Neuroscience made understandable

Dr. A. van Aken

Purkinje cell. Original drawing by Santiago Ramón y Cajal 1899.

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Neuroscience made understandable Within the BSMS curriculum, module 202 (Neuroscience and Behaviour) is generally perceived as difficult. In order to understand how neurons communicate with one another it is necessary to understand the underlying physics and chemistry. This syllabus is written as ancillary learning material for module 202. Who should read this syllabus? If you are interested in the nervous system this syllabus offers you the opportunity to study this subject more in-depth as it goes into more detail than what is covered in the lectures. Also, for those who would like to intercalate and do the Neuroscience BSc this syllabus will give you an idea what to expect. If you don’t feel confident when dealing with physics and chemistry or if you find it hard to relate scientific concepts to the practice of medicine you should definitely take a look at this syllabus. Throughout the text medical case studies are used to relate “abstract” scientific concepts to clinical practice. Also, all concepts are explained in a step by step fashion. Other relevant materials. A series of module tutorials runs throughout module 202 covering a wide range of topics. Within the Module Tutorials section you can find the ‘guide to module tutorials’ in which the more difficult aspects of the MTs are explained in more detail. If you find science a difficult subject you may also want to take a look at ‘Physics and Chemistry for medical students’ available on StudentCentral. You can help to improve this text This document is a work in progress and will be updated periodically on StudentCentral. Your views and comments are highly appreciated. If you feel some subject(s) need more clarification or if you have found any mistakes please send your feedback to A.F.J.van-Aken@sussex.ac.uk If you have any questions about physics, chemistry or mathematics subjects which have not been addressed in this syllabus feel free to write to the same e-mail address and I will try to reply as soon as possible. Maybe it will make it into the next version of this syllabus. Alexander van Aken

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Index

1. Introduction – Neuronal signalling 4

2. Receptors 13

3. Movement of ions across membranes 24

4. The action potential

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Case studies

- Multiple sclerosis 42

- Cystic fibrosis 43

- Euthanasia 45

- Myasthenia gravis 47

- Excitotoxicity 48

- Death by puffer fish

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Sensory neurons

- Receptor cells of the eye 52

- Receptor cells of the ear 54

- Taste receptor cells 56

- Smell receptor cells 57

- Touch and pain receptor cells 58

Appendix – Logarithms 59

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1. Introduction - Neuronal signalling Most neurons communicate by generating electrical signals (action potentials) and through the release of chemicals (neurotransmitters). Figure 1.1 shows a typical representation of two communicating neurons within the central nervous system (CNS). The top one innervating the bottom one. The cell body has numerous small extensions, called dendrites and one long extension called the axon. In most cases the end of an axon is in very close proximity to a dendrite, without actually touching it. A neuron can send a signal to another neuron by generating an electrical signal which travels down its axon. When this signal reaches the end terminal it triggers the release of neurotransmitters which bind to specific receptors found on dendrites of another neuron1. The binding of neurotransmitters can lead to the generation of another electrical signal. The point where two neurons interact is called the synapse and the neuron sending the signal is the pre-synaptic neuron whereas the receiving cell is called the post-synaptic neuron.

Figure 1.1 – Communication between two neurons within the CNS

1 Sometimes neurotransmitters bind to the same neuron from which they were released, e.g. to

modulate neurotransmitter release.

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The most common pathway for neuronal communication can be described as: neurotransmitter release by pre-synaptic neuron which triggers action potentials in the post-synaptic neuron, which leads to release of

neurotransmitters by the same neuron: chemical electrical chemical. This is however altogether different for the sensory neurons in the nervous system. These are the cells which detect physical stimuli, e.g. sound, light, smell and pressure. These cells convert a physical or chemical stimulus into an electrical signal. This process of converting a physical/chemical stimulus into an electrical signal is called transduction. Basically, sensory neurons receive signals from the environment (both internally and externally) and pass this information onto other types of neurons for further processing within the brain. In order to understand how the nervous system works it is very important that you appreciate the difference between the different types of neurons. We will therefore now focus a bit on the anatomy of neurons. Our nervous system can be divided into two parts: the central nervous system, which consists of the brain and the spinal cord and the peripheral nervous system (PNS) which consists of all the nerves outside of the CNS. The neurons in Figure 1 can be immediately identified as CNS neurons because of the presence of oligodendrocytes which wrap themselves around the axon. Oligodendrocytes are glial cells and have a supportive function within the CNS. Insulating the axon with layers of myelin increases the conductivity of electrical signalling within the nervous system (A decrease in myelination is the underlying cause for multiple sclerosis, see the case study on M.S.). Within the PNS myelination of axons also occurs because Schwan cells wrap themselves around the axons of PNS neurons. Oligodendrocytes and Schwan cells are confined to the CNS and PNS respectively. Also, one oligodendrocyte can myelinate several neurons whereas Schwan cells are in a one to one relationship with PNS neurons. Neurons consist of a cell body and one or more cellular extensions called neurites, see figure 1.2. Figure 1.2 – Neurons classified on the basis of their neurites.

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Intermezzo - some nervous system nomenclature The nomenclature used to describe the structures within the nervous system can be a bit confusing. The nervous system is divided into the central nervous system (CNS) which consists of the brain and the spinal cord and the peripheral nervous system (PNS) which consists of all the nerves outside the CNS. Here are some important structures you should know: Grey matter: The cell bodies of neurons in a freshly dissected brain have a grey appearance. White matter: A generic term for a collection of CNS axons, in a freshly dissected brain the axon bundles appear white. Nucleus: A clearly distinguishable mass of neurons, usually deep within the brain, e.g. the lateral geniculate nucleus. Substantia: A group of related neurons with less distinct borders than nuclei, e.g. the substantia nigra. Locus: A small, well-defined group of cells, e.g. the locus coeruleus. Ganglion: A collection of neurons in the PNS, e.g. the dorsal root ganglia. Mind you, within the CNS there is one group of cells which is referred to as a ganglion, the basal ganglia. Nerve: A bundle of axons in the PNS. There is one collection of axons within the CNS that is referred to as a nerve, the optic nerve. Tract: A collection of CNS axons which have a common site of origin and a common destination. Bundle: A collection of axons that run together that do not necessarily have the same origin and destination. Capsule: A collection of axons that connect the cerebrum with the brain stem. Lemniscus: A tract which meanders through the brain like a ribbon, e.g. the medial lemniscus. Commisure: Any collection of axons that connect one side of the brain with the other side.

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There are two types of neurites: axons and dendrites. Axons are specialized ‘long’ extensions which conduct the electrical signals. Dendrites are ‘short’ extensions which express receptors. These are transmembrane proteins to which neurotransmitters (released by axon terminals) can bind. First we will look at a generalised scheme of neuronal communication followed by an example of signal transduction in a sensory neuron. Figure 1.3 – Two neurons communicating. Figure 1.3 shows two neurons. The presynaptic neuron generates an electrical signal called the action potential. This signal travels down the axon. The signal is carried by ions which flow within the cytoplasm of the axon (axoplasm). To improve the conductivity of the axon oligodendrocytes or Schwan cells wrap themselves around it. This prevents leakage of ions from the axon (We will discuss the electrical properties of neurons in more detail later). When the signal reaches the end of the axon it induces influx of calcium ions which then leads to release of neurotransmitters into the synaptic cleft. The released neurotransmitters traverse the synaptic cleft and bind to receptors on the post-synaptic cell. This can then induce another electrical signal in the post-synaptic cell. It is important to note that the neurons do not actually touch each other, i.e. there is no direct communication between them. The axon of the pre-synaptic cell gets close to the membrane of the post-synaptic cell. In the majority of the cases the axons make contact with dendrites which is called axodendritic. It is also possible for axons to make contact with the cell body which is called axosomatic and sometimes axons make contact with other axons to modulate their output, which is referred to as axoaxonic.

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We will now take a closer look at the synapse, see figure 1.4. Figure 1.4 – the synaptic cleft. When the action potential arrives at the axon terminal (which is shaped somewhat like a button) voltage-gated calcium channels open. The concentration of Ca2+ in the extracellular fluid is 10,000 times higher than in the cytosol, i.e. there is a very large inward driving force for calcium ions2. In close proximity to the membrane vesicles loaded with neurotransmitters are stored. Increased intracellular levels of Ca2+ induce the fusion of these vesicles with the membrane and the neurotransmitters are then released into the cleft.

