepithelium. At this early stage, any cell from the ectoderm can develop into either epidermis or neural tissue. However, by the end of gastrulation, cells are committed to either fate [19]. Seminal work from Hans Spemann (awarded the Nobel prize in physiology and medicine in 1935) and Hilde Mangold in the newt (genus Triton) embryo was critical to dissecting the neural induction process [20] (published in English in 2001 [21]). In their experiments, a fragment from the dorsal lip of a gastrulating embryo was grafted in the ventral side of a host at the same stage. As a result, hosts developed a secondary neural plate as well as a secondary notochordal process, somites (paired blocks of paraxial mesoderm), and a gut [22]. Taking advantage of pigmentation differences between donor—Triton cristatus (unpigmented)—and host—Triton taeniatus or Triton alpestris (pigmented)—the origin of the tissues was traced. Grafted tissues differentiate into the notochord, floor plate, and some somites, while the ectopic neural plate cells derive from the host [20,21] (see also [22,23]). In the authors’ own words, A piece taken from the upper blastopore lip of a gastrulating amphibian embryo exerts an organizing effect on its environment in such a way that, following its transplantation to an indifferent region of another embryo, it there causes the formation of a secondary embryo. Such a piece can therefore be designated as an organizer. [21]. In mammalians, grafts of gastrulating mouse nodes can also induct the development of a secondary neuroaxis in recipient embryos at the same stage [24]. In addition to primary organizers like the Spemann’s (or Spemann-Mangold), the Hensen’s node (in the avian embryo; [25]), or the mammalian node: secondary organizers, notably the notochord, the floor and roof plates, the zona limitans interthalamica (ZLI), or the anterior neural ridge/border (ANR/ANB) will emerge at later stages inducing and patterning the development of the nervous system (consult for authoritative reviews [22,26–31]). The molecular pathways underlying the process of neural induction are complex and not completely clarified. Dorsal ectodermal cells have a default proneural potential. Indeed, the organizer secretes agents that inhibit the action of bone morphogenic proteins (BMP) with two major consequences: the proneural potential of the cells in the vicinity of the organizer (dorsal ectoderm) is uncovered, whereas ectoderm cells that are more distant develop to form epidermis; other signaling proteins such as fibroblast growth factors (FGFs), Wnts, noggin, chordin, and follistatin can also act as direct neural inducers [19,32,33]. These neural inducers are potent extracellular inhibitors of TGFβ family, binding with high affinity to the ligands and thereby preventing their ligation to the respective receptors [19].

1.2.3 Neurulation

Neurulation (or primary neurulation) starts in the fourth week of gestation and is the process that leads to the formation of the neural tube from the neural plate, the rudiment of the CNS [34]. It encompasses four essential and overlapping events: (1) neural induction, (2) shaping, (3) bending of the neural plate, and (4) closure of the neural tube. Neural induction (1) is the process of neural plate formation, that is, the beginning of the development of the CNS. It occurs during gastrulation starting on day 18. As described earlier, cells of the ectoderm differentiate into the neuroectoderm, a pseudostratified, single-layered columnar neuroepithelium (neural induction). Shaping (2) consists of the cranial-to-caudal expansion of the neural plate, becoming broad cranially and tapered caudally. These regions will later originate, respectively, the brain and the spinal cord. Indeed, at this point, the lining of the primary brain vesicles are already recognizable (see Section 1.2.4). The lateral parts of neural plate then start to elevate, creating neural folds, during bending (3). This extension begins in the cranial region, extending into the future spinal cord region, and then starts to bend in the direction of the midline. Such bending allows closure (4) at the end of the neural folds, creating a hollow tube. This process starts in the future cervical region (around day 22) and progresses in both rostral and caudal directions, creating the neural tube. The extremities, cranial and caudal neuropores, close on days 24 and 26, respectively. Also, cells at the border between the ectoderm and neuroectoderm will give rise to neural crest cells. These cells emerge from the folds of the closing neural tube (trunk level) or even before the fusion of the neuronal folds (head level) forming a mass of premigratory cells on both sides of the neural plate. These cells express specific markers—Snai 1, Snai 2, and Sox E, for instance—that make them distinct from ectoderm and neuroectoderm cells very early, even before any morphological event [35]. Neural crest cells differentiate into many types of neurons and glia of the somatic and autonomous peripheral nervous system (PNS) as well as melanocytes or chromaffin cells, for instance.

