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Relationship between nerves and axons

Relationship between nerves and axons


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I just wanted to get a realistic viewpoint of our nervous system. I understand arteries and veins, but I wanted to know how similar our nervous system is to that?

I understand we have neurons (please correct me if I am wrong) all over the surface of body. Whenever we feel a touch a neuron fires a response, and that response travels through axons (myelin sheath).

My main question is what a nerve exactly is. Is it a long axon? How many axons (same thing as neuron body?) are in a nerve? I am sure it depends on different nerves.


I will go through your list of questions below:

  • I wanted to know how similar is our nervous system to [the circulatory system]?
    They are very different, but as in every comparison of very complex systems, there is some overlap. The circulatory system carries fluids, the nervous system electrical signals so they are functionally not alike. However, both systems run throughout the body and have a more or less central control unit (the brain and heart respectively). So there is a structural similarity. I reckon they are as much alike as a city's sewer system and electricity grid.

  • I understand we have neurons (please correct me if I am wrong) all over the surface of body.
    That is correct

  • Whenever we feel a touch (stimulus arises) a neuron fires a response, and that response travels through axons (myelin sheath).
    Tactile stimulation may result in firing of one or more neurons, dependent on the intensity of the stimulus. Larger stimuli will, obviously, recruit more fibers. Stronger stimuli will evoke stronger responses (increased firing). Axons indeed conduct the neuronal responses, like an electrical wire conducts electrical current. Longer axons are often myelinated (insulated). Not all neurons related to touch are myelinated, however.

  • So my main question is that what exactly is a nerve?
    A nerve is a bundle of axons that carry related functional information. Typically, nerves conduct information originating from locations closely located together in the body, and generally they convey this information to a localized spot in the body as well. For example, the optic nerve carries information from the photoreceptors (related information) from the retina (localized source) to the brainstem (localized target).

  • Is [a nerve] a long axon?
    A nerve contains many axons. 'Long' or 'short' is rather subjective.

  • How many axons (same thing as neuron body?) are in a nerve? I am sure it depends on different nerves.
    Neuronal cell bodies are typically located outside a nerve. Nerves contain varying numbers of fibers. For example, the auditory nerve harbors the axons of the spiral ganglion cells that transmit auditory information from the inner ear to the brain. It contains between 31k - 32k (myelinated) fibers in normal-hearing humans (Spoendlin & Schrott, 1989). In contrast, the optic nerve, which carries visual information from the retina to the brain, contains some 1.3 million (myelinated) fibers in young human adults (Jonas et al., 1992)

References
- Jonas et al., IOVS 1992;33:2012-8
- Spoendlin & Schrott, Hear Res 1989;43:25-38


Biology and Behavior

Plays a role in Pain relief and response to stress. This neurotransmitter also regulates eating behavior.

  • a. Neurotransmitters allow impulses to flow from one neuron to another.
  • b. Neurotransmitters prevent impulses from flowing from one neuron to another.
  • c. Neurotransmitters are stored in the cell bodies of neurons.
  • d. Each neurotransmitter is associated with a unique receptor.
  • e. Unused neurotransmitter are recycled by neurons and used again.

Plays a central role in learning of fear responses.

  • C convoluted covering of the cerebrum
  • A membrane that connects the cerebral hemispheres
  • B right and left halves of the cerebrum

Which is the best description of the language abilities of a person who suffers damage to Broca's area?

a. They cannot understand spoken language.
b. They can only understand the speech of others who speak very slowly.
c. Their speech cannot be understood by others.
d. They must put forth a great deal of effort to speak.


Type of Neurons

Sensory neurons

These run from the various types of stimulus receptors, e.g., to the central nervous system (CNS), the brain and spinal cord.
Link to CNS

The cell bodies of the sensory neurons leading to the spinal cord are located in clusters, the dorsal root ganglia (DRG), next to the spinal cord. Their axon extends in both directions: a peripheral axon to receptors at the periphery and a central axon passing into the spinal cord. The latter axon usually terminates at an interneuron.

The diagram is a simplified view of the relationship between sensory and motor neurons running to and from the spinal cord.

Interneurons

Interneurons are also called association neurons.

It is estimated that the human brain contains 100 billion (10 11 ) interneurons averaging 1000 synapses on each that is, some 10 14 connections.

The term interneuron hides a great diversity of structural and functional types of cells. In fact, it is not yet possible to say how many different kinds of interneurons are present in the human brain. Certainly hundreds perhaps many more.

Motor neurons

Most motor neurons are stimulated by interneurons, although some are stimulated directly by sensory neurons.

Synapses

Differentiation: one axon but many dendrites

Recent work (Shelly, M., et al., Science, 327:547, 29 January 2010) has provided a clue to the mechanism by which a neuron precursor cell develops only one axon but many dendrites. Working with isolated hippocampal neuron precursors (from rat embryos), these workers showed that the cyclic nucleotide cAMP accumulates at one spot on the developing neuron, and it is here that the axon sprouts. However, the rising level of cAMP at that spot suppresses cAMP elsewhere in the cell, allowing the related cyclic nucleotide, cGMP, to accumulate. cGMP drives the formation of dendrites.

Still to be discovered is the signal (or signals) that cause the localized accumulation of cAMP at one spot in the cell. Perhaps it is a cell-intrinsic signal (see other examples), or a cell-extrinsic signal (see other examples), or perhaps both.


Myelin facilitates conduction

Myelin is an electrical insulator however, its function of facilitating conduction in axons has no exact analogy in electrical circuitry. In unmyelinated fibers, impulse conduction is propagated by local circuits of ion current that flow into the active region of the axonal membrane, through the axon and out through adjacent sections of the membrane (Fig. 4-1). These local circuits depolarize the adjacent piece of membrane in a continuous, sequential fashion. In myelinated axons, the excitable axonal membrane is exposed to the extracellular space only at the nodes of Ranvier this is the location of sodium channels [2]. When the membrane at the node is excited, the local circuit generated cannot flow through the high-resistance sheath and, therefore, flows out through and depolarizes the membrane at the next node, which might be 1 mm or farther away (Fig. 4-1). The low capacitance of the sheath means that little energy is required to depolarize the remaining membrane between the nodes, which results in local circuit spreading at an increased speed. Active excitation of the axonal membrane jumps from node to node this form of impulse propagation is called saltatory conduction (Latin saltare, “to jump”). Such movement of the wave of depolarization is much more rapid than in unmyelinated fibers. Furthermore, because only the nodes of Ranvier are excited during conduction in myelinated fibers, Na + flux into the nerve is much less than in unmyelinated fibers, where the entire membrane is involved. An example of the advantage of myelination is obtained by comparison of two different nerve fibers, both of which conduct at 25 m/sec at 20ଌ. The 500-mm diameter unmyelinated giant axon of the squid requires 5,000 times as much energy and occupies about 1,500 times as much space as the 12-mm diameter myelinated nerve in the frog.

Figure 4-1

Impulse conduction in unmyelinated (top) and myelinated (bottom) fibers. Arrows show the flow of action currents in local circuits into the active region of the membrane. In unmyelinated fibers, the circuits flow through the adjacent piece of membrane, (more. )

Conduction velocity in myelinated fibers is proportional to the diameter, while in unmyelinated fibers it is proportional to the square root of the diameter. Thus, differences in energy and space requirements between the two types of fiber are exaggerated at higher conduction velocities. If nerves were not myelinated and equivalent conduction velocities were maintained, the human spinal cord would need to be as large as a good-sized tree trunk. Myelin, then, facilitates conduction while conserving space and energy [3].


Axon degeneration: Linking axonal bioenergetics to myelin

Bogdan Beirowski, Elisabetta Babetto, Lawrence Wrabetz Axon degeneration: Linking axonal bioenergetics to myelin. J Cell Biol 21 November 2016 215 (4): 437–440. doi: https://doi.org/10.1083/jcb.201611010

The mechanisms by which axonal degeneration occurs, even in the presence of apparently normal myelin sheaths, remain unknown. In this issue, Yin et al. (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201607099) study mutant mice in which proteolipid protein is replaced by the peripheral myelin protein P0 and describe a number of early axonal abnormalities, which together suggest that aberrant mitochondrial energy metabolism precedes axonal degeneration.

Axons, the long cellular projections of neurons extending from the cell body all the way to the distal synapse, are essential for neuronal wiring. This arrangement is facilitated by the intimate association of axons with Schwann cells or oligodendrocytes. These glial cells form compact myelin sheaths around axons in the peripheral nervous system (PNS) or the central nervous system (CNS). Myelin sheaths enable saltatory, and therefore greatly accelerated, conduction of electrical impulses and have been proposed to support axonal integrity. Still, because of their incredible length—several meters for certain axons in larger vertebrate species—axons are at continuous risk of damage. Axons also have an extraordinary demand for adenosine triphosphate to support intensive energy-consuming processes, including axonal transport and generation of ion gradients. The question of how these seemingly fragile cellular processes are maintained throughout life has puzzled neuroscientists for many decades.