2 We will discuss driving forces in the next section.

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Inside the cleft neurotransmitters flow to the membrane of the post-synaptic neuron. Within the membrane of the post-synaptic cell many transmembrane proteins are expressed, receptor proteins. In this example two molecules of neurotransmitter bind to one receptor protein which induces opening of an ion channel3 which allows the influx of ions, in this case sodium. The concentration of Na+ in the extracellular solution is about 10 times higher than the cytosolic concentration. Also, the inside of the cell normally has a negative potential with respect to the outside (around -70 mV). That means that there are two inward driving forces for Na+. A chemical driving force due to the concentration difference and an electrical driving force, due to the electrical potential difference across the membrane. The combination of these driving forces is known as the electrochemical gradient for Na+. Every ion has its own electrochemical gradient. In our example we see that upon binding of neurotransmitter to the receptor an ion channel opens which leads to influx of sodium. This makes the inside of the post-synaptic neuron more positive, which is called a depolarization. As a consequence the post-synaptic cell is now able to generate an action potential of its own. This will then lead to release of neurotransmitters onto another cell. In this way neurons can relay signals to one another. Releasing

neurotransmitter (chemical) generating an action potential (electrical) releasing neurotransmitter (chemical). In this example the binding of neurotransmitters leads to depolarization of the post-synaptic cell. These neurotransmitters are therefore said to be excitatory, glutamate is an example of this type. There are other neurotransmitters like GABA and glycine which make the post-synaptic cell more negative (due to influx of chloride ions). They are inhibitory and therefore decrease the chance that the post-synaptic cell will generate an action potential. There are a few other processes occurring within the synaptic cleft worth mentioning. Neurotransmitters do not stay bound to their receptors. After some time they dissociate back into the synaptic cleft. Some neurotransmitters (e.g. acetylcholine) are broken down by enzymes in the synaptic cleft. Some neurotransmitters passively diffuse away. A large proportion of the neurotransmitters in the synaptic cleft however are actively transported back into the pre-synaptic neuron (or in some cases also into glial cells). You can see in Figure 1.4 that neurotransmitters are transported back into the axon terminal where they are then loaded into neurotransmitter vesicles, ready to be used again. All these processes, the movement of ions, the binding of neurotransmitters, opening of ion channels and loading of vesicles will be discussed in detail further on. But before moving on we will look at an example of neuronal signalling in sensory neurons. As an example we will use a nociceptor (a pain receptor). In the appendix you can find the molecular and cellular mechanisms of all the senses explained.

3 The ion channel is part of the receptor protein, binding of the neurotransmitter induces a

conformational change of the protein which leads to opening of the channel.

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1.1 - Sensory neurons

Figure 1.5 shows a nociceptor, i.e. a sensory neuron which detects painful stimuli. You can see that this neuron only has one neurite and therefore is unipolar. It has one long axon which for the most part is insulated with myelin, the gaps between the myelin sheets are called the nodes of Ranvier. Their significance will be explained later. This neuron has free nerve endings which can detect various painful stimuli. Imagine stepping on a thumb tack with your bare feet. The skin gets punctured and nociceptors in the skin of your foot are mechanically stimulated. Figure 1.5 – a nociceptor This mechanical stimulus directly opens mechanosensitive ion channels through which positive ions flow into the axon generating an action potential. This electrical signal now travels down the axon from the free nerve endings down to the axon terminal depicted on the right side of the diagram which leads to the release of neurotransmitters within the spinal cord. These neurotransmitters will now bind to receptors on so called projection neurons which will relay the pain signal to the brain. As you can see the signalling pathway in sensory neurons is quite different from the previous example. In figure 1.4 we saw a:

chemical electrical chemical pathway. Whereas in this sensory neuron we see a:

mechanical electrical chemical pathway. Very often in textbooks only the first type of neuronal signalling is discussed in the introductory chapters. In later chapters the specific mechanisms usually are explained but it is hardly ever pointed out from the outset that sensory neurons work very differently from most of the other neurons in the nervous system. Because of this we will discuss the specific mechanisms of all the senses in the chapter on sensory neurons.

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Intermezzo – Some neurotransmitter and receptor nomenclature Neurons are classified on the basis of what type of neurotransmitters they release. Cholinergic neurons release acetylcholine, serotonergic neurons serotonin, GABAergic neurons release GABA etc. Neurons are not classified on the basis of what type of receptors they express. The type of neurotransmitter released does not determine what type of receptors are expressed on that same neuron, e.g. a cholinergic neuron may express any type of receptor (including the acetylcholine receptor). Neurons are classified on the basis of what type of small neurotransmitter they release. All the small neurotransmitters are synthesized locally in the axon terminal. These are the monoamines (adrenaline, noradrenaline, dopamine4 and serotonin), amino acids (glutamate, GABA, glycine) and small molecules such as acetylcholine. There is a second category of neurotransmitters called the neuropeptides, the name already tells you that these transmitters are synthesized in the cell body and therefore they need to be transported along the axon to the terminal. The neuropeptides are much larger molecules than the small neurotransmitters, some examples are substance P, bradykinin and the endorphins. Neurons release only one type of small neurotransmitter but they can also release one or more types of neuropeptides. Therefore the nomenclature is not completely correct. Neurons should perhaps be classified as for instance glutamatergic and bradykinergic. But the convention is that neurons are classified only on the basis of which particular small neurotransmitter they release. Receptors are classified on the basis of which neurotransmitter they bind and on the basis of their selectivity for certain agonists. For instance, there are two types of acetylcholine receptors. The nicotinic acetylcholine receptor and the muscarinic acetylcholine receptor. Both receptors bind acetylcholine, however they are different types of receptors. The nicotinic acetylcholine receptor is ionotropic, i.e. it is a transmembrane protein which consists of a receptor part and an ion channel part. The muscarinic acetylcholine receptor is a G-protein coupled receptor (the difference between ionotropic and G-protein coupled receptors is explained in section 2). Nicotine is an agonist for the ionotropic acetylcholine receptor (present on somatic cells) whereas muscarine is an agonist for the G-protein coupled acetylcholine receptor (present on heart cells). Nicotine has a very low affinity for the muscarinic acetylcholine receptor and muscarine equally also has a very low affinity for the nicotinic acetylcholine receptor. So the acetylcholine receptors are classified on the basis of which agonist binds selectively to one acetylcholine receptor, but not to the other.

4 the monoamines can be further divided into indolamines (serotonin) and the catecholamines

(dopamine, adrenaline and noradrenaline).

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This same principle applies to other receptors as well. For instance, there are three types of glutamate receptors each of which is selective for a specific agonist, namely: NMDA, kainate and AMPA. What would be the point of knowing all these pathways? Here is the dopamine synthesis pathway:

You can see that the precursor of dopamine is L-dopa and that the enzyme dopa decarboxylase converts L-dopa into dopamine. This information is useful. Patients suffering from Parkinson’s disease have low levels of dopamine in the basal ganglia due to degeneration of the substantia nigra. It is important to increase the levels of dopamine in the basal ganglia of these patients, dopamine however does not cross the blood brain barrier (BBB). L-dopa on the other hand can cross the BBB. So administration with L-dopa is a good idea. Unfortunately, L-dopa is readily converted to dopamine in the bloodstream before it can cross the BBB. In order to deal with this problem L-dopa is administered in conjunction with a dopa decarboxylase inhibitor. This allows for L-dopa to cross the BBB where it can then be converted to dopamine in the brain. Clearly, understanding the synthesis pathways of neurotransmitters allows for a more effective pharmacological treatment.

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2 – Receptors There are two types of receptors involved in neuronal signalling: ionotropic and metabotropic receptors. The ionotropic receptors are transmembrane proteins which consist of both a receptor part (to which the neurotransmitter binds) and an ion channel. Metabotropic receptors are transmembrane receptors as well but they do not have an ion channel. When a ligand5 binds the metabotropic receptor is activated which then leads to intracellular signalling. We will first discuss ionotropic receptors.

2.1 – ionotropic receptors Binding of a ligand to an ionotropic receptor leads to a conformational change of the protein which causes its ion channel to open. Most ionotropic receptors have extracellular facing binding sites to which neurotransmitters which are released in the synaptic cleft can bind. Some ionotropic receptors have intracellular binding sites to which ligands can bind, see Figure 2.1. Binding of intracellular ligands to ionotropic receptors typically serves to modulate the electrical properties of the neuron, we will return to this in the section on metabotropic receptors. As a general rule ther e are two different categories, an ionotropic receptor either has binding sites facing the extracellular solution or facing the cytosol. Ionotropic receptors are a subset of the family of ion channels. The function of ion channels is to flux ions in order to change the membrane potential. Ionotropic receptors require a ligand to open (or sometimes close) their ion channel. There are two other types of ion channel which cannot be opened through the binding of ligands. These are the voltage-gated ion channels and the mechanical-gated ion channels.