Secondary neurulation is a much-less-understood process. In this case, the medullary cord forms from the condensation of tail bud cells at the caudal limit of the embryo. This solid mass of undifferentiated mesenchymal cells will cavitate to form multiple lumina, which will coalesce into a single lumen surrounded by neuroepithelial cells [34]. Despite the differences regarding cell origin and mechanisms, trunk and tail tubes become continuous.

Neural tube defects can affect up to 1 in 1000 established pregnancies. While open malformations like anencephaly or craniorachischisis are lethal, others like spina bifida occulta are less severe or even asymptomatic [36,37].

1.2.4 Development of brain vesicles

After neuropore closure (see Section 1.2.3), expansions of the neural tube start to develop, creating the primary brain vesicles: the prosencephalon (forebrain), mesencephalon (midbrain), and rhombencephalon (hindbrain). These further develop into secondary brain vesicles: the prosencephalon divides into the telencephalon [that later differentiates into the basal ganglia (Section 1.3.2.4) and cortex (Section 1.3.2.5)] and the diencephalon (Section 1.3.2.3), later forming the thalamus and hypothalamus among other structures; the mesencephalon enlarges keeping the designation in the adult brain; the rhombencephalon divides into metencephalon, later forming the pons and cerebellum, and more caudally into the myelencephalon, later forming the medulla oblongata. The mesencephalon, pons, and medulla oblongata are the brainstem (Section 1.3.2.1). The neural tube caudal to the myelencephalon will originate the spinal cord (Section 1.3.1). Also, as a result of the differential expansion of some cell populations, between the fourth and the eighth week, the neuroaxis folds. The mesencephalic (also cranial or cephalic) flexure is the first and is then followed by the cervical flexure, at the transition between the myelencephalon and the spinal cord, both occurring ventrally. Within each vesicle, the neural canal also expands, creating the primitive ventricles. After the closure of the neuropores (see Section 1.2.3), these ventricles, as well as the central canal of the spinal cord, become filled with CSF (see Section 1.3.3.2).

1.3 General organization of the nervous system

The vertebrate nervous system is divided according to functional or structural features. Functional divisions include the somatic and the autonomic (or visceral) nervous system, responsible for conscient (tactile sensation, pain perception, voluntary movements, etc.) and involuntary processes [38], respectively. The autonomic nervous system is strictly motor (responsible for visceral movements) and has three divisions: sympathetic, parasympathetic and enteric – for more information on the sympathetic and parasympathetic divisions consult for instances;[39] comprehensive reviews and perspectives on the enteric system can be found here [40,41]. Structurally, the nervous system is divided into CNS and PNS. The PNS consists of cranial, spinal, and autonomic nerves and respective ganglia, as well as the enteric nervous system. The CNS, which is the main focus of this chapter, is constituted by the spinal cord and brain. These are further described in the next subsections.

1.3.1 Spinal cord

The spinal cord develops from the neural tube caudal to the myelencephalon. The lumen of the neural tube develops into the central canal, which is continuous with the ventricular system (Section 1.3.3.2). The spinal cord is segmentally organized in five levels—cervical (C), thoracic (T), lumbar (L), sacral (S), and coccygeal (Co)—each further divided in segments that vary across species: human: C1–C8, T1–T12, L1–L5, S1–S5, and Co1; rat/mice: C1–C8, T1–T13, L1–L6, S1–S4, and Co1–Co3. Each segment contains a bilateral pair of nerves (31 pairs in the human) with an anterior/ventral and a posterior/dorsal root with motor and sensitive functions, respectively, as initially described Magendie and Bell (see Introduction). The gray matter occupies the core of the spinal cord with an H shaped form and has in its center the central canal [42]. The posterior horns of the H, contain neurons that receive peripheral inputs from the dorsal roots, thta is, sensory information, and whose cell bodies are located in nearby ganglia [the dorsal root ganglia (DRG)]. These peripheral neurons develop from the neural crest (Section 1.2.3) and are the primary afferents to the CNS conveying information related to mechanical and chemical stimuli, temperature, light touch, and pain. Motor neurons occupy the ventral horns and project through the ventral root to enervate skeletal muscles.