Even focal axonal damage in the CNS can result in irreversible interruption of axonal continuity and produce impairment of fundamental neuronal functions such as sensation, ambulation, memory, and cognition. In fact, axon degeneration is an early event and pathological hallmark in a broad range of acquired and hereditary neurodegenerative disorders, especially those primarily affecting the myelin sheaths of axons (Taveggia et al., 2010 Beirowski, 2013). Moreover, the demise of axons and not the degeneration of myelin in these conditions is the most important predictor of morbidity, despite that demyelination can be the most prominent histopathological feature. This is probably most pronounced in multiple sclerosis, where there is significant evidence that axon degeneration may sometimes parallel or even precede the onset of demyelination (Trapp and Nave, 2008).

It is intuitive that the immune-mediated attack on the myelin sheath may result in neurotoxicity harmful for axons. However, it is now also clear that axon degeneration can occur in the absence of demyelination when individual molecules in myelin sheaths are missing (Nave and Trapp, 2008). For example, deletion of the proteolipid protein (PLP) from myelin sheaths in the CNS leads to a progressive axonopathy in both humans and rodent models (Garbern et al., 2002). Mice deficient for PLP display grossly normal myelin sheaths that are slightly thin and uncompacted. Nonetheless, they subsequently develop a late-onset axonopathy mimicking the loss of long CNS axons that occurs in patients suffering from hereditary spastic paraplegia type 2 (HSP2) or the leukodystrophy Pelizaeus-Merzbacher disease. Both neurodegenerative conditions are caused by mutations in the PLP1 gene coding for PLP.

Further support for dissociation of axonal degeneration from changes in myelin comes from mutant mice, P0-CNS, in which the myelin compaction defect secondary to PLP ablation has been largely rescued. Although the functionally related P0 glycoprotein is expressed in Schwann cells in the PNS, these transgenic mice also express P0 in oligodendrocytes (Yin et al., 2006). Similar to PLP in the CNS, P0 promotes membrane compaction in myelin sheaths in the PNS. Strikingly, P0-CNS mice, which are the subject of study in this issue by Yin et al. show accelerated axonopathy in the form of axonal transport deficits and axonal swellings, despite the stabilization of myelin (Yin et al., 2006). These findings support the model that PLP exerts axon-supportive functions independent from myelination, and those functions cannot be substituted by the related protein, P0.

What is the etiology of axonal degeneration in mutant oligodendrocytes or their PLP-deficient myelin sheaths? To answer this question, one needs to expand the traditional view of glia as mere myelin insulators of axons. Several recent studies have provided evidence for another key function of myelinating glia: metabolic support of axons. According to this concept, metabolic deficits in Schwann cells or oligodendrocytes, or impaired metabolite transport, are thought to account for axonal degeneration (Lee et al., 2012 Beirowski et al., 2014). In fact, energy-rich products of glycolysis (pyruvate) or fermentation (lactate) in oligodendrocytes appear to support CNS axons when shuttled into them via monocarboxylate transporters (Fünfschilling et al., 2012 Lee et al., 2012). Modeled after intercellular lactate shuttling proposed to operate in various tissues, these intermediates may then be used by axonal mitochondria for ATP production.

Impairment of metabolic exchange between axons and glia in mice lacking a single myelin protein like PLP could suggest a direct role for the myelin protein in metabolic coupling, or could be an indirect effect of the myelin protein loss of function. In this respect, it is important to note that compact myelin sheaths contain an elaborate system of channel-like structures (broadly known as noncompact myelin), which contain conduits of glial cytoplasm that are likely essential for conveying metabolites from glia to axons. In addition, these channels are also likely required for transport of the molecular machinery that enables metabolite exchange at axo–glial junctions (i.e., monocarboxylate transporters). Indeed, based on indirect experimental evidence, abnormalities in these structures have been hypothesized in PLP-deficient mice (Edgar and Nave, 2009). Moreover, P0-CNS white matter fibers show substantially shorter myelin sheath internodes and formation of altered noncompact myelin in the form of Schmidt-Lanterman incisures that are normally formed exclusively in the PNS (Yin et al., 2006, 2008). Thus, it is tempting to hypothesize that the metabolic exchange system between oligodendrocytes and axons may be compromised in P0-CNS mutants, resulting in deficient axonal bioenergetics.

Such bioenergetic deficits might be expected to manifest early as attenuated axonal transport resulting in organelle accumulation at juxtaparanodal axon sites. This is because “slow” organelles would not be able to pass the narrow nodes of Ranvier (Fig. 1 constriction is exaggerated for clarity). Accumulation of organelles including mitochondria would then lead to axonal swelling because of the space restraints. Indeed, such pathological features have been well documented in PLP-deficient mice as well as in P0-CNS mutants (Griffiths et al., 1998 Edgar et al., 2004 Yin et al., 2006). Notably, this pattern of axon pathology is remarkably similar to the axonal swellings in CNS axons undergoing an energetic crisis during early Wallerian degeneration (a program of axonal autodestruction) after physical separation from the neuronal cell body (Beirowski et al., 2010).

Yin et al. (2016) do not address compromised axon–glia communication but focus on the downstream bioenergetic consequences in axons resulting from this putative impairment of metabolic coupling in P0-CNS mice. Applying elegant serial block-face EM (SBEM) reconstructions of optic nerves, which enables 3D assessment of cytoskeleton and organelle shape and distribution, the authors demonstrated marked mitochondrial accumulations in swollen juxtaparanodal axon segments of 1-mo-old P0-CNS mice. At this age, only a small amount of axonal degeneration is observed in P0-CNS optic nerves. The authors found that the increase in mitochondrial content was caused by elevated mitochondrial numbers accompanied by drastic changes in mitochondrial shape with a shift toward a much more rounded appearance and reduced volume of individual mitochondria. Moreover, they observed alterations in the ultrastructure of the mitochondrial matrix. These changes were accentuated in the distal juxtaparanodal axon segment (on the opposite side of each node of Ranvier from the cell body), suggesting a potential problem of retrograde axonal transport (Griffiths et al., 1998). Together, these results suggested altered mitochondrial motility and structural adaptations at sites of energetic depletion in mutant axons.

To correlate these static observations with direct assessment of mitochondrial dynamics, Yin et al. (2016) subsequently performed time-lapse imaging of mitochondrial transport in P0-CNS Purkinje cell axons using cerebellar organotypic slice cultures. The mean velocity of the motile mitochondrial fraction was severely reduced in P0-CNS preparations as compared with controls with an accentuation of retrograde transport deficits. This resulted in a preferential standstill of mitochondrial movement at the distal juxtaparanodal region in P0-CNS fibers, thus explaining the organelle accumulations at these sites. These results are reminiscent of the deficits observed in PLP-deficient mice in which retrograde transport in optic nerve axons has been studied by measurements of dynein/dynactin levels and injections of cholera toxin B (CTB Edgar et al., 2004).

Are these abnormalities in mitochondrial motility associated with reduced axonal energy content as a prelude to axonal degeneration? To answer this, Yin et al. (2016) used a functional approach. Electrophysiological activity analysis as a surrogate for axonal energy content after induction of metabolic stress (oxygen-glucose deprivation) demonstrated that optic nerve axons from 1-mo-old P0-CNS mice showed an accelerated loss of electrical function and recovered more slowly after stress. This can be best explained by a reduced ATP content in P0-CNS axons. In agreement, the authors showed reduced ATP concentrations in optic nerve extracts using a bioluminescence assay. In the future, it will be interesting to study ATP levels specifically in the axonal compartment within this model.

To further probe the mechanisms for compromised mitochondrial dynamics, Yin et al. (2016) next studied the ultrastructural organization and biochemical features of microtubules, the tracks that carry mitochondria as they move along axons. No loss of microtubules or abnormalities in their orientations along axons could be detected in optic nerves from young P0-CNS mutants. However, in 6-mo-old mutants, an age with more prominent axonal degeneration, microtubule orientation at paranodes/juxtaparanodes was disorganized, and microtubule length was significantly shorter as visualized by EM tomography. This was paralleled by abnormal acetylation of α-tubulin, which, together with β-tubulin, are the polymerizing constituents of microtubules. Tubulin acetylation is considered a marker for microtubule stability and regulates the anchoring of molecular motors for mitochondria. To test whether the microtubule binding protein, tau, participates in these abnormalities, Yin et al. (2016) studied its phosphorylation status in P0-CNS samples and found prominent hyperphosphorylation, likely mediated by stimulation of glycogen synthase 3 (GSK3) signaling. Tau has a critical role in the regulation of microtubule dynamics, and also binds to motor proteins. Collectively, these data suggest early energetic depletion in P0-CNS axons that leads to later disruption of the axonal cytoskeleton and their associated motors with involvement of upstream regulators of microtubule stability. Future studies will be needed to address the mechanistic relationship between axonal energy status and features such as microtubule acetylation, its polymerization, and the association with tau and motor proteins.

The abnormal mitochondrial dynamics raised the possibility that the interaction of mitochondria with other organelles in the axon is compromised. The tethering of mitochondria to specialized sites of the ER (i.e., the mitochondria-associated ER membrane [MAM]) plays a significant role for the maintenance of intracellular calcium homeostasis. Importantly, intraaxonal calcium overload is a convergent step of distinct pathways that lead to axon degeneration (Beirowski et al., 2010). Yin et al. (2016) extended their beautiful SBEM reconstructions to visualize MAM associations with a novel kind of mitochondrial outer membrane extension and found an 86% reduction in the associations between mitochondrial membranes and the axonal smooth ER (SER) tubular system in axons from 1-mo-old P0-CNS optic nerves. Furthermore, they noted a rather fragmented geometry of the SER tubules in P0-CNS axons. Thus, it is likely that calcium homeostasis is perturbed in P0-CNS axons, although this aspect was not examined in this study.