Figure 2.1 – ion channels

5 A ligand is a more generic term for any molecule which can bind to a receptor.

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The function off all ion channels is to change the membrane potential and the mechanism which causes them to open or close is called gating. Although voltage-gated and mechanically-gated channels do not open through the binding of ligands they do respond to signals. Opening of the voltage-gated sodium channel is an essential step in the generation of the action potential. Typically mammalian cells, including neurons, maintain a resting membrane potential of around -70 mV. When the membrane potential is depolarised to about -35 mV the channel opens. It ‘senses’ the voltage across the membrane. The change in membrane potential is the signal. The sensor for this signal is situated within the hydrophobic part of the protein, see Figure 2.2.

Figure 2.2 – The voltage-gated sodium channel The voltage-gated sodium channel is one large protein which consist of 4 domains each of which has six transmembrane regions. Region 4 in each domain consists of amino acids with positive R-groups. At the resting membrane potential of -70 mV the voltage-gated sodium channel is closed. When the membrane is depolarized, inside become more positive, the positively charged R-groups within the protein experience a change in electrostatic force and regions 4 are pushed outward, which causes the channel to open. We will return to the topic of voltage-gated ion channels in the section 4 – the action potential. Mechanically-gated ion channels We already encountered a mechanically-gated ion channel in our discussion of the nociceptor in section 1. Many of the sensory neurons of the nervous system express mechanically-gated ion channels which can detect mechanical stimuli. We look at different types of mechanically-gated ion channels in the section on sensory neurons.

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2.2 Metabotropic receptors All metabotropic receptors consist of 7-transmembrane domains, see Figure 2.3. These receptors have a neurotransmitter binding site facing the extracellular solution. When a ligand binds to the receptor the binding affinity for a so called G-protein increases. Metabotropic receptors are also referred to as G-protein coupled receptors. Activation of a G-protein coupled receptor (GPCR) leads to intracellular effects.

Figure 2.3 – metabotropic receptor6

The G-protein is a protein consisting of 3 subunits labelled and , see

Figure 2.4. The -subunit has a molecule of either GDP or GTP bound to it, hence the name G-protein. Figure 2.4 – the G-protein coupled receptor7

6 Neuroscience – exploring the brain by Bear et al

7 Image source: http://www.csuci.edu/alzheimer/images/gprotein.jpg

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In the absence of a bound neurotransmitter the GPCR cannot bind a G-protein. A G-protein floats along the inner leaflet of the cell membrane. When it comes across a GPCR which does not have a ligand bound to it, nothing will

happen. When the G-protein is freely floating alongside the leaflet the -subunit has a molecule of GDP bound to it. If a ligand binds to the GPCR its affinity for G-proteins will increase. If now a G-protein happens to collide with the activated GPCR they will associate. You can see from this that there is an element of chance to this signalling pathway. Once the GPCR and G-protein are associated several things happen, see Figure 2.5

Figure 2.5 – Activation of the G-protein8. When a G-protein binds to an activated G-protein coupled receptor the GDP

molecule attached to the -subunit is replaced by a molecule of GTP. Also,

the G-protein splits up into two components: the -complex and the -subunit. Both complexes go their own way (alongside the inner leaflet of the membrane) to induce intracellular effects (discussed later). Whether or not GDP is replaced with GTP before the G-protein splits up is not clear. Possibly

these events occur simultaneously. The -complex and the -subunit induce different effects which we will discuss separately:

The -complex

The -complex induces relatively fast effects by associating with ion channels and modulating their activity. In Figure 2.6 you can see a muscarinic acetylcholine receptor. These metabotropic receptors serve as the main end-receptors stimulated by acetylcholine released by the parasympathetic nervous system. For instance, when acetylcholine binds

to muscarinic acetylcholine receptors expressed by heart cells the -complex associates with a potassium channel causing it to open (Figure 2.6). Because the concentration of potassium in the cytosol is much higher than in the extracellular solution the positively charged potassium will start to flow out of the cell. Because of this the cell becomes more negatively charged on the inside, hence the membrane potential becomes more negative,( i.e. hyperpolarised) and this slows down the heart as hyperpolarization prevents cells from generating action potentials or contracting.

8 Neuroscience – exploring the brain by Bear et al

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Figure 2.6 – -complex pathway9 Remember from figure 2.1 that certain ligand-gated ionotropic receptors have their binding sites facing the cytosol. The potassium channel shown in Figure 2.6 is an example of this type of ionotropic channel (ionotropic because it conducts ions). You may have noticed that for this type of channel we do not use the word neurotransmitter but the more generic term ligand. That is because neurotransmitters are released by pre-synaptic neurons into the synaptic cleft where they bind to ligand-gated ionotropic receptors which have their binding sites facing the synaptic cleft. Activation of a metabotropic receptor does not have a direct effect on the membrane potential since these receptors do not contain an ion channel part. From the example however you can see that activation of the metabotropic acetylcholine receptor indirectly changes the membrane potential through

interaction of the -complex directly with an ion channel.

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The -subunit

The -subunit (with bound GTP) associates with a membrane-bound enzyme called adenylyl cyclase, see Figure 2.7. Adenylyl cyclase catalyzes the conversion of ATP to cyclic AMP (cAMP). When adenylyl cyclase is stimulated the rate with which cAMP is produced increases. cAMP itself is a signalling molecule which can activate other intracellular proteins. In the example of Figure 2.7 cAMP activates protein kinase A (PKA)10 which can phosphorylate other proteins, e.g. ion channels (changing their activity) or transcription factors in the nucleus which will affect gene expression. In this example the GPCR is referred to as a Gs-Coupled Receptor. The s stands for stimulatory and activation of this particular GPCR leads to stimulation of adenylyl cyclase. The Gi-Coupled Receptor (I stands for inhibitory) leads to inhibition of adenylyl cyclase. It is common that cells express both the Gs and the Gi type. Which means that adenylyl cyclase is inhibited and stimulated at the same time. The outcome (increase or decrease of cAMP production) will depend on the number of Gs and Gi type GPCRs active at the same time. This tug of war type of signalling is a common mechanism used to modulate cellular behaviour.

Figure 2.7 – The -subunit pathway11.

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A kinase is a protein which phosphorylates other proteins by attaching a phosphate group to the

molecule. 11

Image source: http://www.biocarta.com/pathfiles/h_gsPathway.gif

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When a neurotransmitter binds to the GPCR the G-protein splits up in the -

complex and the -subunit. The GDP molecule associated with the -subunit

is replaced with a GTP molecule. The -subunit the associates with adenylyl cyclase to either stimulate or inhibit it (depending on whether the GPCR is of

the Gi or the Gs type). As long as the GTP-bound -subunit is associated with

adenylyl cyclase it will stimulate/inhibit it. The -subunit is autocatalytic and will after some time dephosphorylate the GTP molecule, converting it into

GDP. Now the GDP-bound -subunit will dissociate from adenylyl cyclase and

combine with the first -complex it encounters. The G-protein is now ready for another round of cell signalling. One of the main differences between ionotropic and metabotropic receptors is their response to the binding of signalling molecules. Binding of a ligand to an ionotropic receptor leads to opening or closing of an ion channel which will have an immediate effect on the membrane potential. Binding of a ligand to a metabotropic receptor will lead to a delayed effect because activation of metabotropic receptors leads to the generation of intracellular signalling. This is an example of a second messenger signalling pathway. The neurotransmitter is the first messenger and then

within the cytosol the -complex or the -subunit is the second messenger. Another main difference between ionotropic and metabotropic receptors is signal amplification. When a neurotransmitter binds to an ionotropic receptor it induces a short and immediate change in the membrane potential locally, i.e. only in the direct vicinity of the ionotropic receptor. This is a transient and short-lived effect. Whereas with metabotropic receptors binding of one signalling molecule can generate thousands of intracellular signalling molecules with long-lasting effects within the whole cell. Let’s look at an example of second messenger signalling, see Figure 2.8 where a Gs-type GPCR is shown. You can see that one ligand molecule binds to the GPCR. As long as the ligand remains bound the GPCR can activate several G-proteins (sequentially, not several at the same time). So this will amplify the signal

because there are now several activated -subunits. These -subunit will bind to one adenylyl cyclase (so no amplification in this step) which will lead to the generation of many molecules of cAMP. These molecules will activate PKA molecules which in turn can phosphorylate many potassium channels which will have a longer lasting and global effect on the cell membrane potential.

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Figure 2.8 – Signal amplification.12

Summary Ionotropic receptors act fast. When a ligand binds an ion channel is opened or closed which leads to an immediate change in the membrane potential across the patch membrane in the immediate vicinity of the receptor. Metabotropic receptors lead to a delayed response. When a ligand binds intracellular signalling molecules are activated. There are two main pathways.

The -complex pathway is relatively fast associating directly with ion

channels and the -subunit pathway is relatively slow generating a cascade of intracellular signals which can lead to various effects such as ion channel activity modulation and up or downregulation of gene expression.