An additional group of neurons, the intermediolateral cell columns, present at T1–L3 levels (human), provide sympathetic enervation. The axons of these cells also project through the ventral roots branching soon after to form the white ramus, which then enters in the corresponding ganglia from the sympathetic chain ganglia. There, these preganglionic neurons will synapse on postganglionic neurons that project via the white ramus, then reaching the dorsal and ventral nerves from the somatic division on the spinal nerve and providing sympathetic enervation on the target organs. The preganglionic neurons of the parasympathetic division, on the other hand, are located in the brainstem and sacral levels S2–S4. Contrary to the sympathetic division, the postganglionic neurons are located in the walls of the target organs.

As stated earlier, the spinal cord develops from the caudal portion of the neural tube. There, neuroepithelial cells proliferate in the ventricular layer, that is, adjacent to the lumen of the central canal. These neurons will migrate peripherally, forming the mantle layer, and then their axons will grow even more peripherally, forming the marginal layer. The mantle and marginal layers correspond to the gray and white matter of the mature spinal cord. Neurons organize in dorsal and ventral plates/columns, which will develop into sensory and motor neurons, respectively. This functional diversity in spinal cord emerges early in development, and is strongly conditioned by the dorsal-to-ventral position of the developing neurons and the resulting exposure to a number of morphogenes, notably Sonic Hedgehog (Shh) produced by nearby organizers (floor plate, notochord; Section 1.2.2) [18]. At the periphery, surrounding the gray matter core, a number of ascending and descending tracts convey information into/from other spinal levels and into/from the brain (consult [43,44] for an overview of the human spinal cord anatomy).

1.3.2 Brain

The human brain is a 1.5 kg organ located inside the cranium. Like in the spinal cord (Section 1.3.1), the brain has twelve pairs of nerves, the cranial nerves. These are designated with roman numerals I–XII following the rostrocaudal order from which they emerge from the brain. With the exception of the I (olfactory) [45] and II (optic) [46] nerves, the cell bodies of these neurons are located in sensory and parasympathetic ganglia that connect with key regions within the brain. An overview of the main brain areas is presented later.

1.3.2.1 Brainstem

The brainstem is involved in the regulation of basic homeostatic functions as heart rate and respiratory control, sleep, and vigilance cycles control as well as sensory motor functions to the face (see later). It comprises three main subdivisions: medulla oblongata, pons, and midbrain; some authors also include the diencephalon. The medulla oblongata is the most caudal portion of the brainstem and is continuous with the spinal cord. Its organization resembles that from the spinal cord though more complex. Indeed, during development some cells migrate forming nuclei away from their origin. In the brainstem development, alar (dorsal) and basal (ventral) cranial nerve nuclei organize in seven cell columns (or six in some literature) with sensory and motor functions, respectively. These include (1) general visceral afferent column, which receives interoceptive information from the glossopharyngeal (IX) and vagus (X) nerves; (2) first special afferent column (tractus solitarius nuclei), which receives information (e.g., taste-related) via the facial (VII), glossopharyngeal (IX), and vagus (X) nerves; (3) general afferent column receives sensory information from the face via the trigeminal (V) and facial (VII) nerves (face), as well as from the oral, nasal, external ear, pharyngeal, and laryngeal cavities via the V, VII, IX, and X nerves; (4) second special afferent column (cochlear and vestibular nuclei) receives balance and hearing inputs, via the vestibulocochlear nerve (VIII); (5) somatic efferent column neurons enervate extrinsic ocular muscles via oculomotor (III), trochlear (IV), and abducens (VI) nerves and the tongue muscles via hypoglossal nerve; (6) special visceral (or branchial) efferent column neurons enervate striated muscles derived from pharyngeal arches mesoderm via V, VII, IX, and X nerves, and the trapezius and sternocleidomastoid muscles via the accessory (XI) nerve; (7) general visceral efferent, which provides parasympathetic enervation to several smooth muscles and glands via III, IX, and X nerves [18] (see also [47,48–53]). Rostrally, at the mesencephalon, only two columns and respective nuclei are present: (3) general afferent column/trigeminal (V) and (5) somatic efferent column neurons/oculomotor (III). The motor nucleus of trochlear (IV) nerve has a metencephalic origin and then migrates to the mesencephalon (references earlier).