Altogether, these results from a mouse model with grossly normal myelin, yet accelerated axonal degeneration, begin to shed light on how bioenergetic deficits can elicit complex alterations in axonal structure, ultimately leading to axonal loss. This paper also has important implications for our understanding of disease pathogenesis. Because the features of damage in P0-CNS axons are very similar to those of older PLP-null mice (Griffiths et al., 1998 Edgar et al., 2004 Yin et al., 2006, 2016), the mechanisms of axonal degeneration in P0-CNS mice are likely to be relevant to the pathogenesis of HSP2. Confirmation in older PLP-null optic nerves of reduced ATP concentration, microtubular disorganization, and altered MAM associations will strengthen the relevance. In addition, future studies are needed to demonstrate that P0-CNS axons are indeed deprived of metabolic support by their oligodendrocytes. Although a formidable task, conducting careful structural and functional studies of axon–glia communication through noncompact myelin sites in this model may be a viable starting point. The results will provide a mechanistic framework integrating both glial and axonal energy metabolism and may reveal therapeutic targets for the many neurodegenerative conditions with prominent axonal degeneration.


How Nerves Work

Consider this. You touch a hot object and immediately drop it or withdraw your hand from the heat source. You do this so quickly you don't even think about it. How does this happen? Your nervous system coordinated everything. It sensed the hot object and signaled your muscles to let it go. Your nervous system, which consists of your brain, spinal cord, peripheral nerves and autonomic nerves, coordinates all movements, thoughts and sensations that you have. In this article, we'll examine the structure and functions of your nervous system, how nerve cells communicate with each other and various tissues and what can go wrong when nerves become damaged or diseased.

  • Senses your external and internal surroundings
  • Communicates information between your brain and spinal cord and other tissues
  • Coordinates voluntary movements
  • Coordinates and regulates involuntary functions like breathing, heart rate, blood pressure and body temperature.

The brain is the center of the nervous system, like the microprocessor in a computer. The spinal cord and nerves are the connections, like the gates and wires in the computer. Nerves carry electrochemical signals to and from different areas of the nervous system as well as between the nervous system and other tissues and organs. Nerves are divided into four classes:

  1. Cranial nerves connect your sense organs (eyes, ears, nose, mouth) to your brain
  2. Central nerves connect areas within the brain and spinal cord
  3. Peripheral nerves connect the spinal cord with your limbs
  4. Autonomic nerves connect the brain and spinal cord with your organs (heart, stomach, intestines, blood vessels, etc.)

The central nervous system consists of the brain and spinal cord, including cranial and central nerves. The peripheral nervous system consists of the peripheral nerves, and the autonomic nervous system is made of autonomic nerves. Fast reflexes, like removing your hand quickly from a heat source, involve peripheral nerves and the spinal cord. Thought processes and autonomic regulation of your organs involve various parts of the brain and are relayed to the muscles and organs through the spinal cord and peripheral/autonomic nerves.

The Spinal Cord and Neurons

The spinal cord extends through hollow openings in each vertebra in your back. It contains various nerve cell bodies (gray matter) and nerve processes or axons (white matter) that run to and from the brain and outward to the body. The peripheral nerves enter and exit through openings in each vertebra. Within the vertebra, each nerve separates into dorsal roots (sensory nerve cell processes and cell bodies) and ventral roots (motor nerve cell processes). The autonomic nerve cell bodies lie along a chain that runs parallel with the spinal cord and inside the vertebrae, while their axons exit in the spinal nerve sheaths.

Nerve Cells

The brain, spinal cord and nerves consist of more than 100 billion nerve cells, called neurons. Neurons gather and transmit electrochemical signals. They have the same characteristics and parts as other cells, but the electrochemical aspect lets them transmit signals over long distances (up to several feet or a few meters) and pass messages to each other.

Neurons have three basic parts:

  • Cell body: This main part has all of the necessary components of the cell, such as the nucleus (which contains DNA), endoplasmic reticulum and ribosomes (for building proteins) and mitochondria (for making energy). If the cell body dies, the neuron dies. Cell bodies are grouped together in clusters called ganglia, which are located in various parts of the brain and spinal cord.
  • Axons: These long, thin, cable-like projections of the cell carry electrochemical messages (nerve impulses or action potentials) along the length of the cell. Depending upon the type of neuron, axons can be covered with a thin layer of myelin, like an insulated electrical wire. Myelin is made of fat, and it helps to speed transmission of a nerve impulse down a long axon. Myelinated neurons are typically found in the peripheral nerves (sensory and motor neurons), while nonmyelinated neurons are found within the brain and spinal cord.
  • Dendrites or nerve endings: These small, branchlike projections of the cell make connections to other cells and allow the neuron to talk with other cells or perceive the environment. Dendrites can be located on one or both ends of the cell.

Neurons come in many sizes. For example, a single sensory neuron from your fingertip has an axon that extends the length of your arm, while neurons within the brain may extend only a few millimeters. Neurons have different shapes depending on what they do. Motor neurons that control muscle contractions have a cell body on one end, a long axon in the middle and dendrites on the other end sensory neurons have dendrites on both ends, connected by a long axon with a cell body in the middle.

Neurons also vary with respect to their functions:

  • Sensory neurons carry signals from the outer parts of your body (periphery) into the central nervous system.
  • Motor neurons (motoneurons) carry signals from the central nervous system to the outer parts (muscles, skin, glands) of your body.
  • Receptors sense the environment (chemicals, light, sound, touch) and encode this information into electrochemical messages that are transmitted by sensory neurons.
  • Interneurons connect various neurons within the brain and spinal cord.

In peripheral and autonomic nerves, the axons get bundled into groups, based on where they're coming from and going to. The bundles are covered by various membranes (fasciculi). Tiny blood vessels travel through the nerves to supply the tissues with oxygen and remove waste. Most peripheral nerves travel near major arteries deep within limbs and close to the bones.

Next, we'll learn about neural pathways.

Neural Pathways and Action Potentials

Neural pathways

The simplest type of neural pathway is a monosynaptic (single connection) reflex pathway, like in the knee-jerk reflex. When the doctor taps a certain spot on your knee with a rubber hammer, receptors send a signal into the spinal cord through a sensory neuron. The sensory neuron passes the message to a motor neuron that controls your leg muscles. Nerve impulses travel down the motor neuron and stimulate the appropriate leg muscle to contract. Nerve impulses also travel to the opposing leg muscle to inhibit contraction so that it relaxes (this pathway involves interneurons). The response is a quick muscular jerk that does not involve your brain. Humans have lots of hardwired reflexes like this, but as tasks become more complex, the pathway "circuitry" gets more complicated and the brain gets involved.

We have talked about nerve signals and mentioned that they are electrochemical in nature, but what does that mean?

To understand how neurons transmit signals, we must first look at the structure of the cell membrane. The cell membrane is made of fats or lipids called phospholipids. Each phospholipid has an electrically charged head that sticks near water and two polar tails that avoid water. The phospholipids arrange themselves in a two-layer lipid sandwich with the polar heads sticking into water and the polar tails sticking near each other. In this configuration, they form a barrier that separates the inside of the cell from the outside and that does not permit water-soluble or charged particles (like ions) from moving through it.

So how do charged particles get into cells? We'll find out on the next page.

Because ions are charged and water-soluble, they must move through small tunnels or channels (specialized proteins) that span the cell membrane's lipid bilayer. Each channel is specific for only one type of ion. There are specific channels for sodium ions, potassium ions, calcium ions and chloride ions. These channels make the cell membrane selectively permeable to various ions and other substances (like glucose). The selective permeability of the cell membrane allows the inside to have a different composition than the outside.

For the purposes of nerve signals, we are interested in the following characteristics:

  • The outside fluid is rich in sodium, a concentration about 10 times higher than the inside fluid
  • The inside fluid is rich in potassium, a concentration about 20 times higher inside the cell than outside.
  • There are large negatively charged proteins inside the cell that are too big to move across the membrane. They give the inside of the cell a negative electrical charge compared to the outside. The charge is about 70 to 80 millivolts (mV) -- 1 mV is 1/1000th of a volt. For comparison, the charge in your house is about 120 V, about 1.2 million times more.
  • The cell membrane is slightly "leaky" to sodium and potassium ions, so a sodium-potassium pump is located in the membrane. This pump uses energy (ATP) to pump sodium ions from the inside to the outside and potassium ions from the outside to the inside.
  • Because sodium and potassium ions are positively charged, they carry tiny electrical currents when they move across the membrane. If sufficient numbers move across the membrane, you can measure the electrical currents.

When nerves grow, they secrete a substance called nerve growth factor (NGF). NGF attracts other nerves nearby to grow and establish connections. When peripheral nerves become severed, surgeons can place the severed ends near each other and hold them in place. The injured nerve ends will stimulate the growth of axons within the nerves and establish appropriate connections. Scientists don't entirely understand this process.