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Neuroscience – exploring the brain by Bear et al

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Intermezzo – Of special interest: the Neuro-Muscular Junction

The neuromuscular junction (NMJ) is similar to the synaptic cleft between two neurons. The pre-synaptic cell (in this case a motoneuron) releases neurotransmitter molecules (acetylcholine) which traverses the cleft and binds to ionotropic acetylcholine receptors present on somatic muscle fibres, see Figure 1.

Figure 1 – the neuromuscular junction13 When two molecules of acetylcholine bind to the receptor it induces opening of its ion channel. The ionotropic acetylcholine receptor can flux both Na+ and K+. At the resting membrane potential the driving force for Na+ to flow in is considerably larger than the driving force for K+ to flow out. Because of this opening of the ion channel will lead to an influx of sodium and a depolarisation of the muscle fibre. If the driving force for K+ to flow out would be larger than the NMJ could not function which would lead to life threatening conditions. It is therefore important to understand how ionic driving forces are maintained within the nervous system, this will be discussed in section 3.

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Image source: http://thebrain.mcgill.ca/flash/d/d_06/d_06_m/d_06_m_mou/d_06_m_mou_2a.jpg

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The neuromuscular junction has a specialisation which increases its efficiency considerably, see Figure 2. The muscle fibre membrane facing the axon terminal of the motoneuron has many invaginations which considerably increases the membrane surface area where acetylcholine receptors can be expressed. Due to this the NMJ is fail safe system, i.e. when acetylcholine is released the muscle will contract.

Figure 2 – The neuromuscular junction14 Figure 3 shows an electron micrograph of the NMJ, notice the axon terminal being loaded with neurotransmitter filled vesicles.

Figure 3 – Electron micrograph of the NMJ (M: muscle T: axon terminal)15

14

http://jessicaz.me.cmu.edu/molecular_data/NMJ_files/image002.jpg 15

http://upload.wikimedia.org/wikipedia/commons/thumb/2/23/NMJ.jpg/300px-NMJ.jpg

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The acetylcholine receptor in the NMJ is ionotropic and it is commonly referred to as the nicotinic acetylcholine receptor, this to distinguish it from the muscarinic acetylcholine receptor which is metabotropic. Since neurotransmitters can bind to more than one type of receptor normally we need a classification system to identify receptors. The nomenclature for receptors is explained in: intermezzo – Some neurotransmitter and receptor nomenclature. Ionotropic acetylcholine receptors have a preference for the agonist nicotine, whereas muscarine binds only weakly. Metabotropic acetylcholine receptors strongly bind the agonist muscarine whereas nicotine binds only weakly.

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3 – Movement of ions across membranes The function of neurons is to convey signals within the nervous system. When neurons are at rest no signals are generated. When a neuron is stimulated, for instance by the release of neurotransmitters by another neuron, the neuron becomes active and generates an action potential. An action potential is a temporary change in the voltage across the cell membrane. In order to understand how exactly neurons can signal one another we first need to understand how neurons behave at rest. Figure 3.1 Figure 3.1 shows two water filled compartments of equal volume. To both compartments KCL is added and initially both compartments are electrically neutral. The compartments are divided by a semi-permeable membrane, which will allow K+ to diffuse through, but not Cl-. The concentration of KCL in compartment A is higher than in compartment B. As a consequence of the laws of thermodynamics ions in solution will have the propensity to diffuse from A (high concentration) to B (low concentration) until the concentration of KCL in both compartments is equal. It was already mentioned that the membrane will not allow Cl- to diffuse so only the potassium ions can diffuse down their concentration gradient from A to B. Do you think that the concentration of potassium ions in compartments A and B will become equal? Figure 3.2 shows what will happen. Figure 3.2

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The concentrations of potassium ions in compartments A and B will not equilibrate. Some potassium ions will diffuse from A to B but essentially the concentrations of ions in compartments A and B will be left unchanged. Because of the presence of a concentration gradient there is a large driving force for the potassium and chloride ions to diffuse from A to B. The chloride ions are blocked by the membrane, but why is it that the potassium ions will not diffuse until the concentration of K+ on both sides of the membrane are equal? What is opposing the flow of potassium ions from A to B? If we could replace potassium with a neutral molecule such as glucose and let’s assume our membrane is permeable for glucose you would see that after a while the concentration of glucose in compartments A and B will be equal. The reason why potassium ions will not equilibrate in our system is due to the fact that chloride and potassium ions are charged and that chloride is not allowed to diffuse from A to B. At the start of our experiment A and B were electrically neutral but when a few K+ ions start to diffuse from left to right compartment A becomes slightly negative and compartment B becomes slightly positive. A separation of charges is brought about and this establishes an electrical potential difference across the membrane, see Figure 3.3. Figure 3.3 At some point there will be no net transfer of potassium ions across the membrane because the ion fluxes (from A to B driven by the concentration gradient and from B to A driven by the electrical potential difference) will become equal. It is a misconception that the transfer of ions across the membrane stops, there is a continuous flux of K+ ions in both directions, the concentrations of potassium in both compartments however remains constant. To summarize: Due to the presence of a concentration gradient there is a driving force which leads to the diffusion of K+ ions from A to B (Cl- ions cannot diffuse because the membrane is impermeable to them). Both compartments were electrically neutral at the outset, however when positive ions diffuse from A to B, compartment A becomes negative and compartment B becomes positive. This generates another driving force, an electrical potential difference across the membrane, which leads to a flux of K+ ions from B to A, as the

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positive ions are attracted to the negatively charged compartment A. The combination of a concentration gradient and an electrical potential difference is commonly referred to as an electrochemical gradient. The driving forces for K+ ions in this example are opposing each other. What are the driving forces for the Cl- ions and are they opposing each other as well? (Try to answer the question first before reading the answer below). Like the potassium ions the chloride ions experience two driving forces. There is a concentration gradient, if the membrane was completely permeable the chloride ions would diffuse from A to B down their concentration gradient. Because compartment A has become negative and compartment B positive (electrical potential difference) the negative chloride ions are attracted to compartment B. Therefore the driving forces for chloride ions are additive in this example. Let us now see how these so far rather abstract concepts apply to real neurons. Apart from potassium and chloride a real neuron has to deal with various other ions as well, sodium and calcium being the most important ones. We represent the real neuron in its natural environment as a two compartment system comparable to the system described in the previous section, with the inside of the neuron as compartment A and the extra cellular solution as compartment B, see figure 3.4. Figure 3.4 Mammalian cells have an unequal distribution of ions across their membranes (see table 1) and it is essential that these concentration gradients are actively maintained at all times.

27

Outside Inside ratio outside / inside

[K+]o = 5 mM [K+]o = 100 mM 1:20

[Na+]o = 150 mM [Na+]o = 10 mM 10:1

[Ca2+]o = 2 mM [Ca2+]o = 0.0002 mM 10,000:1

[Cl-]o = 150 mM [Cl-]o = 13 mM 11.5:1

Table 1 taken from [1] Figure 3.15 page 65. If concentrations of ions were allowed to equilibrate neurons would not be able to fire action potentials. If the nervous system cannot generate electrical signals the human body will shut down immediately. You have to understand how ions are transported across the cell membrane. Figure 3.5 shows a schematic representation of a neuron. When neurons are not actively generating electrical signals they maintain a stable electrical potential difference across the membrane which is commonly referred to as the resting membrane potential (Vm)16. Normally neurons maintain a membrane potential value of around -65 mV. The reason why there is an electrical potential difference across the membrane is because the inside of the cell is negatively charged with respect to the outside. It is the charge difference which determines the value of Vm. Ca2+ and Cl-, although they are involved in many important physiological processes, do not have a big influence on the value of Vm. Figure 3.5

16

Other often used abbreviations which you will come across are ΔΨ and E.

28

It is the charge difference caused by the unequal distribution of Na+ and K+

which sets the value of Vm, not only in neurons but in mammalian cells in general. Learning numbers by heart does not necessarily lead to a better understanding of physiological processes, but if the intra –and extracellular concentrations of Na+ and K+ would be different from the values as shown in table 1 and Figure 3.5 there would not be a single cell in your body which could function normally. So remembering that the concentration of Na+ is higher on the outside of the cell than on the inside and that the concentration of K+ is higher on the inside than outside is essential. In order for any cell to function properly the concentrations of Na+ and K+ have to be stable. There are passive channels in the membrane which will allow Na+ and K+ to flow down their concentration gradient and thereby dissipating their concentration gradients. In order to maintain the concentration gradients all mammalian cells employ an ATP-driven ion pump, the sodium/potassium ATPase, see Figure 3.5. This protein complex actively transports 3 Na+ ions out of the cell and 2 K+ ions into the cell at the expense of one molecule of ATP. The ATPase has to pump ions against their concentration gradient, hence this is not a spontaneous process, it requires energy. ATP (adenosine triphosphate) is the universal cellular energy currency. Generated within the mitochondria these molecules release small packets of energy each time a phosphate bond is hydrolysed where ATP is converted to ADP (adenosine diphosphate). Within the nervous system it is estimated that 70% of all cellular ATP is used just to maintain the activity of the sodium/potassium ATPase.

