EXCITABLE CELLS: YEAR 1 SEMESTER 2LECTURE 1 – INTRODUCTION

LECTURE 2 – IN AND OUT OF THE BAGAll known cells exist in an aqueous environment, so it is necessary to know the properties of water. Water is one of the most complex molecules known, oxygen is electronegative so draws charge from the hydrogen atoms, giving them a δ+ charge and the bonding angle of water is 105°. Water easily forms hydrogen bonds between molecules in a solution and is essentially a dynamic charged, hydrogen bonded polymer. The implications of the structure of water mean that it has an anomalously high boiling point and is an excellent solvent for polar molecules. Solvents are liquids which dissolve solutes. Polar molecules have polar groups such as –OH, NH2, COOH and any ion; non-Polar groups include alkyl chains, benzene rings and cyclohexane.

The important ions for membrane biology can be divided into two groups: those which are physiologically useful, such as Na+, K+ and Cl-, and those which are biochemically useful, such as Mg2+. Some ions also fall into both categories, such as Ca2+. Ions attract water and therefore have hydration shells, the size of which depends on the ion radius; small ions have a high charge density and therefore attract more water molecules than high large ions. This means that the size of an ion depends on its hydration shell radius, the bigger the hydration shell, the less motile it is and hydration therefore impedes ion flow.

Biological membranes are made of water insoluble lipids; phospholipids, glycolipids or cholesterol. The main feature of membrane lipids is that they are amphipathic, having regions which are polar (head) and non-polar (backbone). Since the head group is polar it attracts water molecules and vice versa, allowing a lipid bilayer structure to be established.

LECTURE 3 – THE PROTEINS: PUMPS, CARRIERS AND CHANNELSThe lipid bilayer is very impervious to polar molecules, especially ions, allowing the concentrations to differ on either side of the membrane. Concentration gradients represent a source of energy and big concentration gradients can do more work than little ones. Concentration gradient energy can be used in the following ways:

Chemical work ATP synthesis, transportMechanical work Rotation of flagella

Cell volume regulation Generation of osmotic potentialsCell homeostasis Efflux of toxic compounds

Chemiosmosis Generation of ATP with H+ ions in ETCSignal transduction Action potentials, Ca2+ signalling

Cells use special protein pumps to transport ions against their gradient, which requires energy in the form of ATP. They work fairly slowly and nearly always move cations, such as Ca2+ and Mg2+. The sodium (Na+/K+ ATPase) pump is extremely important and cells expend 25% ATP to sustain these pumps. It extrudes 3Na+ and 2K+ for every molecules of ATP hydrolysed and generates a gradient as shown to the left. Inhibitors of this pump, such as digoxigenin, are therapeutically and toxically important. Since it blocks the pump, Na+ concentration builds up in heart cells, causing increased [Ca2+] to accumulate, thus increasing the contractility of the heart; however too much digoxigenin can cause heart and nerve failure.

Ion channels use an electrochemical gradient and are often selective. Voltage gated ion channels are tetramers which have a central pore, a voltage sensor and a coupling mechanism, as well as an inactivation mechanism to close the channel. Many toxins block voltage-gated channels, such as conotoxins from the venom of marine cone snails, which plug the channel pore mouth. Ligand gated on channels are pentamers which have a central pore, a ligand binding site, a coupling mechanism and desensitisation mechanisms, which close the channel if the ligand remains bound for too long.

LECTURE 4 – DIFFUSION, PERMEABILITY AND ELECTRICITYDiffusion is the spontaneous movement of molecules from regions of high to low concentration, which dissipates the concentration gradient. Molecules that punch holes in membranes are said to facilitate diffusion and examples include:

Detergents

Saponin

Antibiotics Gramicidin, Valinomycin, AlamethicinToxins Mellitin (bee stings)

Viruses Influenzavirus M2 protein

Molecules in liquids are in constant motion due to thermal agitation, for water molecules the average centre to centre distance is about 2.8Å. Fick showed that the number of molecules (N) moving across an interface is proportional to the area of the interface (A) and the concentration gradient.