For unknown reasons, nerve regeneration appears most often in the peripheral and autonomic nervous systems but seems limited within the central nervous system. However, some regeneration must be able to occur in the central nervous system because some spinal cord and head trauma injuries show some degree of recovery.

The nerve signal, or action potential, is a coordinated movement of sodium and potassium ions across the nerve cell membrane. Here's how it works:

  1. As we discussed, the inside of the cell is slightly negatively charged (resting membrane potential of -70 to -80 mV).
  2. A disturbance (mechanical, electrical, or sometimes chemical) causes a few sodium channels in a small portion of the membrane to open.
  3. Sodium ions enter the cell through the open sodium channels. The positive charge that they carry makes the inside of the cell slightly less negative (depolarizes the cell).
  4. When the depolarization reaches a certain threshold value, many more sodium channels in that area open. More sodium flows in and triggers an action potential. The inflow of sodium ions reverses the membrane potential in that area (making it positive inside and negative outside -- the electrical potential goes to about +40 mV inside)
  5. When the electrical potential reaches +40 mV inside (about 1 millisecond later), the sodium channels shut down and let no more sodium ions inside (sodium inactivation).
  6. The developing positive membrane potential causes potassium channels to open.
  7. Potassium ions leave the cell through the open potassium channels. The outward movement of positive potassium ions makes the inside of the membrane more negative and returns the membrane toward the resting membrane potential (repolarizes the cell).
  8. When the membrane potential returns to the resting value, the potassium channels shut down and potassium ions can no longer leave the cell.
  9. The membrane potential slightly overshoots the resting potential, which is corrected by the sodium-potassium pump, which restores the normal ion balance across the membrane and returns the membrane potential to its resting level.
  10. Now, this sequence of events occurs in a local area of the membrane. But these changes get passed on to the next area of membrane, then to the next area, and so on down the entire length of the axon. Thus, the action potential (nerve impulse or nerve signal) gets transmitted (propagated) down the nerve cell.­

There are a few things to note about the propagation of the action potential.

­When an area has been depolarized and repolarized and the action potential has moved on to the next area, there is a short period of time before that first area can be depolarized again (refractory period). This refractory period prevents the action potential from moving backward and keeps everything moving in one direction.

  • The action potential is an "all-or-none" response. Once the membrane reaches a threshold, it will depolarize to +40 mV. In other words, once the ionic events are set in motion, they will continue until the end.
  • These ionic events occur in many excitable cells besides neurons (like muscle cells).
  • Action potentials are propagated rapidly. Typical neurons conduct at 10 to 100 meters per second. Conduction speed varies with the diameter of the axon (larger = faster) and the presence of myelin (myelinated = faster). The rapid nerve conductions throughout the neural circuitry enable you to respond to stimuli in fractions of a second.
  • The channels can be poisoned and prevented from opening. Various toxins (puffer fish toxin, snake venom, scorpion venom) can prevent specific channels from opening and distort the action potential or prevent it from happening altogether. Similarly, many local anesthetics (e.g. lidocaine, novocaine, benzocaine) can prevent action potentials from being propagated in the nerve cells in an area and temporarily prevent you from feeling pain.
  • The propagation of the action potential is also sensitive to temperature in experimental settings. Colder temperatures slow down the action potential, but this usually doesn't happen in an individual. However, you can use cold-block techniques to temporarily anesthetize an area (like putting ice on an injured finger).

So, if the size of the action potential does not vary, how does an action potential code information? Information is encoded by the frequency of action potentials, much like FM radio. A small stimulus will initiate a low frequency train of a few action potentials. As the intensity of the stimulus increases, so does the frequency of action potentials.

On the next page, we'll learn about how nerves communicate with each other.

Like wires in your home's electrical system, nerve cells make connections with one another in circuits called neural pathways. Unlike wires in your home, nerve cells do not touch, but come close together at synapses. At the synapse, the two nerve cells are separated by a tiny gap, or synaptic cleft. The sending neuron is called the presynaptic cell, while the receiving one is called the postsynaptic cell. Nerve cells send chemical messages with neurotransmitters in a one-way direction across the synapse from presynaptic cell to postsynaptic cell.

Let's look at this process in a neuron that uses the neurotransmitter serotonin:

  1. The presynaptic cell (sending cell) makes serotonin (5-hydroxytryptamine, 5HT) from the amino acid tryptophan and packages it in vesicles in its end terminals.
  2. An action potential passes down the presynaptic cell into its end terminals.
  3. Serotonin passes across the synaptic cleft, binds with special proteins called receptors on the membrane of the postsynaptic cell (receiving cell) and sets up a depolarization in the postsynaptic cell. If the depolarizations reach a threshold level, a new action potential will be propagated in that cell. Some neurotransmitters cause the postsynaptic cell to hyperpolarize (the membrane potential becomes more negative, which would inhibit the formation of action potentials in the postsynaptic cell). Serotonin fits with its receptor like a lock and key.
  4. The remaining serotonin molecules in the cleft and those released by the receptors after use get destroyed by enzymes in the cleft (monoamine oxidase (MAO), catechol-o-methyl transferase (COMT)). Some get taken up by specific transporters on the presynaptic cell (reuptake). In the presynaptic cell, MAO and COMT destroy the absorbed serotonin molecules. This enables the nerve signal to be turned "off" and readies the synapse to receive another action potential.
  5. There are several types of neurotransmitters besides serotonin, including acetylcholine, norepinephrine, dopamine and gamma-amino butyric acid (GABA). Any given neuron produces only one type of neurotransmitter. Any one nerve cell may have synapses on it from excitatory presynaptic neurons and from inhibitory presynaptic neurons. In this way, the nervous system can turn various cells (and subsequent neural pathways) "on" and "off." Finally, nerve cells synapse on effector cells (muscles, glands, etc.) to evoke or inhibit responses.

Next, we'll learn about the different types of sensory neurons.

Neurological activity is an important phase in coordinating digestion. Neurobiologist Dr. Michael Gershon of Columbia University has written about a layer of 100 billion nerve cells in the stomach. This "second brain" coordinates digestion, works with the immune system to protect you from harmful bacteria in the gut, uses the neurotransmitter serotonin and may be implicated in irritable bowel syndrome and feelings of anxiety (like butterflies in your stomach) [source: Psychology Today].

The nervous system has many types of sensory neurons. Nerve endings on one end of each neuron are encased in a special structure to sense a specific stimulus.

  • Chemoreceptors sense chemicals. The olfactory bulb that monitors your sense of smell has chemoreceptors that sense odors (chemicals in the air). Taste buds have chemoreceptors to detect chemicals dissolved in liquids. Chemoreceptors in the brain also monitor the concentration of carbon dioxide in the blood and cerebrospinal fluid to help control your rate of breathing.
  • Mechanoreceptors sense touch, pressure and distortion (stretch). Stretch receptors in your muscle tendons are the first link in the knee-jerk reflex.
  • Photoreceptors, which sense light, are found in the retinas of your eyes.
  • Thermoreceptors are free nerve endings that sense temperature, but we're not sure exactly how they do this. Changes in temperature could affect the movements of ions across the cell membrane and influence action potentials in that way.
  • Nociceptors are free nerve endings that sense pain. They respond to a variety of stimuli (heat, pressure, chemicals) and sense tissue damage.
  • Auditory receptors in the inner ear sense vibrations from sound waves.

Typically, a stimulus causes ionic changes in the receptor neuron's dendrites, which lead to the formation of action potentials in the receptor neurons. These action potentials travel the sensory neuron, which connects to a motor neuron (and possibly an ascending neuron) in the spinal cord. The action potential causes neurotransmitter release within the presynaptic cell. The neurotransmitter binds to the postsynaptic cell and elicits an action potential there. The action potential will travel the length of the postsynaptic cell to another synapse on the effector cell (like a muscle cell, skin, blood vessel, gland), where its neurotransmitter will cause a response in the effector cell (like a muscle contraction). Alternatively, the postsynaptic cell may be another neuron that transmits the signal to another neuron in the brain or spinal cord.

What happens when nerves are damaged or diseased? We'll find out on the next page.

A physician may evaluate your nerves by testing how well you sense touch, pain or position when a limb is manipulated. This information can tell him that a functional connection exists. In some cases, he may conduct a nerve conduction velocity test to evaluate how well the nerve conducts an impulse. In this test, two small electrodes are placed a fixed distance apart from each other on the surface of the skin above a nerve. One electrode electrically stimulates the underlying nerve while the other records the corresponding electrical activity in the nerve. The recording shows the time it takes for the nerve to conduct the electrical impulse across the distance. By dividing the distance by time, the physician (or the machine) calculates the conduction velocity. The test is often performed when a conduction block or demyelinating disease (like multiple sclerosis) is suspected.


Relationship between nerves and axons - Biology

Neurons the cells composing the nervous system.
There are more than 10 billion nerve cells in the human body.

Neurons are composed of:

Nerves are organized into two major subsystems in your body:

The central nervous system:

Your brain contains about 1 X 10 11 neurons, making up about 2% of your body weight and using 20% of your body's oxygen. The cortex of the brain is folded into grooves and bumps which increase the surface area of the brain. The total surface area of the brain's cortex is about the same as a full size sheet of newspaper.