The equilibrium potential At this stage we understand that the concentrations of Na+ and K+ and several other ions such as Ca2+ and Cl- are fixed under normal physiological conditions. We know that cells maintain a membrane potential of about -65 mV (inside being negative with respect to the outside). We know that is caused due to the charge separation across the membrane of Na+ and K+ ions. But why does Vm settle at this specific value? The answer is that cell membranes have a high conductance for K+ ions which results in Vm being closer to the potassium equilibrium potential than to the sodium equilibrium potential. This answer does not make sense right now but what you should understand at this point is that under resting conditions the flow of potassium ions across the membrane strongly determines the value of the membrane potential17.

17

As we will see later on, during the action potential it is the flow of sodium ions which strongly

determines the value of the membrane potential.

29

It was shown in figures 3.1-3.3 that in a two compartment system separated by a semi-permeable membrane K+ does not equilibrate across the compartments, i.e. the concentration of K+ on one side is different from that on the other side. K+ ions are driven from left to right down their concentration gradient and at the same time K+ ions are driven from right to left down their electrical gradient. So there is constant movement of K+ ions across the membrane in both directions but at some point the flow of K+ ions from left to right will exactly cancel out the flow of K+ ions from right to left. You remember that due to the movement of potassium both compartments became charged. Compartment A being negative and compartment B positive. Because of this an electrical potential difference is generated across the semi-permeable membrane and as you can see in figure 3.3 ions are ‘glued’ to the membrane on both sides. The width of biological membranes is in fact so thin that positive and negative ions can sense the electrical fields from the ions on the other side of the membrane. There is one specific value of this electrical potential difference across the membrane where the flow of ions from left to right and from right to left equal out, this is the equilibrium potential. Under physiological conditions each ion has its own equilibrium potential. The potassium equilibrium potential (EK

+) is -80 mV. So if in a neuronal cell we would set Vm to -80 mV the influx and efflux of potassium would be exactly equal. For sodium the equilibrium potential (ENa

+) is +60 mV. To calculate these numbers we use a relatively straightforward equation called the Nernst equation:

i

oion

ion

ion

zF

RTE

][

][ln

[1]

We will pick this equation apart in this paragraph18. As explained earlier, in a two compartment system separated by a semi-permeable membrane, ions will experience two driving forces: the electrical driving force, drawing negative ions to the positive compartment (or positive ions to the negative compartment) and the chemical driving force which results in ions flowing from the compartment with high concentration to the compartment with lower concentration. At some point both driving forces will be equal and there will be no net movement of ions across the membrane (but remember that the ions move constantly across the membrane). This steady state occurs at one specific value of the membrane potential19 across the membrane. The Nernst equation describes this steady state by equating the membrane potential (E) to the concentration gradient of the ion across the membrane.

18

For those interested in deriving the Nernst equation see the appendix. 19

Technically the name membrane potential is incorrect, it really should be membrane potential

difference but this term is commonly used in text books.

30

We shall take a short look at the components of the Nernst equation: E: membrane potential R: universal gas constant T: absolute temperature z: valence of the ion F: Faraday constant [ion]o : the ion concentration on the outside [ion]i : the ion concentration on the inside ln: natural logarithm (see the intermezzo on page x to read up on logarithms) R is the gas constant (8.314 J K-1 mol-1) which is used here to describe the movement of ions. T is the absolute temperature (K) and is important since the movement of particles in solution is temperature dependent. z is the valence (number of charges per ion) this is important for our concentration because the valence determines how strongly an ion is attracted by the membrane potential. F is the faraday constant (96485 C mol-1) this constant tells you how much charge (in Coulombs) there is on one mole of a univalent ion because in this equation we are not dealing with the behaviour of single ions but with moles of ions. You need not worry to much about understanding R,T,z and F in detail, we use them because our equation is all about movement of ions. Movement is affected by temperature, entropy and charge. The Nernst equation can only be used for one type of ion at the time. Let us use potassium as an example. That means that z = 1 (K+) and we will use

body temperature (37 C) this converts to 310.15 K. Now the RT/zF term becomes 0.026756 V which is 26.76 mV.

i

o

K K

KE

][

][ln mV 76.26

Notice that the unit mV is already put into the equation. Because EK

+ is expressed in mV and the ratio of [K+]o/[K

+]i is has no units.

31

Figure 3.6 Figure 3.6 shows a situation described by the Nernst equation. Two compartments labelled o (outside) and i (inside) separated by a semi-permeable membrane. Potassium can move across the membrane but chloride cannot. You can see from the equation that the membrane potential (E) only depends on the ratio of the potassium concentrations on either side of the membrane. We will use physiological values for [K+] where [K+]o is 5 mM (representing the extracellular situation) and [K+]i is 100 mM (representing the intracellular situation). If we now calculate EK

+ using these numbers we arrive at the value of -80 mV. The Nernst equation allows us to calculate the exact membrane potential where the net flux of an ion across the membrane is zero. The value of E only depends on the ratio of the steady state concentrations of the ion on both sides of the membrane. Summary The membrane potential is determined by the concentrations of ions across the membrane. That is why it is important for you to know the distribution of Na+ and K+ across the membrane because it is the combined driving forces of these ions which determine the resting membrane potentials of neurons.

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4 – The action potential The action potential is a transient change in membrane potential which travels down the axon to the axon terminal. In this section we will take a look at the underlying mechanism of the action potential. Then we will see how the signal originates in the dendrites and subsequently spreads across various regions of the neuronal cell membrane. Figure 4.1 shows the characteristic shape of the action potential.

Figure 4.1 – the different phases of the action potential20. During resting conditions the membrane potential of mammalian cells, including neurons, is about -70 mV. In section 3 we discussed the underlying mechanisms which maintain the membrane potential at this value. When excitatory neurotransmitter, e.g. glutamate, bind to ionotropic receptors on the dendrites positive ions will flow in. As a consequence the membrane potential will start to depolarise. At some point a threshold will be reached and an action potential is generated21. The membrane potential starts to depolarise quickly (rising phase) until it peaks at a positive potential, i.e. the inside becomes positive with respect to the outside, this is called the overshoot. After reaching the maximum depolarisation the membrane potential starts to hyperpolarise (falling phase), i.e. the membrane potential is getting more negative again. In fact, the membrane potential gets more negative than the resting potential for about a millisecond. This is called the undershoot. Then the resting membrane potential is restored. Figure 4.1 indicates that during the falling phase and the undershoot phase there is a refractory period, during this time it is not possible to generate another action potential.

20

http://encefalus.com/wp-content/uploads/2010/07/neuron_action_potential.jpg 21

If the threshold value is not reached the signal will dissipate and no action potential will be

generated.

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4.1 The underlying mechanisms of the action potential. The action potential is a transient change in membrane potential, travelling along the axon. The change in membrane potential is due to the activity of two types of voltage-gated ion channels, the voltage-gated sodium channel (which we already discussed in section 2.1) and the voltage-gated potassium channel. Let’s look at the structure of the voltage-gated sodium channel again, see Figure 4.2.

Figure 4.2 – The voltage-gated sodium channel22 The voltage-gated sodium channel is a transmembrane protein with four large domains. Within each of those domains there is a region containing amino acids with positive R-groups. These regions are the voltage sensors of the channel and when the membrane depolarises these voltage-sensing regions are pushed outward (towards the extracellular side) thereby opening the ion channel. Due to the very large inward driving force sodium ions will rush in and depolarise the membrane. The voltage-gated potassium works in a similar way, i.e. it also has a voltage sensor but when this channel opens potassium ions rush out, hyperpolarizing the membrane. The voltage-gated sodium channel has some interesting characteristics, namely that during the resting potential it is closed. When opened due to a depolarization the channel quickly inactivates. It will stay inactivated whilst the membrane is depolarised and the cell needs to be hyperpolarised before the voltage-gated sodium channel can be deinactivated, see Figure 4.3.

22

Image source:

http://journals.cambridge.org/fulltext_content/ERM/ERM1_19/S1462399499001349sup022.gif

34

Figure 4.3 – gating mechanism of the voltage-gated sodium channel During resting conditions where the membrane potential is around -70 mV the voltage gated sodium channel is closed. As you can see the channel has two gates, one facing the extracellular solution and the other facing the cytosol. When the membrane depolarises and the threshold is reached the channel opens and sodium ions flow in. This influx causes the inside of the membrane to become positive. After about a millisecond the channel inactivates (the cytosolic gate closes) and the channel closes (outside facing gate closes). The channel will remain inactivated as long as the cytosolic membrane potential is positive. Only after the resting membrane potential is restored, due to the activity of the sodium/potassium ATPase (see section 3) will the voltage-gated sodium channel deinactivate.