1.3.2.2 Cerebellum

The cerebellum has been classically associated with motor function and balance. This resonates from the early works of Luigi Rolando (1773–1831) and others, particularly the seminal Saggio Sopra le Vera Struttura del Cervello Dell’uomo e Degli Animali e Sopra le Funzioni del Sistema Nervoso [54], where the motor impact of ablation and stimulation of the cerebellum is described (see also [55]). Cerebellar function is, however, much more complex and heterogenous, and it is now consensual that it plays an important role in cognitive and emotional processes (see [56–58] for review). It consists of two hemispheres, joined in the midline by the vermis. Each of these hemispheres can then be divided into three lobes: anterior, posterior, and flocculonodular. Connection to the brainstem is achieved via the inferior, middle, and superior cerebellar peduncles, which respectively reach the medulla oblongata, pons, and mesencephalon. The organization of the cerebellum consists of an outer layer of gray matter, the cerebellar cortex, and an internal region of white matter, which consists of fibers that run to, and from, the brainstem [59]. The cerebellar cortex itself has a consistent layered organization: molecular, Purkinje cells, and granule cells layers (outermost to innermost). The cerebellar circuitry is relatively simple. Cerebellar afferents are constituted by climbing fibers projecting from the inferior olive in the pons and synapsing on Purkinje cells, and by mossy fibers with origin in precerebellar nuclei other than the inferior olive, synapsing predominantly on granule cells dendrites. The information on this pathway is then conveyed to Purkinje cells by granule cells’ axons via the so-called parallel fibers on the molecular layer. Purkinje cells then project to deep cerebellar nuclei neurons, which in turn project back to other CNS areas [60].

1.3.2.3 Diencephalon

The diencephalon is located between the brainstem and the cortex. It is constituted by the thalamus, subthalamus, hypothalamus, and epithalamus [61]. It receives all the pathways that ascend from the spinal cord and brainstem, thus having a core role in sensory information awareness. The thalamus is a heterogenous assembly of nuclei whose main function is to relay sensory and motor information from subcortical structures to the cortex [62]. Its role in tactile discrimination and pain [63–65], visual [66–68], and auditory [69,70] processing is well documented, but it also plays a role in higher cognitive functions [71], mood [72], arousal [73], or addiction [74,75].

The subthalamus, although anatomically included in the diencephalon, is functionally part of the basal ganglia. Its main input comes from the medial globus pallidus. In turn, the subthalamus projects back to the globus pallidus, substantia nigra, and thalamus. Its glutaminergic cells form the main excitatory projection from the basal ganglia, playing an important role in movement inhibition [61].

The hypothalamus is constituted by several nuclei and it is a major center of hormonal regulation, thus influencing mood and behavior. It is closely connected with the hypophysis (pituitary gland) through the hypothalamic-hypophyseal tract and portal system. Indeed, the neurohypophysis is formed by axons originated in the paraventricular and supraoptic hypothalamic nuclei, being responsible for the release of two neuropeptides: oxytocin and vasopressin. The adenohypophysis releases several hormones of importance for homeostasis, growth, and reproduction [76].

The epithalamus includes the habenula and the pineal gland. The habenula is a notably conserved structure connecting fore- and midbrain regions. It has been associated with motivated behavior, but its role appears to be more heterogeneous, and its dysfunction is well recognized in the context of depression [77–80]. The pineal gland releases melatonin at the night period and has been associated with the regulation of circadian rhythms [81].

1.3.2.4 Basal ganglia

As the telencephalon expands caudally, it covers the dorsal and lateral diencephalon. The pallium (the thin dorsal aspect of the telencephalon) will develop into the cerebral cortex (Section 1.3.2.5) and olfactory bulbs. Also, it will form the large white matter tracts connecting the cerebral hemispheres. The ventral portion, the subpallium, will protrude into the lumen of the neural canal forming the ganglionic eminences (GE) that will then develop into the basal ganglia, parts of the amygdala, and septum [18,82]. In a strict sense, the basal ganglia refer to the striatal and pallidal nuclei of the telencephalon. However, mesencephalic nuclei of the substantia nigra and ventral tegmental area, as well as the subthalamic nucleus are also considered an integrant part of the basal ganglia due to their close anatomical and functional relations [83]. The basal ganglia have been for a long time associated with the initiation and execution of movements. This view was stimulated by the symptomatology manifested in basal ganglia-related pathologies, particularly Parkinson’s disease [83]. Recent views on basal ganglia expand its functions to time processing, reward evaluation, transitions between habit-based and goal-directed decision making, response inhibition/impulsivity, and conflict monitoring, among other functions [84–89].