  • Contains 75% of your neurons.
  • Composed of two equal hemispheres.
    • Frontal lobe reasoning, speech, movement, and emotions
    • Parietal lobe touch, pressure, temperature, and pain
    • Temporal lobe hearing and memory
    • Occipital lobe vision
    • Located at the lower back of the head.
    • Controls muscle coordination and balance.
    • Connects the cerebrum with the spinal cord.
    • The 3 main sections of the Brain Stem:
      • Midbrain - controls reflexes and changes pupil size.
      • Medulla oblongata - controls heart rate, breathing rate, and flow of blood through the blood vessels.
      • Pons - relays signals between the cerebrum and the cerebellum.
      • The limbic system - composed of the Thalamus and Hypothalamus. These structures work together to regulate emotions.
      • The reticular formation - a network of nerves running through the brain stem and the thalamus. These nerves filter incoming sensory impulses, enabling a person to sleep.

      The spinal cord: composed of a column of nerve tissue through the vertebral column.

      • The dura mater (outer layer): consists of connective tissues, blood vessels, and nerves.
      • The arachnoid layer (middle layer): elastic and weblike.
      • The pia mater (inner layer): contains nerves and blood vessels.
      • Cerebrospinal fluid, a clear watery liquid, separates the middle and inner layers and cushions the brain and spinal cord from shock.

      The peripheral nervous system:

      Nerves that connect the central nervous system to the rest of the body.

      • Ganglion - a mass of nerve cells outside the central nervous system.
      • Receptors - nerve cells that receive information from internal and external stimuli.
      • Conductors - nerve cells that transmit information from receptors to the central nervous system.
      • Effectors - nerve cells that receive information from the central nervous system and transmit to the body. These cells activate muscles and glands.

      Autonomic nervous system controls involuntary actions.

      • Sympathetic Nervous System - controls internal organs during high stress activity.
      • Parasympathetic Nervous System - controls internal organs during normal activity.

      Impulses move from one nerve cell to another because of a difference in electrical "action potential" caused by ions inside and outside the cell. The cell membrane is selectively permeable to K + and highly impermeable to Na + .

      To simplify the process, think of the following steps:

    • Resting state:
      • A neuron is not conducting an impulse.
      • The K + concentration is much higher inside the cell than out.
      • The Na + concentration is much higher outside the cell than in.
    • Depolarization:
      • A nerve cell is stimulated.
      • At the point of stimulation, the membrane becomes permeable to Na + for an instant and they quickly move into the cell.
      • The inner surface of the cell membrane is now more positively charged than the outside.
    • Repolarization:
      • When the cell membrane becomes depolarized, K + automatically leave the cell until the cell is back to its resting state.
    • The impulse travels:
      • This quick movement of ions causes a similar change or wave all across the cell and down the axon.
      • Vertebrate nerves are covered by a myelin sheath with openings called Nodes. The myelin sheath is an insulator and causes the ion exchange to occur only at the nodes which speeds up the process.
    • Transmission across a synapse:
      • Neurons to not actually touch. The axon terminals of one neuron stop before reaching the dendrite of the next neuron. This gap between the two cells is called a Synapse.
      • Impulses are carried across a synapse by chemical messengers called neurotransmitters.
      • Approximately 30 different neurotransmitters have been identified, but they all do one of two things:
        1. Stimulate the action potential in a second cell.
        2. Inhibit the action potential in the next cell.
        • The period of time it takes a neuron to return to its resting potential after being stimulated.
        • A neuron cannot be stimulated during this period.
        • This period of time is about 0.0004 of a second.

        Test your reaction time.

        Play Neuroscience Hangman.

        • This diagram shows the areas of the brain responsible for touch. The size of the hands in this drawing indicates a large number of receptors.
        • There is a relationship between touch and pain.
        • Your ear can distinguish more than 300,000 tones.
        • For more information.
        • For more information.
        • For more information.

        Together, your eyes produce a sterioscopic view of the world.
        Study this diagram of the workings of the human eye.

        In the diagram above, notice the exit of the optic nerve to the brain. Any light falling directly on this point does not stimulate the retina, making this point blind. You can use the figure below to demonstrate the blind spot. Cover your right eye. Look at the "+" with the left eye and move your head toward or away from the screen. Although you are looking at the plus mark, concentrate on the spot. At some point in your head movement, the spot will disappear . it is being projected onto the area of the blind spot where the optic nerve exits the retina. Since the blind spot in each eye is not aimed at the same spot, their images do not overlap. If you uncover your right eye, the spot is projected onto the retina of that eye and you "see" it.


        The Dynamic Duo of the Nervous System

        Don Quixote and Sancho Panza. Michael Jordan and Scottie Pippen. Holmes and Watson. Characters bound together by circumstance, opportunity, or fate. Sometimes they’re friends other times, the relationship is more antagonistic. Whatever the reason and dynamics of the union, it’s always clear that one needs the other. They’re just better together. Who would Butch Cassidy be without the Sundance Kid? Could Han Solo be Han Solo without Chewbacca?

        There’s something alluring about the dynamic duo narrative. It pervades literature and movies. We seek it out in sports coverage. And we try to find it in our own lives. Maybe we gravitate to the story of the dynamic duo because it’s an essential part of who we are. Maybe it is who we are.

        Dynamic duos can be found all around the body. Mitochondria—the little cellular machines generating most of the energy needed by our living cells—are the descendants of engulfed prokaryotes. Bacteria line the skin, respiratory tract, and gut providing essential protective and faciliatory functions. Even viruses (despite their bad reputation) are critical to our existence. It’s estimated that at least 8 percent of the human genome contains the ghosts of ancient retroviruses that worked their way into our DNA. Viruses can provide us with helpful genes that make us more capable of functioning in our environment.

        Four hundred and twenty-five million years ago, a synergy developed within the nervous system of the hinge-jawed fish, a placoderm. The pressures of adaptation and natural selection forged a "dynamic duo" relationship that set the evolutionary stage for our brains to become what it is today. Millennia ago, a cell—maybe some type of primitive phagocyte—wrapped itself around a nerve fiber. That cell wrapping was essential for the increased processing power, enhanced coordination between brain regions, and the ability to adapt that has allowed our species to thrive.

        But the relationship is delicate. When the scale tips toward dysfunction, maladies of the nervous system arise. Multiple sclerosis, Alzheimer’s disease, Parkinson’s disease, depression, and anxiety are attributable, or linked to, perturbations to this dynamic duo of the nervous system.

        This article, Part I of this series on the dynamic duo of the nervous system, examines the increased brain processing power allotted by this relationship between brain cells.

        Increased Processing Power

        Electricity is the information currency of the brain. Little bursts of it travel from neuron to neuron and neuron to cell, allowing brain regions to communicate with one another and with different parts of the body. These little bursts are called action potentials or impulses. Action potentials racing throughout the body make possible the conscious movement of our arms, legs, and other extremities. They allow us to think, reason, and remember. They allow us to feel.

        Action potentials are generated by the movement of tiny particles called ions. Ions lie in wait inside and outside the neuron until it’s stimulated. They then flow across the membrane barrier of the neuron and into adjacent regions, triggering the entry of more ions. As ions in subsequent areas enter, the electrical impulse can travel.

        Action potentials travel along wiry, cable-like extensions called axons. When the end of an axon is reached, a chemical messenger carries the electric signal on to the next cell. The speed the impulse can travel is, in part, dictated by how easily ions can flow without being impeded or lost to the exterior of the cell.

        Speed, along with the sheer number of operations the brain can perform simultaneously, dictate the brain’s processing power. An operation can be simple—like moving your finger or computing 2 + 2—or it can be complex, say abstract reasoning or figuring out what your next move is going to be during a chess game. The speed component of processing power is limited by the speed of the action potential. The number of simultaneous operations reflects the number of elements capable of communicating using action potentials.

        Hundreds of millions of years ago, the ancestors of the hinge-jawed fish were under evolutionary pressure to get bigger and faster—likely so they wouldn’t be such easy targets for predators and could become better predators themselves. But an increase in size poses a structural problem, one that can only be overcome by increasing the brain’s processing power. If the body gets bigger, neurons have to get longer. A fish cannot command the movement of its tail fin without electrical signals being passed from the brain, along the length of the body, and to the muscles responsible for the back-and-forth movement of the tail.

        If the neuron gets longer and the speed of the action potential doesn’t change, the time it takes for the signal to travel from its origin to its destination has to increase. In the case of the hinge-jawed fish, it means signals commanding the tail fin won’t get there very fast and the fish won’t be able to move as quickly as it needs to.

        Biology has developed two ways to increase the speed of signal transduction. The first is to increase the diameter of the axon. An axon is a bit like a garden hose with a bunch of gunk in it (except that in the axon, the gunk is necessary cellular machinery). Water flowing through the garden hose will be impeded by all the gunk it runs into. If you increase the diameter of the garden hose, you give the water more room to get around the gunk, allowing it to move faster.

        Cephalopods like the squid and octopus have adopted this approach to increase the speed of signal transduction. But it’s limited to the few neurons involved in the rapid escape response.