Einstein showed that diffusion was due to a random walk of molecules and that the time it takes for a molecule to travel away from its starting point depends on whether the molecule moves in 1/2/3 dimensions. A molecule can diffuse much faster if it travels in three dimensions because the chances of bumping into other molecules are lower. This is why catalysts work; by providing a surface area to allow reactions on. Signalling molecules therefore have longer ranges if they are not bound to membranes.

one dimension : t= d2

2Dtwodimensions : t= d2

4Dthree dimensions : t= d2

6D

Where t is time, d is the root mean square distance in cm, and D is the diffusion constant in cm2/sec.

The movements of ions under the influence of an electric field are called electrophoretic movements, which add to, or subtract from diffusion. The total gradient is called the electrochemical gradient.

Electrochemical gradient=Gradient ¿diffusion−Gradient ¿elecrophoreticmovement

The rate of ion movement across a membrane depends on four key factors; the size of the electrochemical gradient, the nature of the ion, the number of open ion channels and the ion channel’s properties, such as its selectivity and permeability for specific ions.

LECTURE 5: RADIOTRACERS, DYES AND ELECTROPHYSIOLOGYWe need to be able to detect channels, assays show that cells make channel proteins, but to determine whether or not the channel is actually functional, we need to be able to assay its activity. Ion flux can be measured using radioactive dyes, imaging dyes or currents using electrophysiology. Radioactive dyes show increased radioactivity in proportion to ion flux through the channels and radiotracer measurements can also be performed on cells:

Types of radioactive ions Channel IonCalcium 45Ca2+

Potassium 86Rb+

Sodium 22Na+

Chlorine 36Cl-

45Rb2+ is used instead of a radioisotope of K+ because it has the same function whilst being safer to use than radioactive potassium.

The disadvantages of radioactive ion flux assays is that radioisotopes are hazardous and have poor spatial resolution, meaning specific areas in cells or tissues are hard to resolve clearly, whilst also having poor sensitivity, meaning we can’t look at a few channels. Another major problem is time resolution. If the system is left long enough, vesicles will still fill even if the protein channel is not fully functional. “Long enough” generally refers to a length of time in milliseconds, which is far too fast to measure manually, so rapid kinetic millisecond events need to be recorded, meaning horrific mathematics and expensive equipment.

Ion sensitive dyes bind ions and change colour (wavelength) or brightness (intensity), a change which can be detected optically using an imaging microscope or spectophotometry. In the example below, the colour of the solution changes according to the calcium ion concentration. Knowing the fluorescence intensity we can determine the Ca2+ concentration through comparison before and after the Ca2+ was added.

The fluorescence intensity in the absence of Ca2+ is 10 units and in 50nM Ca2+ is 60 units. What is the [Ca2+] when the fluorescence is 40 units?

Equation of a straight line : y=mx+c

¿ this case :F=m∙ ¿¿

At Fmin :10=m∙0+10∴Fmin=10units

Where¿¿

At F=40 :40=1∙¿¿

Other dyes are also being developed; the idea is to get them to be selective for specific ions. So far dyes to measure Na+, K+ and Cl- have been created, but none are good as Ca2+ selective dyes. These dyes are so important so calcium fluxes can be looked at in real time and so the spatial distribution of ion flux can be determined. The problem with indicator dyes is that they are polar molecules and so cannot get into cells! The polar molecule is therefore made into a non-polar molecule by adding an ester group, and then cleaved by naturally occurring esterases in the cell, allowing the polar dye to be liberated and to bind Ca2+.

Electrophysiology allows currents in physiological samples, like cells, to be measured using recording equipment. Ion flow is equivalent to current and voltage is potential difference, there can be no ion flow (current) if no potential difference exists. A higher resistance (R) produces smaller currents for the same voltage. This principal is Ohm’s law (V=IR), but electrophysiologists use a re-arranged equation:

Current ( I )=Volts (V )×Conductance( 1R )

Electrophysical recordings are extremely fast events, ranging from the sub-millisecond timescale upwards, they are extremely sensitive; as little as one ion channel can be detected, the spatial resolution is good and it allows the details of individual channels to be recorded, such as activation, inactivation and pore properties.

