1.1 Building a Nervous System

Brain cells are eukaryotic cells

The first cell is thought to have arisen on earth at least 3.8 billion years ago. Today we classify cellular life into three domains: Bacteria, Archaea and Eukarya. All cells on this planet contain a barrier to the outside world called a cell membrane and genetic material (DNA). However, unlike prokaryotic cells, which are members of the Bacteria and Archaea domains, eukaryotic cells belong to the Eukarya domain which has as its major cellular feature membrane-bound organelles. Membrane-bound organelles allow for functional compartmentalization and specialization. DNA is segregated in the nucleus where it is copied (via a process called replication) and/or converted into mRNA (messenger RNA). Once converted (transcribed) into mRNA, this material exits the nucleus. It is outside of the nucleus that the translation of mRNA to protein occurs in eukaryotic cells Figure 1.2.

Figure 1.2 The Central Dogma

Brain cells, neurons and glia, also adhere to the characteristics specified above and are fundamentally just eukaryotic cells. However, neurons have additional features that make them uniquely specialized for communication, a feature which defines nervous systems. Before we get into the specifics of neurons and glia, let us establish a few fundamentals of eukaryotic cells.

Introduction to the central dogma: cell identity is determined by gene expression

The human body is thought to consist of trillions of cells. Because a single fertilized egg gives rise to all the cells in the adult body, every cell within an organism contains basically the same unique DNA genome. Yet, the human body is not made of identical cells. Depending on the organ system, cells differ in their shape, size, function, and modes of interaction. What makes a heart or liver cell different from a brain cell is not the DNA contained in its nucleus, which is identical in both. Rather, the difference is due to the selective process of transcription of DNA into RNA. Which parts of the DNA are read, and by extension which parts of the DNA are excluded, is the first step in making proteins. The chosen regions of DNA are transcribed into mRNA and translated into specific proteins.

One way to envision the genome is as a comprehensive cookbook containing all the written instructions of life and found in every cell. However, only certain recipes (DNA parts called genes) are chosen to ultimately be cooked (made into proteins) in different kitchens (different cells). Cell identity therefore arises from cell-specific gene choices. This is called gene regulation and is controlled by both intrinsic (within cells) and extrinsic factors (the environment that the cell matures in). For example, a neuron may require a specific channel protein that allows electrical communication while a pancreatic cell requires the expression of an insulin gene to regulate glucose levels in our body. Furthermore, different types of neurons found in different brain regions require different proteins to produce unique chemical messages known as neurotransmitters (e.g. dopamine versus acetylcholine). Proteins do all the work in our cells. This information flow of DNA to RNA to protein is called the central dogma. The central dogma is universal and is a feature of all living cells on our planet: DNA is transcribed into RNA which can be translated into proteins. The process of translation is facilitated by ribosomes which are large macromolecular complexes found floating inside cells or attached to organelles like the endoplasmic reticulum (see below).

Organelles: carry out functions of cells

Brain cells, like all eukaryotic cells contain a complement of organelles necessary for the proper cell function. This includes a nucleus (houses the DNA), endoplasmic reticulum, Golgi apparatus, lysosomes, mitochondria (singular is mitochondrion) and a few others (Figure 1.3). Cells store genetic material in their nucleus, transport materials across the cell membrane and within the cell, produce energy in the form of ATP, breakdown and build macromolecules, and communicate and interact with other cells. Organelles serve in these multiple functional roles. Of notable importance are the mitochondria, which facilitate most of the processes of cellular respiration that produce ATP. While the human brain only takes up about 2% of body mass, it uses about ~25% of daily energy production. Neurons can have up to 2 million mitochondria each and thus have the capability to produce a large amount of ATP. This high ATP demand is needed for the basic function of neurons and the communication between neurons (see Chapter 2 Neurophysiology).

Figure 1.3 Typical eukaryotic cell

Neurons

The human brain is made up of ~86 billion neurons. Neurons are highly specialized for communication that occurs via electrical and chemical signals. With some exceptions, neurons have special structural features that branch from the cell body (soma), called axons and dendrites (Figure 1.4). Functionally, neurons receive and integrate signals, and send information in both electrical and chemical forms. Neurons communicate with their partners at regions called synapses.