        The second way to increase the speed of signal transduction is to insulate the axon. As ions move along the nerve fiber, some leak out, weakening the strength of the signal. If the signal isn’t strong enough to trigger the movement of ions across the membrane in neighbouring areas, the signal dies. Wrapping an axon in a material that preserves signal strength for greater lengths along the nerve fiber allows the impulse to travel faster.

        In our brains, myelin is the substance wrapping axons to preserve signal strength and speed up signal transduction. Myelin is the evolutionary progeny of a cell that serendipitously wrapped an axon in the hinge-jawed fish millions of years ago.

        Why did evolution choose myelin over increasing axon diameter to speed up signal transduction? It's likely because of space.

        The brain and spinal cord are confined within the skull at the top of our head and the vertebrae running down the midline of our backs. This is true for us and true for much of our evolutionary line. Being tucked away behind these rigid structures introduces special constraints—which means there’s a limit to how big the brain and spinal cord can get. And this limit is reached far quicker than the increase in axon diameter needed to accommodate a modest increase in body size.

        We humans need a signal transduction speed of about fifty meters per second. To attain that speed by increasing axon diameter, the spinal cord (which is made up of tracts of axons) would need to be one meter in diameter—about ten times what it is now! This isn’t exactly space-efficient.

        By wrapping axons in myelin, we’re able to dramatically increase signal transduction speed while maintaining an axon diameter that is spatially viable. This allows us to maintain a high number of neurons (and other brain cells) while also being able to increase signal transduction speed. Maintaining a high number of potential contacts in the brain leaves room for increased processing power by upping the number of operations that can be performed simultaneously. In other words, it leaves room for high order brain processes like complex perception, judgment, and planning to develop. These are brain functions that require the use and coordination of many operations simultaneously.

        The next part of this series is going to explore how the interaction between myelin and axons (our dynamic duo of the nervous system) paved the way for enhanced coordination between brain regions.

        ADDIN ZOTERO_BIBL <"uncited":[],"omitted":[],"custom":[]>CSL_BIBLIOGRAPHY Zalc B (2016) The acquisition of myelin: An evolutionary perspective. Brain Res 1641:4–10.


        The Three-Dimensional Distribution of Nerves Along the Entire Intrapulmonary Airway Tree of the Adult Rat and the Anatomical Relationship Between Nerves and Neuroepithelial Bodies

        Using airway microdissection and three-dimensional confocal microscopy techniques in combination with the immunomarkers protein gene product (PGP) 9.5 and calcitonin gene-related peptide (CGRP), we defined the distribution of small afferent nerves fibers and all nerves throughout the intrapulmonary airways, along with the distribution of airway neuroendocrine cells and neuroepithelial bodies. We found (i) the presence of CGRP-and PGP 9.5-positive structures along the entire intrapulmonary airway tree of adult rats, (ii) decreasing nerve density from more proximal to more distal generations of conducting airways, (iii) the presence of nerve fibers in terminal bronchioles, (iv) the asymmetrical distribution of nerves within a single generation of intrapulmonary airway with regard to associated vessels, (v) the frequent interchange of single nerve fibers across epithelial and sub-epithelial compartments without termination, and (vi) a definably intimate relationship between afferent nerves and neuroepithelial bodies (NEBs) (i.e., 58% of NEBs studied were observed to have nerve fibers coursing through them, indicating direct connections). We conclude that the distribution of nervous elements (nerve fibers and neuroendocrine cells) within the intrapulmonary airways is highly heterogeneous, varying between airway levels and locally within a specific airway level.

        It has long been suspected that small sensory nerves play a central role in a number of airway pathophysiologies, including airway hyper-responsiveness and inflammation. As early as 1967, activated vagal sensory nerve endings were linked to stimuli causing bronchoconstrictive responses in patients with asthma (1). Soon after, the neuropeptides contained within these small sensory nerves were implicated as potentiators of inflammation (2–6). Although it is tempting to draw conclusions regarding the functional significance of individual fibers within the airway, the role of these nerves in the development of airway hyper-reactivity and inflammation cannot be assessed before the development of a firm appreciation for the distribution of these fibers. Furthermore, the anatomical associations between nerve fibers and other airway structures should be explored in detail before speculations functionally linking nerves to other airway cell types, such as neuroendocrine cells (NECs).

        Pulmonary NECs and pulmonary neuroepithelial bodies (NEBs) are distributed throughout the pulmonary airways of rats and are often found in close proximity to nerve fibers. Pulmonary NECs and NEBs, which are essentially aggregates of pulmonary NECs, contain a number of neuropeptides also produced and contained in small sensory nerves. As early as 1949, Frolich observed NEBs in close association with nerves (7). Lauweryns later confirmed the relationship by electron microscopy for a limited number of NEBs (8). The scattered positioning of NECs throughout the airway, their ability to degranulate in response to changing airway conditions, and their association with nerve fibers suggest that airway nerve/NEC interactions may play an important role in the exaggerated airway reflexes associated with airway diseases (9, 10). Previous work draws attention to the close associations between NEBs (aggregates of NECs) and nerves but fails to define the frequency of such relationships.

        Weichselbaum and colleagues have created three-dimensional maps of the nerve plexus overlying the smooth muscle on the adventitial surface of intrapulmonary airways in the fetal pig (11). Although their study demonstrated the relationship between nerves and smooth muscle, their approach from the adventitial side of the airway wall hampered the definition of nerve fibers distributed within the epithelial compartment. Baluk and colleagues followed immunoreactive nerve fibers into the epithelial and lamina proprial compartments using two-dimensional techniques. This allowed them to estimate the relative length of nerve axons per surface area of epithelium, lamina propria, or muscle (12). However, it did not enable them to define the continuity of nerve fibers across airway wall compartments. In addition, although their study did recognize epithelial cells that were not nerves but were immunoreactive to protein gene product (PGP) 9.5, a nonspecific neuronal marker that stains NECs, NEBs, and nerves, their study did not identify NEB connections to the local nerve network (12). Shimosegawa and Said, who attempted to define the frequency of NEB interactions with calcitonin gene-related peptide (CGRP)-positive nerve fibers in thick sections, succeeded only in nonpulmonary airways (13). Finally, none of the three followed the distribution of neural structures into the most distal conducting airways or compared differences in local distribution within a single airway generation.

        Using airway microdissection and three-dimensional confocal microscopy techniques in combination with the immunomarkers PGP 9.5 (a nonspecific neural marker that stains NEBs and nerves) and CGRP (a vesicular stain that labels rat NEBs and small afferent nerves), we attempted to define (i) the abundance and distribution of small afferent fibers and all nerves along the entire adult rat pulmonary airway tree, including terminal bronchioles (ii) the differences in airway nerve distribution with regard to associated vessels (proximity to pulmonary artery verses pulmonary vein) (iii) nerve fiber continuity within and across the epithelial layer and (iv) the relationships between nerves and other airway epithelial cell types, specifically neuroepithelial bodies. Our results demonstrate (i) the presence of CGRP- and PGP 9.5-positive structures along the entire pulmonary airway tree of adult rats, (ii) decreasing nerve density from more proximal to more distal generations of conducting airways, (iii) the presence of nerve fibers in terminal bronchioles, (iv) the asymmetrical distribution of nerves within a single generation of pulmonary airway, (v) the frequent interchange of single nerve fibers across epithelial and sub-epithelial compartments without termination, and (vi) a definably intimate relationship between nerves and NEBs, of which there was a clear direct connection to 58% of the NEBs studied.

        Male Wistar rats (300–350 g) were obtained from Charles River Breeding Laboratories (Wilmington, MA). All animals were housed in animal facilities for at least 5 d before use and were provided food and water ad libitum. Animals were anesthetized with sodium pentobarbital (200 mg/kg), tracheotomized, and killed by exsanguination. The lungs were fixed in situ via infusion through tracheal cannula with 2% paraformaldehyde at 30 cm of pressure for 90 min. After fixation, the right middle (cardiac) lobe was removed to phosphate-buffered saline (PBS) and microdissected to expose the axial path of the intrapulmonary airway tree. Costal and mediastinal halves were preserved for immunohistochemistry.

        After fixed tissue had been microdissected and excess parenchyma trimmed, the airway halves were washed three times in dimethyl sulfoxide (10 min per wash). After a 10-min wash in PBS (pH 7.2), the tissue was immersed in solutions of primary antibody (dilutions: rabbit α PGP 9.5, 1/100 and/or goat α CGRP, 1/100) overnight at 4°C. The samples were washed with PBS over 4 h with at least four changes in solution and then immersed in fluorochrome-labeled immunoglobulins (1/50 dilution) for 16 h at room temperature. After being washed in PBS, the preparations were mounted on cover slips using Cyanoacrylate tissue glue (Nexaband Veterinary Products, Phoenix, AZ) and immersed in PBS. A few specimens (not used for quantitative measurements) were covered with a 3% N-propyl gallate in glycerol before immersion in PBS to reduce bleaching of fluorescein isothiocyanate (FITC) fluorochromes.