1.3.2.5 Cortex

Several cortical structures are present in the vertebrate brain. Major divisions include the isocortex (neocortex; see Section 1.3.2.5.1) and the allocortex, that evolutionary precedes the former and is subdivided into paleocortex, comprising olfactory regions (see Section 1.3.2.5.3), septal nuclei, piriform regions, and a minor part of the amygdala (and archicortex) comprising the hippocampal formation (see Section 1.3.2.5.2), retrosplenial cortex, and a cortical band in the cingulate gyrus [90].

1.3.2.5.1 Neocortex

The neocortex develops from the dorsal-most aspect of the telencephalon. While at the early stages, cerebral cortex development is similar to other regions of the neural tube, corticogenesis is more complex (see [91,92] for comprehensive reviews). The neuroepithelial cell pool initially expands by symmetrical division (for a recent review on (a)symmetrical cerebral cortex development consult [93]). These cells transform into radial glial cells which, like the highly related neuroepithelial cells, maintain their bipolar morphology with apical and basal processes but expressing glial hallmarks. At this stage, radial glial cells divide asymmetrically, generating at each division a radial glial cell, thereby contributing to the cell pool maintenance and an intermediate progenitor that then migrates from the apical surface to the subventricular zone [91–93]. Radial glia later switch to generate mature glia either directly as it the case of proliferating astrocytes or via intermediate progenitors (e.g., oligodendrocyte progenitor cells [94]; see also Section 1.4). The intermediate progenitors can then symmetrically divide generating two projection neurons that migrate into the cortical plate; this process of indirect neurogenesis has been suggested as an essential factor of cortical expansion in gyrencephalic species (see later) [95–97]. Early-born neurons move to integrate the basal layers of the forming cortex. Later-born neurons adopt a bipolar morphology and then attach to radial glial cells that assist migration to upper layers [98]. Cajal-Retzius neurons are a transient cell population pivotal in the orchestration of this process. These neurons have multiple origins at the developing pallium and very early in development migrate tangentially occupying the outermost layer (layer I; see earlier) of the entire cortex [99] (see also [100,101]). Cajal-Retzius cells produce reelin [102,103], a large glycoprotein, that is essential for cortical laminar structure [99–101,104–106]. In addition to projection neurons that migrate radially, it was found in the mid-1990s that cortical GABAergic interneurons originate in the GE (see Section 1.3.2.4) [107]; this heterogenous population migrate tangentially in a manner similar to the Cajal-Retzius neurons to populate the developing neocortex (see for review [108,109]). Defects on neural migration during development have been associated with epilepsy and several neuropsychiatric disorders [110,111]. At the end, a six-layer neocortex is formed, from the most superficial to the deepest layer: molecular (I), external granular (II), external pyramidal (III), internal granular (IV), internal pyramidal (V), and plexiform (VI). The molecular layer is positioned immediately beneath the pia matter (see Section 1.3.3.1). It is characterized by a very low cellular density composed essentially of glial cells and few neurons; also apical dendrites from pyramidal neurons synapse with afferent axons. Pyramidal neurons predominate in layers III and V and stellate neurons in layers II and IV. Layer VI contains a wide variety of neurons including small pyramidal cells, Martinotti, stellate, and fusiform cells. Layer IV receives projections from the thalamus being more prominent in primary sensory areas (e.g., the primary visual cortex). Layers V and VI project to subcortical regions and are more prominent in motor areas. In a general manner, upper layers are involved in the cortical communication either with the ipsilateral or the contralateral hemisphere. Prior to the advent of neuroimaging (see Section 1.5) these cytoarchitectonic variations served to map the cortex. The Brodmann’s classification in 43 regions based on Nissl-stained samples is among the most well-known [112]. At the macro level, the human cortex presents an intricate pattern of sulci (grooves) and gyri (ridges). This organization allows not only to maximize cortical size but also to optimize wiring and functional organization [113]. Gyrification starts around the 16th gestational week with the formation of the lateral cerebral sulcus on each hemisphere [114]. Other primary sulci appear later, namely the central and occipital sulcus, dividing each hemisphere in four lobes: frontal, parietal, temporal, and occipital.