Radiotracers Ion-sensitive dyes ElectrophysiologySpeed Poor Fast Fast

Spatial resolution Poor Excellent GoodSensitivity Poor Good ExcellentMajor uses Biochemical assays Imaging ion fluxes in cells Channel properties and distributions

LECTURE 6 – ELEMENTS OF NEURONAL BIOPHYSICSNewton’s first law states that a body remains at rest, or moves in a straight line at a constant velocity, unless acted upon by a net outside force. Newton’s second law states that the acceleration of an object is proportional to the force acting upon it. F=ma. Newton’s third law states that whenever one body exerts a force upon a second body, the second body exerts an equal and opposite force upon the first body.

The SI unit for quantity of electricity or electric charge is the coulomb (C), which represents approximately 6.24x1018 elementary charges (the charge carried by a single proton or electron). An electron can be removed from an atom, which gives it a charge. A positive ion is called a cation (electrons taken) and a negative ion is called an anion (electrons added).

A capacitor is an electronic device that can store energy in the electric field between a pair of conductors ("plates"), and biological membranes behave very much in the same way so the electric potential within a membrane and an ion channel is approximately constant. Electric potential (V) is a measure of how much kinetic energy (½ mv2) can be generated by the electric field acting on a unit charge. Given a static distribution of electric charges, the electric potential is defined for every point in space, such as the difference in electric potential between two points A and B (∆V=V B−V A), which is a measure of how much kinetic energy a charge will acquire when moving freely from point A to point B. Biological membrane voltages are usually best dealt with in millivolts (mV).

Transmembrane potentia l=Intracellular potential−Extracellular potential

Electric current is a flow of charged particles and its rate of flow is measured with an ammeter. The quantity of charge passing through a surface is equal to the rate of charge flow x time. Q = It. When a charge “falls” through an electric field it loses electrical potential energy, which can be maintained using a power source. According to Ohm’s law the resistance of a conductor increases as its temperature increases.

LECTURE 7 – ORIGINS OF MEMBRANE POTENTIALSThe intracellular fluid in cells is neutral; charge can only accumulate on the internal membrane surface and an equal and opposite charge will always appear on the external side of the membrane, which is necessary to ensure the extracellular fluid has the same potential (equipotential). All neurons are negative at rest and typical resting values range from -50mV to -80mV; the electric field is therefore directed from the outside towards the inside of the cell and a cation will follow it, while an anion will go against it. The transmembrane potential gradient is also present within the ion channels that are located across the lipid bilayer, but diffusional processes are also at work within the cell.

All systems move towards a state of equilibrium, thermal agitation causes particles in a solution to move all the time in a random fashion, if non-charged particles are placed in two compartments, only diffusional forces act upon them. Concentrations are measured in moles/litre, and fluxes in moles/second.

A bath is set up with two compartments, separated by a membrane containing pores only permeable to K+. A high concentration of a salt, KA, is introduced into the left side, and a low concentration into the right. At first the voltmeter reads 0mV since both sides are neutral, then potassium ions immediately start to diffuse down their concentration gradient into the right side and a net positive charge builds up; an electrical potential difference appears across the membrane because the anion cannot cross it. The positive side soon begins to repel K+ ions until an equilibrium value (Ek) is established.

At Ek , electrical force=diffusional force , sosystem stops changing

Equilibrium potentials are essential in any description of biological membrane potentials. A formula can be found for Ek, the equilibrium potential for K+ ions. This is the Nernst equation. Ek must be more positive on the side where there is less K.

EK=RTF

ln[K ]o[K ]i

Where R is the thermodynamic gas constant, T is the absolute temperature and F is the faraday constant.At room temperature (20°C), RT/F is about 25mV

The physical meaning of the Nernst equation: If we have a membrane permeable to K+ ions and we want to maintain a concentration difference between the two sides, we have to apply a transmembrane electric potential difference to counteract the diffusional force which tends to move ions down their concentration gradients. We call this the equilibrium potential. To maintain a concentration difference, the electric field must be directed from the less concentrated side to the more concentrated side. This implies that the electric potential must be larger on the less concentrated side. In neurons the K+ concentration is much more concentrated inside the cell than outside, therefore EK is negative (more positive potential outside).

LECTURE 8 – THE ACTION POTENTIAL IThe equilibrium potential is given by the Nernst equation and depends logarithmically on the ratio of K +

transmembrane concentrations, to maintain the concentrations at a specific ratio, at a given temperature the same transmembrane potential must exist. This cannot be created by potassium ions alone, so other ions must be present on the two sides of the membrane.