        The antibodies to PGP 9.5 and CGRP were obtained from Biogenesis (Poole, UK). The secondary antibodies (anti-rabbit and anti-goat) were conjugated to FITC, tetramethyl rhodamine isothiocyanate, or indocarbocyanine (Jackson Laboratories, West Grove, PA). Fluorochrome-labeled immunoglobulins had been pre-absorbed to minimize nonspecific reactions with rat tissues or serum proteins from the host of the unintended primary antibody. Negative controls were performed by selectively replacing primary antibodies with PBS and incubating as before with secondary antibodies.

        Epifluorescent images of the nerves, NEBs, and NECs labeled with FITC, rhodamine isothiocyanate, or indocarbocyanine were obtained using 4× to 60× water immersion objectives (Olympus W Plan Melville, NY) and confocal laser scanning microscopes (MRC 600 and 1024 Bio-Rad, Hercules, CA) with COMOS software (version 3.0 and 3.2 Bio-Rad). The airway whole mounts were optically sectioned by scanning at increasing depths of focus (steps varied between 1 μm and 20 μm depending on the water immersion objectives used). Aperture settings were chosen to minimize overlap between consecutive optical sections. Image processing was done with Adobe Photoshop 5.5 software.

        In eight male Wistar rats, the ratio of PGP 9.5- and CGRP-positive nerves to luminal epithelium were estimated using an unbiased optical “dissector” in an optical series of confocal images captured using a 40× objective at specified airway locations (14). The surface area of nerves and luminal epithelium were estimated by applying a quadratic lattice to each section and counting the number intersections with each object of interest in x and y directions. Because the points of the lattice are rays in the z direction sweeping through space, intersections in the z direction were recorded by transitions within and without the epithelial or nerve surface between confocal images. Objects that intersected the top section and two sides of the counting frame were excluded. Stacks of serial sections varied from 14–50 images, depending on the orientation of the airway surface in the dissected whole mounts. For statistical comparison between airway generations comprising the length of the airway, only images from the mediastinal side were analyzed. The generation number was determined by direct count of each branch on both halves of the dissections.

        We analyzed the surface area of PGP 9.5-positive nerves per surface area of luminal epithelium and the surface area of CGRP-positive nerves per surface area of luminal epithelium using analysis of variance, where airway generation number was the grouping factor. Post hoc analysis between the seven groups were completed using Fisher's least significant difference test (Statview, Version 5.01 SAS Institute, Cary, NC). We compared the density of PGP 9.5-positive nerves on opposing airway surfaces (mediastinal versus costal) using the Student's t test (Statview, Version 5.01 SAS Institute). All data are presented as mean ± standard error (SE). Statistical significance was considered at P < 0.05.

        To define the density and distribution of nerve fibers and NEBs within pulmonary airways, whole-mount preparations were labeled with antibodies to PGP 9.5, a nonspecific neuronal marker of ubiquitin carboxyl terminal hydrolase (15–18), and CGRP, a neuropeptide product of small afferent nerves and neuroendocrine cells (19, 20). Figure 1

        Figure 1. Projected series of confocal images comparing the distribution of PGP 9.5- (A) and CGRP- (B) positive structures within the epithelial compartment of the airway. Epithelial nerves (arrow), neuroepithelial bodies (asterisk), and neuroendocrine cells (arrowhead) are labeled by PGP 9.5 and CGRP. The colocalization of these immunomarkers was confirmed by merging image A and B to make C.

        In all fields observed, nearly all nerves positive for PGP 9.5 contained a nerve fiber positive for CGRP. Figure 2

        Figure 2. This projected series of confocal images of the entire wall in a midlevel intrapulmonary airway compares the distribution of PGP 9.5 (A) and CGRP (B). The airway NEB (asterisk) was labeled by PGP 9.5 and CGRP. Not all nerves (arrows) contained within the airway wall were positive for CGRP.

        The estimated density of airway nerves varied significantly from airway generation to airway generation. The densest nerve plexuses were observed in more proximal airways, with progressively finer and looser plexuses observed in more distal airways ( Figure 3 )

        Figure 3. The entire intrapulmonary axial airway path of the right middle lobe in the rat lung was exposed by microdissection and labeled with PGP 9.5. Confocal images of specific airway generations matched to the numbered airways in the dissection image show the distribution of PGP 9.5 nerves and NEBs within the airway wall. Mild epithelial PGP 9.5-positive labeling made it difficult to discriminate nerves using a low magnification lens. Nerves are clearly discernable in the terminal bronchioles.

        Figure 4. Morphometric comparison of the estimated surface area of PGP 9.5-positive nerves per surface are of luminal epithelium. Analysis was from stacks of confocal images at specified airway generations. The estimated mean surface area of PGP 9.5-positive nerves per surface area of luminal epithelium ± SE is shown. There were significant differences (P < 0.05) in the mean density between third-generation airways and 5th-generation, 7th-generation and 9th-generation, and 9th-generation and more distal airway locations (mean + SE n = 5–8 per generation).

        Figure 5. Morphometric comparison of the estimated surface area of CGRP-positive nerves per surface area of luminal epithelium. Analysis was from stacks of confocal images at specified airway generations. The estimated mean surface area of CGRP-positive nerves per surface area of luminal epithelium ± SE is shown. There were significant differences (P < 0.05) in the mean density between fifth-generation airways and ninth-generation nerve density and between ninth-generation and terminal bronchioles (mean ± SE, n = 5 to 8 per generation).

        Our technique allowed us to define asymmetries in density between mediastinal and costal sides of the same airway. By documenting the airway position, we could evaluate nerve density on artery versus the artery-deficient airway halves. In the rat, the pulmonary artery and bronchus follow a parallel course (21). The mediastinal half displayed a denser distribution of PGP 9.5-positive nerve fibers ( Figure 6 )

        Figure 6. Confocal images compare opposing halves of a fourth-generation intrapulmonary airway wall labeled for PGP 9.5 and CGRP. The mediastinal half (A), which is not associated with the pulmonary artery, contained thicker central nerve fibers and a denser nerve plexus compared with the costal half (B), which is adjacent to the pulmonary artery.

        To better appreciate the continuity of nerves between the epithelial and interstitial compartments, we used optical sections to infer the location of nerves relative to NEBs and NECs. Because NECs, or a portion of the NECs that make up NEBs, are located on or near the basement membrane, we used their position to estimate where nerves cross from the interstitial to the epithelial compartments based on planar reference to NECs. The projected series of images in Figure 7

        Figure 7. Series of optical confocal sections of CGRP-positive nerves along with a neuroendocrine cell (asterisk) in the wall of a distal airway. The series represents a 20-μm optical section of bronchiolar wall beginning in the epithelial plane (upper left) and ending in the interstitium (lower left). The lower right image shows the composite image of all sections. The position of the basement membrane, the site where nerve fibers cross from the interstitial to the epithelial compartments, was estimated based on planar reference to neuroendocrine cells. Nerves wind through the airway epithelium multiple times but rarely terminate in the epithelium. Arrows point out segments of nerve that appear within the epithelium adjoining segments are located below the basement membrane. The projected series is displayed in the lower right corner.

        All NEBs were observed in close proximity to a nerve fiber however, nerves coursed into or through NEBs in only 15 of the 26 NEBs analyzed in detail (58%). Nerve fibers rarely terminated in close proximity to NEBs. The NEB represented in Figure 1 was positive for CGRP and PGP 9.5, as were all NEBs observed. In fifteen cases, we were able to follow the passage of one or more nerve fibers through a NEB by scanning through the structure ( Figure 8A )

        Figure 8. (A) Projected confocal images taken at a branch point within a midlevel airway wall demonstrate CGRP-positive nerves that wind through a CGRP-positive NEB. (B) A composite image of 113 1-μm optical sections displays a NEB intimately associated with PGP 9.5-positive nerves but lacking direct contact. In 42% of the NEBs studied, a nerve penetrating a NEB could not be identified.

        Most NEBs were identified in close association with the more prominent fibers of their associated airway generation. For this reason, NEBs were often sequentially recorded along the airway in alignment with thicker central nerves ( Figure 9 )

        Figure 9. A projected series of confocal images taken in a proximal intrapulmonary airway display PGP 9.5-positive nerves with three NEBs (asterisks) oriented along the longitudinal axis. A pattern of sequential NEB alignment along central nerve trunks was observed in ∼ 90% of the proximal pulmonary airways studied.

        PGP 9.5 and CGRP colocalized to many nerves found in the walls of the conducting airways, indicating an abundance of afferent fibers within the intrapulmonary airway tree. As reported previously, antibody to PGP 9.5, a nonspecific neural marker for ubiquitin carboxyl terminal hydrolase, uniformly labeled nerve fibers and ganglia (17). Antibody to CGRP, a sensory neuropeptide, had a varicosed labeling pattern indicative of CGRP packaging in synaptic vesicles. PGP 9.5 and CGRP antibody labeled NECs and NEBs. PGP 9.5 antibody evenly labeled NECs, the primary component of NEBs, and CGRP antibody exhibited focal areas of intensity within the cells that compose neuroepithelial bodies. Differences in the distribution of the nonspecific neural marker PGP 9.5 and our indicator of small afferent nerve fibers (CGRP) can be seen in NEBs, NECs, and nerve fibers by examining Figures 1A, 1B, and 1C .