Figure 1.4 Basic neuronal structure

Neurons come in many shapes and sizes. When asked to describe a neuron, most students would draw a multipolar neuron containing one axon and several dendrites branching from the cell body (for example a motor neuron). This is the classic depiction found in the popular press and many biology texts. However, the nervous system contains other neuronal varieties, including bipolar neurons (two processes jetting off from the cell body; one dendrite and one axon; olfactory (smell) neuron being an example) and unipolar neurons (a single process from the cell body; dendrite and axon are continuous; often used for sending sensory information) (Figure 1.5). Neuronal shape and structure can inform function. For example, bipolar neurons are uniquely suited for providing a pathway between input and outputs in the visual system.

Figure 1.5 Neuron heterogeneity

Neuroscientists have used many lenses for classifying neurons, grouping them by size, shape, or the connections they make (e.g. whether those connections have predominantly inhibitory or excitatory effects). For example, afferent neurons (e.g., sensory neurons) carry messages towards the central nervous system (CNS). Efferent neurons (e.g., motor neurons) carry messages away from the CNS. Interneurons allow communication within the nervous system and are located between sensory and motor neurons. Classification of neurons remains difficult, though, in part because they are so dynamic. Huge efforts are underway to catalog all the different types of neurons in the brain

Whatever their morphology or function, neurons have all the typical eukaryotic organelles, all of which are found in the cell body, called the soma (Figure 1.6). Below, we will discuss in more detail the unique cellular features of neurons, using a common multipolar neuron depiction to help visualize them.

Figure 1.6 Parts of a neuron

Dendrites

Dendrites are branches/processes that extend from the cell bodies (or soma) of most neurons. The word dendrite comes from Greek and means tree. We often talk about neurons having tree-like branching, arborization (also a tree analogy). Functionally, dendrites are specialized for receiving information at a synapse and transferring that information toward the cell body. Dendritic branching can be extensive or can be more minimal depending on the type of neuron. In some animal nervous systems, dendrites are studded with bumps called dendritic spines, which provide specialized compartmentalization for synapses and, among other functions, seem to be important in learning and memory (Figure 1.7).

Figure 1.7 Dendritic spines Dendrites are covered in dendritic spines. Spines have many different shapes and sizes. They also change over time. Image credit: Platholi J, Herold KF, Hemmings HC Jr, Halpain S (2014) Isoflurane Reversibly Destabilizes Hippocampal Dendritic Spines by an Actin-Dependent Mechanism. PLoS ONE 9(7): e102978. https://doi.org/10.1371/journal.pone.0102978. CC BY 4.0.

Axons

A neuron can have thousands of dendrites, but only one axon. An axon is a branch that like dendrites, emerges from the soma or cell body of the neuron. At the point of connection with the soma, the axon region is called the axon hillock (Figure 1.6). Functionally, axons are specialized for sending information. Some cells, such as those in the retina, lack axons or have axons that are very hard to identify. An axon emerging from the soma can branch out at its terminal end to communicate with multiple cell partners. These branches are called axon collaterals and at the ends of these collaterals are axon terminals. Axons can vary in length depending on their location. For example, axons extending from the spinal cord to your toes are very long in comparison to axons found within the brain. Axons can be surrounded by a fatty substance called myelin, which helps insulate axons and allows more efficient/faster sending of signals over long distances in the body. Multiple sclerosis (MS) is an autoimmune disease where an individual’s immune system attacks myelin, causing neuronal communication slow-down and disruption. This leads to severe motor problems in the affected individual.

Not all axons in our nervous system are myelinated, especially those that travel shorter distances. Interestingly, there are many types of animals including some vertebrates that do not have myelin at all. Other biological adaptations, such as larger diameters of axons, are used in these cases to create efficient communication.

The Synapse

The synapse is the place where one neuron communicates with its partner. Neurons do not touch other neurons/cells directly, but rather interact via the space between them called a synaptic cleft. Neurons can form synapses not only with neurons, but also glands (neuroglandular junctions) and muscles (neuromuscular junctions). (Figure 1.8).