The frontal lobe is home to the primary motor and premotor cortices, responsible for movement of the contralateral side of the body. Also, it includes the prefrontal cortex, responsible for higher cognitive functions. The parietal lobe includes the somatosensory cortex and the parietal association cortex, in which sensory information (also from the contralateral side of the body) reaches a conscious level, as well as language-related regions. Within the temporal lobe lays the primary auditory and auditory association cortices, necessary for conscious perception of sound. It also includes regions necessary for the understanding of language as well as the hippocampus and the amygdala. Main functions of the former include memory, spatial navigation, cognition, and emotion, while the latter is responsible for conscious smell and plays an important role in fear and aggression. Finally, the occipital lobe includes the primary visual cortex, responsible for visual perception, and the visual association cortex, necessary for interpretation of visual stimuli [59].

1.3.2.5.2 Hippocampal formation

The hippocampal formation comprises the dentate gyrus, hippocampus (subdivided in CA1-3; Cornu Ammonis/Ammon's horn), subiculum, presubiculum, parasubiculum, and entorhinal cortex [115]. The hippocampus develops from the dorsomedial region of the telencephalon. The cortical hem, which has on its lateral side the developing cortex and on the medial side the choroid plexus (see Section 1.3.3.2), secretes factors that instruct the formation of the hippocampus, thereby functioning as an organizer (see also Section 1.2.2) [116]. Hippocampal development is in many aspects similar to that described earlier for the neocortex (Section 1.3.2.5.1), for example, [117] (see for review [116,118]) resulting in the formation of a layered structure [119]. The classical circuit of the hippocampus is a trisynaptic loop starting on the projection from the entorhinal cortex to the dentate gyrus (perforant path), which provides the main cortical input. The dentate gyrus projects then to CA3 (mossy fiber pathway) which then projects to CA1 (Schaffer collateral pathway) and also sends collaterals to other CA3 neurons. The loop is then closed with a projection back to the entorhinal cortex [120]. The hippocampus has a well-established role in memory and spatial navigation; see also for place and grid cells [121,122]. However, functional subdomains have been identified, the dorsal region associated with its classical cognitive role and the ventral with emotion and stress [123].

1.3.2.5.3 Olfactory cortex

The olfactory cortex is an evolutionarily conserved three-layered paleocortex involved in odor processing. It is located in the ventrolateral telencephalon and is subdivided in several structures including the piriform and entorhinal cortices. While visual, auditory, and somatosensory information is relayed to the thalamus and from there to the respective primary cortical areas (see Section 1.3.2.3), the olfactory cortex receives direct input from the olfactory bulbs (see [124]).

1.3.2.5.4 Amygdala

The amygdala is a complex of nuclei located in the medial temporal lobe being morphologically and functionally heterogeneous. It is essentially of telencephalic origin although a diencephalic contribution was identified [125]. Classically, the amygdala and related structures have been associated with the emotional response, particularly fear and anxiety [126]. The amygdala receives and integrates sensory information, and projects back to the prefrontal cortex (via thalamus). Neurons in the amygdala encode aversive stimulus (see for pain [127]) and trigger the response associated with an imminent threat (see also [128]).

1.3.3 Meninges and the ventricular system

The CNS is protected by different systems. The brain and the spinal cord are enclosed within bone structures—skull and vertebra, respectively—in close contact with a system of connective tissue membranes, the meninges. The CSF circulating within the meninges act as a cushion providing mechanical protection against impacts.

1.3.3.1 Meninges

The meninges are, from the most superficial to the deepest layer, dura mater, arachnoid, and pia mater [59]. The dura mater is a tough fibrous membrane which is adherent to the periosteum in some locations, while in others it allows a small extradural space. Immediately below is a small subdural space, in which venous irrigation passes by and then lays the soft translucent arachnoid membrane. Under the arachnoid, and over the brain-adherent pia mater, the subarachnoid space can be found. It contains strands of connective tissue, as well as circulating CSF [129].