        Using three-dimensional technology to image along the length of the entire intrapulmonary airway tree, we observed a reduction in the density of nerve fibers as we progressed from proximal to more distal airway positions. At selected airway locations, we captured stacks of high-magnification images and used these to estimate the surface area of nerves per surface area of luminal epithelium. Previous research has quantified an inverse relationship between airway nerve density and airway position (with regard to distance from proximal trachea). In proximal airway locations, including the rostral trachea, caudal trachea, and main stem bronchus, a significantly lower nerve density was calculated from sections of more distal nonpulmonary airway positions (12, 13). Our study extends past airway nerve analysis studies and shows dramatic changes in nerve density/positional relationships within intrapulmonary airways.

        We examined opposing surfaces of an airway within a single generation and observed distinct differences in nerve density. The mediastinal half the rat airways had a 72% increase in nerve fiber density and thicker central fibers compared with the costal airway half. These differences in nerve distribution could easily contribute to differences in the biology of airways within the same airway generation. Any function dependent on innervation or neurochemical modulation would be affected by this heterogeneity in nerve distribution to include mucus secretion, glandular function, smooth muscle tone, vascular permeability, inflammatory recruitment, and epithelial repair (2, 3, 22, 23). Furthermore, in diseased airways, a state usually associated with imbalances, one might expect additional divergence in nerve distribution. For this reason it is critical that investigators and clinicians carefully define their site of sample or biopsy collection, especially when the mechanism of the disease under investigation might involve the influence of local nerve networks. Our results lead to the question of whether other cells types, including mucous cells, vascular plexuses, glands, and inflammatory cells, are influenced by the asymmetrical distribution of nerve fibers.

        Our study emphasizes the continuity of the nerve network between epithelial and submucosal layers throughout the intrapulmonary airway tree. Individual nerve fibers frequently enter the epithelial compartment and then return to the submucosal compartment. Based on the early discovery of intra-epithelial nerve endings, investigators have predicted the termination of submucosal nerve fibers within the epithelial layer or within the lumen of the airway (24). Although bulb-like nerve endings were present within the epithelium, more frequently epithelial nerve fibers traversed the basement membrane, returning to the submucosal layer without termination. Often, a single nerve fiber crossed the basement membrane several times as it moved between epithelial and submucosal layers. It is possible that the varicosed nature of many epithelial nerves may have given the impression of nerve termination within the epithelium in two-dimensional images. The infrequency of nerve fiber termination within the epithelium does not discount their sensory role within the epithelium. Receptors have been clearly identified on unmyelinated sensory axons (25, 26). The continuity of the airway nerve network among airway levels makes it the perfect intermediary for communication between a number of cell types and tissue systems.

        In the rat, PGP 9.5 and CGRP are present in NEBs and individual NECs of the intrapulmonary airway. The distribution of NEBs in the rat has been mapped previously (9, 13). We emphasized the relationship between NEBs and nerves first described by Frolich using light microscopy (7) and later by Lauweryns using electron microscopy (8). The close proximity of nerves to NEBs has led a number of investigators to suggest close communication between the two, but a definitive explanation of the nature of the interaction in not available. Because NEBs morphologically resemble carotid bodies and because they release vesicular products in response to hypoxia, a sensory (chemoreceptor) function has been postulated for NEBs (8, 27, 28). If this were the case, then one would expect that all NEBs would be innervated by sensory fibers and that degranulation of NECs within NEBs would produce a measurable systemic response. Ultrastructure studies in rabbits indicate that only one third of NEBs are innervated, and denervation studies suggest that only two thirds of those NEBs are innervated by nerves whose cell bodies are housed in the vagal ganglia (29). Assuming that the innervating vagal afferents synapse within the nucleus tractus solitarius (like carotid body vagal afferents), then NEB degranulation would be associated with adjustments in respiratory center function to maximize the partial pressure of oxygen within the airways. Current studies do not provide evidence of a systemic response to NEB degranulation. There does seem to be a local compensatory response to chronic hypoxia, NEB hyperplasia, which is evident in children with hypoventilation syndrome (30). It is unclear whether nerves, afferent or efferent, play a role in this airway NEB hyperplasia.

        Although past studies predict only a 22% innervation rate of intrapulmonary NEBs by vagal sensory nerve fibers (29), NEB analysis in denervation studies do suggest close interactions between NEBs and afferent nerves. Studies by Lauweryns indicate that NEB activity may be modulated by local reflexes within the airway. When airways containing biogenic amine-positive NEBs are unilaterally deprived of their afferent and efferent innervation via vagal ligation below the nodose ganglia, NEBs become less responsive to airway hypoxia within 3 d, which is sufficient time to deplete axon terminals of neurotransmitter. However, the response of NEBs to hypoxia is not affected when the vagus is ligated above the nodose ganglia (10). These studies suggest that the release of neuropeptides from afferent nerves whose cell bodies are located in the nodose ganglia modulate NEB degranulation in response to hypoxia.

        Based on detailed three-dimensional analysis of 26 NEBs in male adult rats, we found a definable direct connection between NEBs and nerve fibers in slightly over one half of the NEBs studied. Most of these nerves were positive for CGRP and PGP 9.5. Earlier work by Van Lommel using anterograde neural tracer injected into the nodose ganglia established the existence of NEB/afferent nerve fiber complexes (31). However, the frequency of such relationships was not determined. In neonatal dogs, several NEBs have been found to be deficient of any kind of anatomic innervation (32). Moreover, the aberrant nerve morphology, possibly indicative of degeneration, associated with 40% of the NEB/nerve complexes studied led the investigators to propose that NEB-associated nerves are lost with age. Determining whether rats lose NEB innervation with age or whether such innervation never existed in 40% of the pulmonary airway NEBs was not the purpose of our study. However, we do believe that the paucity of NEBs with definable innervation in adult rats brings into question the contribution of NEB/nerve interactions in normal respiratory function. Conversely, although formidable distances between NEBs and nerves may make rapid communication more difficult, these distances do not necessarily discount interactions between NEBs and nerve fibers. Studies by Jan and Jan demonstrate that peptides released from nerve terminals can influence neurons hundreds of micrometers away (33).

        Although we may not fully comprehend the interactions of NEC/NEBs and nerves, we know that the availability of neuropeptides at any loci within the airway is for the most part dependent upon these cells. Changing neuropeptide availability can influence local airway biology, possibly enhancing epithelial cell proliferation (23) or exacerbating defense mechanisms by increased blood flow, mucous secretion, airway smooth muscle tone, and/or the recruitment and activation of inflammatory cells (2, 3). The increasing density of neuropeptide-containing structures (like NECs and NEBs) in a number of pathologies hint of their importance. In infants diagnosed with sudden infant death syndrome (34), adults with bronchial asthma (35), and adults with chronic bronchitis (36), notable increases in airway NECs and NEBs were recorded. Because the innervation of these structures was not considered, we cannot predict their local influence. The activity and growth of NEBs may be normally inhibited by innervating nerves. A loss of innervation might contribute to the uncontrolled activity and growth of neuroendocrine cells. The speculation over regional neuropeptide influence in pathology cannot fully be appreciated until the frequency of direct connections between nerves and NEBs are compared among a number of respiratory pathologies. The ratio of nerves to NECs and NEBs may be more significant in the development of airway pathology rather than either marker independently.

        This work was supported by NIEHS grants 06791, ES05707, ES00628, ES06700, and T32 HL07013. The authors acknowledge the help and advice of Dr. R. Paige and A. Weir.


        Nerves

        Bundles of axons in the PNS are referred to as nerves. These structures in the periphery are different than the central counterpart, called a tract. Unlike tracts, nerves are composed of more than just nervous tissue. They have connective tissues invested in their structure, as well as blood vessels supplying the tissues with nourishment. The outer surface of a nerve is a surrounding layer of fibrous connective tissue called the epineurium. Within the nerve, axons are further bundled into fascicles, which are each surrounded by their own layer of fibrous connective tissue called perineurium. Finally, individual axons are surrounded by loose connective tissue called the endoneurium (Figure 13.2.3). These three layers are similar to the connective tissue sheaths for muscles. Because peripheral axons are surrounded by an endoneurium it is possible for severed axons to regenerated. After they are cut the proximal severed end of the axon sprouts and one of the sprouts will find the endoneurium which is, essentially, an empty tube leading to (or near) the original target. The endoneurim is empty because the distal portion of the severed axon degenerates, a process called Wallerian (anterograde or orthograde) degeneration. Nerves are associated with the region of the CNS to which they are connected, either as cranial nerves connected to the brain or spinal nerves connected to the spinal cord.

        Figure 13.2.3 – Nerve Structure. The structure of a nerve is organized by the layers of connective tissue on the outside, around each fascicle, and surrounding the individual nerve fibers (tissue source: simian). LM × 40. (Micrograph provided by the Regents of University of Michigan Medical School © 2012)

        Figure 13.2.4 – Close-Up of Nerve Trunk: Zoom in on this slide of a nerve trunk to examine the endoneurium, perineurium, and epineurium in greater detail (tissue source: simian). LM × 1600. (Micrograph provided by the Regents of University of Michigan Medical School © 2012)



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