This explains why there is a refractory period during which it is not possible to generate another action potential. The opening of the channel causes the inside of the cell to become positive. This inactivates the voltage-gated sodium channel. In order to ‘reset’ the channel, the inside of the cell has to become negative again before the channel can participate in the generation of another action potential.

The voltage-gated potassium channel is commonly referred to as the delayed rectifier. It opens as a response to depolarisation, be it with a delay of about 1 millisecond. Its activity serves to bring the membrane potential back to its resting potential, hence the name rectifier. The channels slowly close upon hyperpolarisation of the membrane.

35

Now that we understand how the voltage-gated sodium and the voltage-gated potassium channel operate we will see how their interaction causes the action potential, see Figure 4.4.

Figure 4.4 – The action potential dissected23. During the resting state (a) the membrane potential is -70 mV and both the voltage-gated sodium channel and the voltage-gated potassium channel are closed. Upon depolarization (b) the voltage-gated sodium channels open right away whereas the voltage-gated potassium channels remain closed for another millisecond. The influx of sodium ions causes the rising phase of the action potential. The peak of the action potential approaches the equilibrium potential for Na+ where it plateaus (We will explain why this happens in the next section). At the peak of the action potential voltage-gated sodium channels start to inactivate and voltage-gated potassium channels start to open which leads to an efflux of potassium ions. This causes the falling phase (c) of the action potential (the inside of the cell becoming negative again). Because the voltage-gated sodium channels are closed and the voltage-gated potassium channels remain open longer the membrane potential becomes more negative than the resting potential (d).

23

Image source: http://www.cidpusa.org/I10-40-neuron.jpg

36

Common misconceptions An action potential is not generated by the activities of just one voltage-gated sodium channel and one voltage-gated potassium channel. It is the combined activities of many voltage-gated sodium channels and voltage-gated potassium channels which generate the action potential, see Figure 4.5.

Figure 4.5 – the combined activity of voltage-gated sodium and potassium channels generate the action potential.24 The opening of one channel only changes the membrane potential a little, see parts b and d in Figure 4.5.

24

Image source: http://tainano.com/Molecular%20Biology%20Glossary.files/image002.gif

37

The influx of sodium and the efflux of potassium does not change the ionic concentrations of sodium and potassium on either side of the membrane. Under resting conditions the intracellular sodium concentration [Na+]i is 15 mM whereas the extracellular sodium concentration [Na+]o is 150 mM. During the action potential the inside of the cell becomes positive. This is not due to massive influx of sodium however. In order to change the membrane potential only a minute amount (in the order of nanomoles, i.e. 10-9) sodium ions have to flow across the membrane in order to change the potential. Remember that the ATP driven sodium/potassium exchanged continuously pumps 3 Na+ out and 2 K+ in, see Figure 4.6.

Figure 4.6 – Ionic concentrations are stable. The sodium/potassium exchanger works continuously, also during the action potential and ionic concentrations are kept stable at all times.

38

4.2 How electrical signals spread across the neuron In section 1 we discussed how neurons communicate by releasing neurotransmitters into the synaptic cleft which bind to receptors on the post-synaptic neuron (see figure 1.4). Binding of excitatory neurotransmitters induces a depolarisation of the post-synaptic neuron which can then trigger an action potential. The action potential travels down the axon to the nerve terminal. How does the initial depolarization reach the axon? As you can see in Figure 4.7 a neuron has many different regions. A cell body, dendrites, an axon and the axon terminal.

Figure 4.7 – Schematic representation of a neuron.25 In most cases axon terminals are in close apposition to the dendrites of the post-synaptic neuron, i.e. in most cases the electrical signal leading to an action potential originates at the dendritic membrane, see Figure 4.8.

Figure 4.8 – axo-dendritic communication between two neurons.

25

Image source: http://www.mindcreators.com/Images/NB_Neuron.gif

39

Let’s look again at the synapse, see Figure 4.9. The synapse shown is excitatory, i.e. the released neurotransmitters bind to receptors which leads to depolarisations across the post-synaptic membrane. But how much depolarisation? When the contents of one vesicle are released the post-synaptic membrane is depolarised by approximately 1-2 millivolts. In order to generate an action potential the membrane potential needs to be depolarised by 20-30 mV. Look again at Figure 4.7. How could a 1-2 mV depolarization in the dendrites cause an action potential in the axon? This is obviously impossible. Just like you need many voltage-gated sodium channels to generate an action potential you need many depolarisations in the dendrites in order to reach the threshold.

Figure 4.9 – an excitatory synapse. The depolarization caused by the release of one vesicle filled with neurotransmitters is called an excitatory post-synaptic potential (EPSP). One EPSP is not enough to trigger an action potential, but the combination of several EPSPs could depolarize the membrane up until the threshold value at which an action potential is generated, see Figure 4.10. At point A the pre-synaptic neuron fires an action potential, which leads to release of neurotransmitters into the synaptic cleft. The ensuing depolarisation is below threshold and the signal dissipates. At B the pre-synaptic neuron fires 4 action potentials in succession which leads to accumulated EPSPs, but still not enough to trigger an action potential. At C however a series of 5 action potentials by the pre-synaptic neuron generate enough EPSPs in order to reach the threshold and an action potential is generated.

40

Figure 4.10 – a series of EPSPs leading to an action potential. What happens when inhibitory neurotransmitters are released into the synaptic cleft? The release of one vesicle of inhibitory neurotransmitters leads to a hyperpolarisation of the post-synaptic membrane called an inhibitory post-synaptic potential (IPSP). Let’s see what happens when a neuron receives both inhibitory and excitatory input, see Figure 4.11. Figure 4.12 – the effects of combined excitatory and inhibitory innervation26. You can see that EPSPs and IPSPs have opposite effects. It is quite common in the nervous system that neurons receive both inhibitory and excitatory input, the balance between EPSPs and IPSPs determine whether the neuron will fire an action potential or not.

26

Image source:

http://www.mathworks.com/matlabcentral/fx_files/25788/1/content/epsp_ipsp/HTML/epsp_ipsp.jpg

41

Although the action potential is triggered by depolarisations originating in the dendrites the origin of the action potential is in a specialised region of the neuron called the axon hillock, see Figure 4.7. This region of the neuron contains a high density of voltage-gated sodium channels and when the axon hillock is depolarised the voltage-gated sodium channels open. Dendrites and the cell body contain little to no voltage-gated sodium channels. So how does the electrical signal originating at the dendrites reach the axon hillock? The answer is by passive conduction. Action potentials do not occur in the dendrites, nor in the cell body. The passive spread of depolarisation across the membrane is called a graded potential. Summary The electrical signal originates at the dendrites. EPSPs accumulate and this depolarisation passively spreads from the dendrites, along the cell body membrane to the axon hillock. At the axon hillock voltage-gated sodium channels detect the depolarisation and open which leads to the influx of sodium which causes further depolarisation which opens adjacent voltage-gated sodium channels. This process keeps repeating itself until the action potential reaches the axon terminal where it triggers the opening of voltage-gated calcium channels which leads to the influx of calcium which leads to the release of neurotransmitters.

4.3 Saltatory conduction There is one last topic to be discussed in this section. Myelinated axons have little gaps between the myelin sheets, see Figure 4.7. These gaps are called the nodes of Ranvier. There are high densities of voltage-gated sodium and potassium channels in these nodes. The myelin sheets make sure that the axon is well insulated. When voltage-gated sodium channels open a limited amount of sodium ions flows in which then passively will flow down the axon (in both directions), this passive spread of depolarization will then trigger adjacent voltage-gated sodium channels to open and more sodium can flow in, i.e. this signal is self-propagating. The myelin sheets prevent leakage of sodium ions out of the axon. Basically what happens is that voltage-gated sodium channels in the axon hillock open upon depolarization and sodium flows in. Some of the sodium ions flow down the axon until the first node of Ranvier is reached. If the depolarisation is enough to open the voltage-gated sodium channels there new sodium will flow in and the signal is regenerated. In this way the signal seems to ‘jump’ from node to node, hence the name ‘saltatory conduction’. In neurological conditions such as multiple sclerosis the myelin sheets covering the nerve axons are attacked by the immune system which interferes with nerve conduction. This will be discussed further in the case study on multiple sclerosis.

42

Case study – Multiple Sclerosis Multiple sclerosis (MS) is a neurological condition where nerve conductivity is impaired due to demyelinisation of the nerve axons, see Figure 1. Symptoms include weakness, lack of coordination and both impaired vision and speech. MS targets the myelin sheaths covering nerve bundles in the brain, spinal cord and the optic nerve. Sclerosis is derived from the Greek word for ‘hardening’ which describes the lesions found around axon bundles. This hardening is multiple because MS targets many sites in the nervous system simultaneously.