1.3.3.2 Ventricular system

The ventricular system consists of chambers and canals within the CNS, in which the CSF circulates. Within the spinal cord, it is exclusively comprised of the central canal, while in the brain it includes the lateral ventricles, third ventricle, cerebral aqueduct, and fourth ventricle. The fourth ventricle is continuous caudally with the central canal of the spinal cord (Section 1.3.1). As it advances rostrally, the canal opens on the dorsal surface of the medulla, forming the floor of the ventricle. The tela choroideia, the cerebellum (Section 1.3.2.2), and other cerebral structures, form the roof of the fourth ventricle. In this ventricle, three apertures make the ventricular space continuous with the subarachnoid space; these are the two lateral foramina of Luschka at the pons level and the medial foramen of Magendie at the medulla level [130,131]; for a historical perspective and clinical correlates of the foramina, see also [132]. At the mesencephalic level the ventricular space narrows forming the Sylvius (or cerebral) aqueduct which connects rostrally with the third ventricle at diencephalic level (see Section 1.3.3.2). The lateral ventricles are located in both hemispheres and communicate with the centrally located third ventricle.

The CSF is produced in the choroid plexus, which is located in the lateral, third, and fourth ventricles. It is a transparent fluid with reduced amounts of cells and proteins, produced by both secretion and passive diffusion. CSF is continuously circulating, being produced and reabsorbed, and, as most is produced in the lateral ventricle, it normally goes from here to the fourth ventricle, where it enters the subarachnoid space. It then flows superiorly, being reabsorbed into the venous system [133] (see also [134]).

1.4 Cells of the nervous system

There are two main cell types in the nervous system: neurons and supporting cells. While the neuron is the main functional unit, the remaining cells (glial and ependymal) have been classically viewed as secondary, mainly supporting neuronal function. However, new data has shown that this neuron-glial interaction is far more complex. The idea that neurons and glia coexist in a 1:10 relation has been highly propelled in the literature but it is now clear that they exist in similar proportions [135].

1.4.1 Neurons

Neurons (as well as astrocytes, oligodendrocytes, and ependymal cells) derive from the cytodifferentiation of the neuroepithelium lining the neural tube. The process starts after the fusion of the neural folds and proceeds cranially and caudally as the tube zips up. Neurogenesis initiation precedes gliogenesis [92,136] and can persist through adult life in specific neurogenic niches, including the hippocampal dentate gyrus and the subventricular zone [137–139]. New neurons then migrate, differentiate, and establish synapses integrating networks [92].

A neuron’s main function is to receive, integrate, and transmit information to other cells. Neurons possess a cell body (soma) from which two types of processes (neurites) emerge: dendrites, which are specialized in receiving input from other cells or the environment, and an axon, a long projection of the cell body that sends information to other neurons, muscles, or glands; it often branches away from the soma into multiple collaterals, which possess presynaptic boutons. Neurons are classified into three major types according to the type of branching in multipolar, bipolar, and pseudounipolar. Multipolar are the most common presenting multiple dendrites and single axon, which arise directly from the soma. Such is the case of cortical pyramidal cells. Bipolar cells, on the other hand, possess two processes, one dendrite and one axon, which later branch away from the soma. These occur in afferent pathways of visual, auditory, and vestibular systems. Finally, pseudounipolar cells possess a single neurite, which branches into dendritic and axonal branches and are primary afferents of the spinal cord and cranial nerves. Unipolar neurons also exist in invertebrates. Functionally, neurons are also classified in interneurons or projection neurons if projecting within the local circuitry or to distant regions, respectively. They can also be classified based on their neurotransmitter content (e.g., dopaminergic neurons) [59].

Neurons are electrically excitable. At rest, the membrane potential is around −70 mV. When the neuron membrane is depolarized to a certain level, an action potential occurs that can be conducted through the axon [140], inducing release of neurotransmitters at the synaptic terminal. These neurotransmitters diffuse through the synaptic gap, reaching receptors in the postsynaptic cell, changing its membrane potential, and potentially reinitiating the cycle in the postsynaptic neuron.

1.4.2 Glial cells

1.4.2.1 Oligodendrocytes and Schwann cells

Oligodendrocytes (CNS) and Schwann cells (PNS) are the cells that produce the myelin sheath, which coats many axons thereby facilitating current conduction [59]. In the periphery, one Schwann cell wraps around a segment of the axon while in the CNS, oligodendrocyte possesses multiple processes, each being able to myelinate segments (up to about 1 mm)—internodal segments—of multiple axons. Thus, each (myelinated) axon, is covered with multiple myelin sheaths, at which no ionic exchanges occur, and voltage currents spread passively. These regions are separated by small uninsulated gaps, the nodes of Ranvier, enriched in ionic channels that can propagate the action potential from the previous node. This so-called saltatory conduction accelerates the propagation of the action potential. Nodes of Ranvier are larger in the CNS further increasing conduction efficiency. Oligodendrocytes and Schwann cells also differ in the proteins present in the myelin sheaths, for example, myelin oligodendrocyte glycoprotein (CNS), and P0 and P22 (PNS), and that are essential for its integrity. Demyelinating diseases like multiple sclerosis affect the conduction ability of the neurons leading to neurological deficits. For in-depth information, consult, for instance [141–143].