Figure 1.8 Types of synapses

In the case of neuronal synapses, axons can synapse with dendrites (axo-dendritic) (Figure 1.8), with other axons (axo-axonic) or with the soma (axo-somatic). The number of synapses that one neuron can participate in varies from tens to thousands. In chemical synapses, the presynaptic side of the synapse sends chemical signals (neurotransmitters) from one neuron to the postsynaptic side (membrane of the receiving cell). Most synapses are chemical synapses, but another type of synapse exists: the electrical synapse. It allows communication via channel proteins that physically connect adjacent cells and no neurotransmitter is utilized (see Chapter 2 Neurophysiology).

Glia

The name glia comes from the Greek word for ‘nerve glue’. Historically, these cells were first identified as connective cells that “glued” neurons together. However, over many years of research, glia have emerged as key players of the nervous system with a variety of sophisticated functions and not just nerve glue. Glia are non-neuronal cells in the nervous system that make up ~30-60% of the total brain mass. They are found in both the central and peripheral nervous systems. In many parts of the nervous system, they outnumber neurons. Glia are not thought to generate electrical signals, but function to support neuronal signaling (regulate the spread of signals in the brain). They can function to guide developing neurons to their proper destination. They can provide nutrients and other chemicals to the neurons that they surround. There is a rich and evolving literature around the interesting roles that glia play in the nervous system, but we will limit ourselves to a general overview. Below we will discuss the major types of glia: astrocytes, microglia, oligodendrocytes, and Schwann cells (Figure 1.9).

Figure 1.9 Types of glia and example functions

Astrocytes

Astrocytes have a characteristic star shape and represent the most abundant fraction of glial cell type in the adult human brain. Astrocytes have many functions, such as the maintenance of the blood-brain barrier. The blood-brain barrier (BBB) is a structure that filters chemicals and pathogens entering the brain. For example, the larger and less water soluble a molecule is, the less likely it is to cross this barrier (Figure 1.10). In addition, these glial cells provide neurons with metabolic and structural support and help modulate neuronal function in some cases. One very important role for astrocytes involves recycling neurotransmitters by reuptake back into neurons (Figure 1.10). Dysfunction of astrocytes has been associated with pathological conditions including epilepsy, brain tumors and neurodegenerative diseases like Alzheimer’s disease.

Figure 1.10 Astrocytic functions

Microglia

Microglia serve an immunological role in the nervous system and act like phagocytic cells. Phagocytes are cells that ingest foreign particles like bacteria, for example (see Chapter 17 Neuroimmunology). Microglia are named for being very small. They destroy invaders that get through the blood-brain barrier and then clear out damaged neurons and unused synapses and thus play an important role in synaptic pruning and elimination of extra synapses during brain development. Improper synaptic pruning may be involved in neuropsychiatric disorders such as autism and schizophrenia.

Oligodendrocytes

Myelin is the insulating axon wrapping made up of lipid and protein, and oligodendrocytes are the myelinating cells of the central nervous system. Each oligodendrocyte provides myelin for several neurons. The axon of one neuron can be myelinated by multiple oligodendrocytes. As you will see in later chapters, myelin wrapped around axons is necessary for rapid communication in the nervous system (see Chapter 2 Neurophysiology).

Schwann Cells

Schwann cells are the main glial cells of the peripheral nervous system and serve similar functions to oligodendrocytes. Unlike oligodendrocytes, one Schwann cell provides myelin for the axon of a single neuron (see Figure 1.9).

History of Neuroscience: Visualizing Brain Cells: from Cajal to Brainbow

Throughout this section, we have discussed the cellular components of the nervous system and the unique specializations that allow neurons to communicate with each other. In the late 1880s, a debate brewed in the field of neuroscience. Without sophisticated tools and relying on simply observing the human brain, it was almost impossible to ascertain its components. Camillo Golgi, a prominent scientist of his time and the inventor of the Golgi stain (see below), championed a brain model where the building material of the brain was a single, dense, intricate, and continuous network akin to a material like gauze. This was called the reticular theory. On the other side of the debate was another Spanish scientist, Ramon y Cajal. Based on his findings, Cajal championed the Neuron Doctrine—the idea that the brain was composed of individual, unconnected cells. Ultimately, Cajal’s theory proved to be correct. Our ability to visualize the brain has advanced and deepened our understanding of Neuroscience. Beginning with the Golgi stain, this section will outline some common approaches to visualizing brain cells (Figure 1.11).