Figure 1 – demyelinisation is the cause of multiple sclerosis27 Although the aetiology is unclear it is believed that MS is caused by the immune system inappropriately targeting myelin. The degradation of myelin leads to a loss of conduction of the nerve fibres. Saltatory conduction is dependent on having the nodes of Ranvier (which contain high densities of both voltage-gated sodium and potassium channels) interspersed by patches of myelin. See section 4.3.

27

Image source: http://static.howstuffworks.com/gif/multiple-sclerosis-demyelinization.gif

43

Case Study – Cystic Fibrosis Cystic fibrosis (CF) is not directly a disease of the nervous system but the cause is a dysfunctional ion channel which conducts chloride ions. Figure 1 shows the pathology of CF. Due to a salt imbalance mucus starts to accumulate in the lungs which becomes an excellent breeding ground for bacteria. Infections lead to an inflammatory response which adds to the mucus obstruction which provides even more possibilities for bacteria to thrive. Figure 1 – Cystic fibrosis pathology28. The ion channel responsible for this condition is called the Cystic fibrosis transmembrane conductance regulator (CFTR), see Figure 2. Due to a genetic mutation one amino acid is deleted from the protein rendering the ion channel dysfunctional. Treatments can only alleviate the symptoms but not cure this condition. The best approach would be to screen parents for the genetic mutation and if necessary use gene therapy on the embryo as soon as this approach becomes feasible.

28

Image source: http://pics.hpathy.com/cystic-fibrosis.jpg

44

Figure 2 – the Cystic fibrosis transmembrane conductance regulator (CFTR).29

29

Image source: http://www.thepregnancyzone.com/wp-content/uploads/2009/01/cystic-fibrosis.gif

45

Case study – Euthanasia Figure 1 – Injection with potassium chloride30 In sections 3 and 4 it was explained that in order for the cells in the nervous system to function the ionic concentrations on either side of the cellular membrane has to be stable. The intracellular concentration of potassium has to be high (around 100 mM) and the extracellular concentration needs to be low (around 5 mM). If the extracellular concentration of potassium were to increase this would change the equilibrium potential for potassium. Remember the Nernst equation (see section 3).

i

oion

ion

ion

zF

RTE

][

][ln

Let’s rewrite it for potassium and put some numbers into the equation for RT/zF.

30

Image source: http://cdn.wn.com/pd/71/6c/854975bb8e8966ea3983cc28cdc0_grande.jpg

46

i

o

K K

KE

][

][ln mV 76.26

If [K+]o is 5 mM and [K+]i is 100 mM then EK

+ will be: -80 mV If the extracellular concentration increases to say 30 mM EK

+ becomes: -32 mV You know from section 3 that the cellular membrane during resting conditions is highly conductive for potassium ions (and barely conductive for sodium ions). That means that The equilibrium potential for potassium almost completely determines the resting membrane potential. When the extracellular concentration of potassium increases EK

+ deceases, that means that cells in the human body will be depolarised considerably. Remember from section 4 that voltage-gated sodium channels become inactivated during the depolarising phase of the action potential? In order to ‘reset’ these channels the cell has to be hyperpolarised again, however with high concentrations of potassium in the blood stream and extracellular fluid this is not possible. The cells remain depolarised. It is not possible to generate action potentials anymore and the as an immediate consequence the heart would stop beating. In countries where it is legal to assist people in ending their life, called euthanasia one method which is used is the administration of potassium chloride to the blood stream in conjunction with an anaesthetic.

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Case study – Myasthenia gravis

Myasthenia gravis is a disease where the patient experiences muscular weakness and extreme fatigue. Myasthenia gravis is an autoimmune disease where the immune system for unknown reasons produces antibodies targeting the acetylcholine receptors in the neuromuscular junction. If acetylcholine cannot bind to its receptors on the muscle fibres the motoneurons cannot reliably innervate the somatic muscles.

Can you think of treatments which can alleviate this condition?

Figure 1 – Myasthenia gravis is caused by a dysfunctional neuromuscular junction.31 One commonly used treatment is the administration of acetylcholinesterase inhibitors. Acetylcholinesterase is an enzyme present within the cleft of the neuromuscular junction which breaks down acetylcholine. Inhibition of this enzyme will keep levels of acetylcholine in the neuromuscular junction high, which will facilitate the innervation of muscle fibres in patients suffering from Myasthenia gravis. Targeting the immune system by removal of the thymus gland and/or administration of gamma globulin also alleviates the symptoms.

31

http://jama.ama-assn.org/content/298/1/29/embed/graphic-1.jpg

48

Case study – Excitotoxicity Various conditions such as cardiac arrest, stroke, seizures, brain trauma and oxygen deficiency can lead to a vicious cycle of glutamate release. When levels of released glutamate reach a high concentration it can lead to neuronal death by overexcitation, a process known as excitotoxicity. Neurons in the brain, like all other cells, need a continuous supply of oxygen and glucose. During a stroke the blood supply to the brain is disturbed. Within the cells of our body the mitochondria utilize oxygen and glucose to generate ATP. If there is no supply of oxygen cells can still produce ATP anaerobically but this does not yield enough molecules of ATP to keep the cell alive. What happens to the membrane potential when ATP levels drop? In order to understand the relationship between ATP levels and the membrane potential Figure 3.5 is reproduced below. In section 3 you can read in-depth about the establishment of the resting membrane potential but here is a short summary. The resting membrane potential is due to a charge imbalance across the membrane with the inside of the cell being negative with respect to the outside. This charge imbalance is due to the continuous operation of the ATP-driven sodium / potassium exchanger. This leads to a high concentration of sodium outside and a high concentration of potassium inside. When ATP levels drop, the exchanger no longer functions which causes the charge imbalance to disappear and the membrane potential to depolarise.

49

The dissipation of the membrane potential has serious consequences. Below figure 1.4 is reproduced. When an action potential reaches the axon terminal the membrane is depolarised. This leads to the opening of voltage-gated calcium channels and calcium flows in. This then leads to the release of neurotransmitters. When glutamatergic neurons within the brain are depolarised due to the lack of ATP this will also lead to the opening of voltage-gated calcium channels and more glutamate is released. Glutamate leads to excitation of neurons which leads to further depolarization, which leads to further influx of calcium, which leads to further release of glutamate etc. The increased concentrations of calcium leads to cell swelling and activation of intracellular enzymes which catalyse the degradation of lipids, proteins and nucleic acids. When the area of the brain that was deprived of oxygen and glucose has its blood supply restored an even more serious condition arises, reperfusion injury. Under the anoxic conditions many so called reducing equivalents were formed, i.e. highly reduced biomolecules. Oxygen which arrives to anoxic cells readily reacts with these reduced molecules to form reactive oxygen species (ROS), e.g. O2

-. The reduced oxygen species are highly reactive, breaking down lipids, DNA and proteins. To summarise, blood deprivation can cause serious damage to the brain in a two-stage process, cell damage due to calcium overload (excitotoxicity) and cell damage due to the formation of ROS (reperfusion injury).

50

Case study – Death by pufferfish Fugu is a Japanese dish prepared from the meat of pufferfish32. Within its organs the pufferfish contains lethal amounts of the poison tetrodotoxin (TTX). Ingesting minute amounts can be fatal. The meat of this fish is considered a delicacy in Japan and when the dish is well prepared it will induce a numbness of the tongue and lips. If properly cleaned the consumer dies. TTX blocks sodium channels. Can you think of a reason why blocking sodium channels would be lethal? The figure below shows the stages of the action potential (which is explained in-depth in section 4.1). In short, when due to depolarization of the membrane the threshold value is reached voltage-gated sodium channels open which leads to the influx of sodium ions, which are carried along the axon leading to opening of other voltage-gated sodium channels. In this way the action potential is propagated along the axon until it reaches the axon terminal where it induces the release of neurotransmitters. When TTX blocks these sodium channels it becomes impossible to generate action potentials. As a direct consequence there will be a direct paralysis of the muscles and the patient will die of asphyxiation whilst being fully conscious.

32

Fugu is also the Japanese colloquial name for pufferfish.

51

Figure 4.4 reproduced

52

Sensory neurons Sensory neurons are at the periphery of the nervous system and they employ a wide range of mechanisms to detect stimuli from the environment. In this section we will look at the senses in our body. Receptor cells of the eye The sensory neurons of the eye are the photoreceptors in the retina, see Figure 1. Light rays hit the retina at the back of the eye. The photoreceptors can be divided into two categories: rods and cones, see Figure 2.