1.4.2.2 Astrocytes

Astrocytes are star-shaped cells and are the largest neuroglial cells. Two main types of astrocyte are recognized: protoplasmic and fibrous. They differ in their relative abundance—the former being more prevalent in the gray matter and the latter in the white matter—and morphology—protoplasmic present numerous, short branching processes while fibrous have fewer and simpler processes. Astrocytes have diverse functions in the CNS. As stated earlier, they provide scaffolds to assist neurons migration during development in embryonic development (see Section 1.3.2.5.1). Also, they are an important component of the blood-brain barrier, ensheathing capillary vessels with expansions of their processes; perivascular feet cover most outer surface of the capillaries. Astrocytes participate in the exchange of metabolites between the blood and brain having a role in the metabolism and homeostatic regulation CNS microenvironment. Importantly, they are part of what has been called the tripartite synapses, where they are able to sense neuronal activity, elevate Ca²+, and release neurotransmitters and other effectors, playing an active modulatory role in synaptic transmission. Such has been shown to be relevant for behavior and cognition [144] (see also [145–148]).

1.4.2.3 Microglia

Microglia are the immune cells of the CNS, responsible for vigilance and protection from infection and lesion. In opposition to the remaining glial cells, which derive from the neural tube (see Section 1.4.1), microglial cell progenitors arise from the yolk sac and colonize the CNS before the blood-brain barrier is formed. In a physiological state, microglia are typically in a surveillance state, exhibiting a small soma and long ramified processes, which are permanently moving and scouting the environment. Upon stimulation, these cells become reactive (phagocytic), proliferative, and mobile. Their branches retract, and they actively migrate to the lesion/infection site [149].

1.4.3 Ependymal cells

The ependyma is a ciliated epithelium located in the ventricular walls. In the adult CNS, their functions include support of the subventricular zone, barrier functions, and CSF production and movement induction. Thus, these cells play a role in neurogenesis, and in regulating the influx, outflux and movement of the CSF [150].

1.5 Technical approaches to study the nervous system

The study of natural lesions in humans (e.g., trauma, stroke) or experimental lesions in models was among the first strategies to probe the function of specific nervous system areas; the studies of Paul Broca are paradigmatic (see Introduction). The 21st-century neuroscientist has a wide range of pharmacological tools that allow to modulate the activity of specific areas or groups of cells while maintaining the structural integrity of the region of interest. Some of these molecules can have significant therapeutical interest. For instance, L-DOPA can be systemically administered with the aromatic amino acid decarboxylase inhibitor carbidopa. This adjuvant, which does not cross the blood-brain barrier, inhibits the peripheral conversion of L-DOPA into dopamine ensuring that it can reach the CNS, where (after conversion) it activates dopamine receptors (L-DOPA/carbidopa is used in the treatment of Parkinson’s disease). The development of chemo- and optogenetic tools further increased the ability to target brain circuits with great specificity [151–154]. In both cases, channels with specific properties are expressed in targeted neural populations. In chemogenetics, activation/inhibition is achieved by synthetic molecules as clozapine-n-oxide (CNO), typically administrated systemically at times of interest. In the case of optogenetics, receptors are activated by light of a specific wavelength, rather than a ligand. Both strategies provide an unprecedented specificity both at the cell/circuit level and, in the case of optogenetics, temporal precision which, when paired with behavioral analysis and electrophysiological recordings (see later), provide valuable information.

In parallel, knowledge on nervous system anatomy and cell biology was greatly influenced by the development of staining methods. The Golgi method was key in the establishment of modern neuroscience foundations, particularly with the Neuron Doctrine championed by Santiago Ramon y Cajal and others in the early 20th century (see Introduction). Developed by Camilo Golgi, the Golgi method, also known as la reazione nera (black reaction), is a silver staining that stains neurites with great detail, revealing the morphological heterogeneity of the neuron. Also, silver impregnation techniques have been used to