Figure 1.11 Examples of neuronal stains Image credit: Golgi: By MethoxyRoxy, CC BY-SA 2.5, https://commons.wikimedia.org/w/index.php?curid=1325286. Immunofluorescent: By Wei-Chung Allen Lee, Hayden Huang, Guoping Feng, Joshua R. Sanes, Emery N. Brown, Peter T. So, Elly Nedivi - Dynamic Remodeling of Dendritic Arbors in GABAergic Interneurons of Adult Visual Cortex. Lee WCA, Huang H, Feng G, Sanes JR, Brown EN, et al. PLoS Biology Vol. 4, No. 2, e29. doi:10.1371/journal.pbio.0040029, CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=3482256. Brainbow: By Jeff W. Lichtman and Joshua R. Sanes - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2577038/, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=25703326

Chemical Stains: Focus on the Golgi Stain

The driver of scientific progress is often fueled by technical discoveries (a new instrument, a new technique) that allow scientists to visualize, analyze or probe. The Golgi stain (silver nitrate) was first discovered by Camillo Golgi as a way to visualize neurons in postmortem tissue. Its improvement and application to brain tissue by Ramon y Cajal in the late 1880s opened the door to our ability to see brain cells as individual entities for the first time. A unique feature of this technique is that, if applied to a forest of neurons, only a few random cells will become stained and stand out when visualized under a microscope. The reason for this selectivity is still unknown to this day. The neurons that do get stained become visible in a very complete way, allowing visualization of cell bodies/soma, dendrites, and axons (Figure 1.11).

Cajal’s work revolutionized the field of neuroscience. For the first time, it was clear that neurons were separated from other individual cells and that they were morphologically distinct in different brain regions. He used Golgi’s own methods to refute Golgi’s belief that brain cells were a continuous cellular web. Through his careful work, Cajal gained many additional insights, including the fact that neurons were polarized (receiving information on their dendrites and cell bodies and sending information via axons). The Golgi stain continues to be a valuable tool for modern day neuroscientists. In addition to the Golgi stain, we have a number of chemical stains that can be used to visualize neural tissue, including but not limited to Nissl and H&E (Hematoxylin and Eosin) staining for cell bodies and nuclei and Luxol fast blue for myelin.

Immunofluorescence

All the chemical stains mentioned above allow scientists to visualize parts of a neuron such as a nucleus, cell body or processes. However, as we learned in the first section of this chapter, the types of genes expressed within a cell dictate its function. If a scientist is interested in determining the distribution of a specific protein (like an enzyme that produces a certain type of neurotransmitter) or the presence of myelin proteins, a more specific method of visualization is necessary. Immunofluorescence is a technique that uses antibodies (designed against a specific protein of interest) equipped with fluorescent tags (which come in many different colors) to localize and visualize a protein of interest using a fluorescent microscope (Figure 1.11) (see Methods: Immunohistochemistry). There are well established antibodies that can be used as neuronal cell markers that allow scientists to distinguish neurons from glia, visualize synaptic connections, measure protein expression levels in different neurons, etc. We have the technology to generate antibodies to almost any protein that we are interested in studying, giving scientists a large toolkit to answer basic questions in neuroscience.

GFP Labeling/Brainbow

Following its discovery in the 1960s, green fluorescent protein (GFP) has been used to label cells, organelles and specific proteins. Because we know the exact DNA sequence that codes for the green fluorescent protein and because all living cells on this planet use the same genetic code (see central dogma above), we can insert the GFP DNA directly or attach it to any sequence that we may want to express in a cell. Once that DNA is taken up and expressed, the cell will glow green wherever the protein is found in the cell. The GFP DNA can be introduced into cells grown in culture or brain slices using specialized delivery tools like viruses. The GFP DNA can also be genetically engineered into gametes (reproductive cell of an animal or plant) prior to fertilization, which leads to GFP transgenic mice allowing visualization of specific regions in the brain throughout the lifetime of an animal. Beyond just green, genetic manipulations of the GFP DNA have been developed to introduce the genetic machinery to randomly mix colors (such as green, cyan and yellow fluorescent proteins) in individual neurons thereby creating a palette of different color combinations and shades, allowing visualization of whole neuronal circuits. This process produces a Brainbow, where each individual neuron stands out due to a different shade of color (Figure 1.11).