Figure 1 – The eye33

The photoreceptors contain stacks of pigments which are sensitive for light rays. The pigments within the receptor outer segments form part of G-protein coupled receptors, which means that when activated, these proteins induce a cascade of intracellular biochemical reactions. When light hits the pigment molecules the membrane potential of the photoreceptor cell changes due the stimulation of these G-protein coupled receptors called rhodopsin. Neurotransmitters released at the synaptic endings of the photoreceptors bind to bipolar cells which subsequently stimulate ganglion cells, figure 3. There are several other cell types in the retina, the horizontal cells and the amacrine cells that serve to modulate the output of the receptor and the bipolar cells. When light hits the eye the photoreceptors innervate the bipolar cells which innervate the ganglion cells. The axons of the ganglion cells leave the eye through the optic disc and form the optic nerve which carries visual information to higher centres of the brain.

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Image source: http://drugster.info/img/ail/619_622_3.jpeg

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Figure 2 – photoreceptors: rods and cones.34

Figure 3 – Retinal cells35

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http://thebrain.mcgill.ca/flash/d/d_02/d_02_m/d_02_m_vis/d_02_m_vis_1a.jpg 35

http://www.arn.org/docs/glicksman/110104%20fig1.jpg

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Receptor cells of the ear

Figure 4 – The auditory system36 The physical nature of sound is a variation in air pressure. Sound waves enter the auditory canal (see figure 4) and hit the eardrum, subsequently the three smallest bones in the human body (hammer, anvil and stirrup) move and exert pressure on the oval window of the cochlea and the vestibular system. The vestibular system consists of semi-circular canals and is responsible for the sense of balance whereas the cochlea is a long tube, coiled back onto itself, responsible for audition. The cochlea and vestibular system are filled with fluid so air movement is converted to fluid movement when the oval window is pushed. Within the semi-circular canals and the cochlea are the auditory sensory neurons, the haircells, see figure 5. Haircells project stereocilia apically which move in response to the sound induced fluid movements. Within the cochlea there are inner and outer hair cells, the inner hair cells are responsible for the sensorineuronal detection of sound whereas the outer hair cells serve to amplify the sound signals. When the stereocilia of haircells move ion channels open allowing positively charged ions to flow into the cells. This leads to depolarization and the release of neurotransmitters onto the auditory nerve which carries the information higher up into the brain. This mechanism where a mechanical stimulus (sound) is converted to an electrical signal (change in voltage of the haircells) is called mechano-electrical transduction.

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Image source: http://tsi.nacd.org/images/aud1.jpg

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Figure 5 – haircells within the cochlea37 Figure 6 – Inner and Outer hair cells within the cochlea38

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Image courtesy of professor C.J. Kros 38

Image courtesy of professor C.J. Kros

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Taste receptor cells Unlike the hearing cells which convert a physical stimulus into an electrical response the neurons of the gustatory system convert a chemical stimulus into an electrical response. The tongue consist of papillae clearly visible by eye, within each individual papilla one can find between one to several hundred taste buds. Each taste bud typically contains 50-150 taste receptor cells, figure 7. Figure 7 – The gustatory system39 Papillae are classified as sweet, bitter, salt or sour sensitive. A taste receptor will respond to being exposed to a solution which is either sweet, sour, salty or bitter, but only if the concentration is above a threshold level. Within a taste pore are several taste receptor cells, if enough solute flows into the pore the receptor cells will depolarize and fire action potentials. This information, unlike any of the other senses, is taken first to the brain stem before it is relayed to higher level brain centres such as the thalamus. The taste system employs more than one method to detect taste. Salt and sour tastes are detected through ions which directly pass through ion channels, bind and block ion channels (sour), bind to and activate G-protein coupled receptors (sweet, bitter and unami). The reason these stimulations are called chemical is because chemicals in solution directly stimulate the receptor cells. In the cause of salt and sour ions flow through channels so one could argue that this is in fact a biophysical process.

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Image source: http://fau.pearlashes.com/anatomy/Chapter%2026/Chapter%2026_files/image002.jpg

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Smell receptor cells Figure 8 – The olfactory system40 Within our nasal cavity is a small sheet of olfactory epithelium in which the smell receptor cells are situated. The olfactory neurons are one of the few types of mammalian neurons that regenerate. Chemical stimuli present in the air we breathe (and sniff) called odorants. Olfactory receptors have sensory cilia (see figure 8) which express G-protein coupled receptors to which the odorants bind. This leads to intracellular signalling that caused membrane depolarization. If the depolarization crosses the spike threshold the olfactory cells will generate action potentials which travel down their axons into the CNS.

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Image source:

http://upload.wikimedia.org/wikipedia/commons/thumb/2/20/Olfactory_system.svg/800px-

Olfactory_system.svg.png

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Touch and pain receptor cells Figure 9 – Touch and Pain Receptors41 The touch and pain receptors are a mixture of both mechanosensitive and chemically sensitive receptors. Figure 9 shows you the pain and touch receptors present in the skin. There are certain touch receptors that respond to strong pressure, called Pacinian corpuscles. As you can see from the figure these receptors appear to have layers of tissue surrounding a nerve, like layers of an onion. These layers when squeezed (due to pressure) transmit this mechanical stimulus into an electrical one by forcing open mechanically gated ion channels in the nerve. So this again is an example of mechano-electrical transduction. You can also see a pain receptor in the epidermis which is basically a free nerve ending, which can respond to e.g. acids, like HCl. This then leads to the generation of an action potential coding for a painful stimulus. There are also pain receptors which employ G-protein coupled receptors.

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Image source: http://pmr-

science.wikispaces.com/file/view/c7.49.3.skin.jpg/32414373/c7.49.3.skin.jpg

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Appendix 1 – logarithms and the Nernst equation. What are logarithms? I will not bother you with any formal definitions. Logarithms are mathematical functions that you can use to convert numbers to a different scale. Here is an example where the metabolic rate is correlated to the body mass of a range of mammals, see figure 8. Figure 8 taken from Tatsuo Motokawa, "Elephant's Time, Rat's Time". Figure 8 shows that there is a linear relationship between body weight and metabolic rate. The horizontal axis denotes the body mass. The bottom row of numbers represent the weight in kg and you can see that the units are not equally spaced apart. That makes sense because the body weight of a mouse is less than 100 grams whereas an elephant weighs over 1000 kg. You would not be able to plot their weights on the same scale. In order to plot a range of magnitudes in one figure the numbers have been transformed to a logarithmic scale, see the top row. There you can see that the units are equally spaced apart. As you can see from the bottom row each successive unit is 10 times bigger than the previous one. In order to convert the scale a logarithm with base 10 is used.

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Here is the equation necessary to convert normal numbers to a logarithmic scale: blog x = y (where b stands for base) This means that by = x Let’s find the 10log for 1000. 10log 1000 = y 10y =1000 Y = 3 Normally 10log is written as log as it is commonly understood that 10 is used as a base. So log 1000 = 3. You can use any number as a base for a logarithm but in practice you will only encounter two types: the logarithm with base 10 (10 log) and the natural logarithm (nl). The natural logarithm uses the mathematical constant e as its base: elog. e = 2.7182 so nl = 2.7182log. Now is the time to bring out the calculator because this is not a number that you can easily use. But the rules are the same, so let’s see what the natural logarithm is for 1000: ln (1000) = elog 1000 = y ey = 1000 y = 6.91 Here is another example to convince you of the usefulness of logarithms. Remember the definition of pH? pH = - log [H+] The pH tells you how acidic a solution is. Here are some values: gastric acid 0.7 urine 6.0 blood 7.34-7.45 coffee 5.2 – 6.9

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The acidity is determined by the concentration of protons in solution [H+]. Here is the same list again but now with proton concentrations: gastric acid 0.2 M urine 0.000001 M blood 0.000000035 – 0.000000045 coffee 0.000000126 – 0.000000631 You can see that using [H+] can get very cumbersome. That’s why the pH standard was introduced. Let’s apply the equation to the urine example. pHurine = - log [H+]urine = - log 0.000001 - log 0.000001 = pHurine

log 0.000001 = - pHurine

10 – pHurine = 0.000001

- pHurine = -6 pHurine = 6 Working with logarithms might seem a bit tedious but as the pH example illustrates, logarithms make our lives easier. Back to the Nernst equation.

i

oion

ion

ion

zF

RTE

][

][ln

Sometimes the Nernst equation is presented in a slightly different shape:

i

oion

ion

ion

zF

RTE

][

][log3.2

Can you think of a reason why the multiplication factor 2.3 has been introduced here? In the first equation the natural logarithm is used whereas in the second equation 10log is used. In order to convert from ln to 10log you have to multiply by 2.3 Why do you need to multiply by 2.3? We calculated previously that log 1000 = 3 and that ln 1000 = 6.9. 6.9/3 = 2.3

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1. Bear, M.F., B.W. Connors, and M.A. Paradiso, Neuroscience -

Exploring The Brain. second ed. 2001, Baltimore USA: Lippincot Williams & Wilkins. 855.