EK is not necessarily the actual value of transmembrane potential given by K o and Ki, it is the value required for a net flux of potassium through the channels of the membrane. If the membrane potential (Vm) is different from EK, there will be a net ionic movement through the potassium channels. The current caused by this is:

IK=gK (V m−EK )

Where I is the ionic driving force and g is the ionic conductance

To calculate the resting membrane potential of the cell consider a cell with a membrane permeable to K +, Na+

and Cl-. (For these purposes we can leave Ca2+ out, because it is less important in electrical signalling). Electrical

V=Ek

Larger electric

potentialElectric field direction

equilibrium means there must be no net current flowing through the membrane, since the membrane potential does not change at rest. By rearranging the above equation we can obtain a “weighted average” of ENa, EK and ECl.

V m=(ENa×gNa)+ (EK× gK )+(ECl×gCl )

gNa+gK+gCl

In neurons at rest, potassium and chloride conductances are larger than sodium. Although this is a rough model of the membrane potential, a more complete description should also take into account the presence of the Na/K ATPase pump.

The ionic basis of the action potential was understood through experiments on the squid giant axon while the squid was still alive (1939-1950). The large diameter of the axon allowed the use of several intracellular electrodes; the axon could then be excited by an electric shock which was delivered by a stimulating electrode. In 1939 it was shown that the action potential was due to a selective increase in Na+ permeability rather than a generic breakdown in membrane resistance. Membrane resistance breakdown would cause the membrane potential to collapse to

0mV, where an increase in Na+ permeability leads the cell towards ENa, the equilibrium potential for Na+ ions. It was found that reducing the [Nao] decreased the action potential overshoot.

So what is it that causes the falling phase of the action potential? If it were just Na + channels closing, the

membrane potential would slowly return to the resting level. The answer is that there is a second large increase in membrane permeability caused by the opening of voltage-activated potassium channels. The action potential results from a quick increase in the sodium permeability of the membrane where Na+ entry drives Vm

towards ENa. Repolarisation is caused by a slower increase in potassium permeability where K+ exit drives Vm

toward EK.

A neuron’s membrane contains thousands of Na+ and K+ channels, each of which can either be open or closed, their behaviour is probabilistic rather than deterministic and the probability of finding them open depends on the membrane potential (more will be found open at more depolarised potentials). The effect of a depolarisation on sodium conductance is regenerative; a greater depolarisation leads to a greater number of Na+ channels opening, and more Na+ entering as a result (positive feedback). The voltage-dependent activation of potassium conductance is self-limiting; Depolarisation leads to more K+ channels opening, but a greater potassium efflux causes repolarisation which triggers the channels to shut (negative feedback).

LECTURE 9 & REVISION LECTURE – THE ACTION POTENTIAL II When an ion channel opens, ions flow down their concentration gradient and a blip of current is recorded. Ion channels work according to an on or off mechanism, but some channels have a really big conductance which lets through may more ions and produces a greater current. Over long periods of time, channels open many times and activation of channels increases the probability of them opening. Rarely channels may also open spontaneously without any external stimulus being present. If a current leaves the cell, deflection is shown on the graph as going upwards, while current entering deflects the graph downwards. When many channels open, tiny blips build on top of each other and all sorts of shapes may be produced, such as waves or nerve action potentials.

Neher and Sakmann developed a revolutionary method of studying individual channels. This was termed patch clamping and gained them the Nobel prize in 1991. Patch clamping uses a tiny capillary tube, which is heated

in the middle and slowly pulled apart to make a micro-pipette that is finer than a human hair and this is used as an electrode. The pipette (electrode) is filled with an electrical conducting solution and connected to a very fast amplifier and recording equipment. An electric controller is used to set the desired membrane potential. The living cell is kept in a bath solution to keep it alive throughout the experiment. With the current and voltage as known values, conductance can be deduced from a graph. If the voltage is kept constant, the current allows the channel conductance to be determined, which measures how well the channel works, and the various properties of the channel. We can use this technique to see how the channels are regulated by the cells and drugs and toxins can be added to see how the channels are affected.

Whole cell configuration is used to record currents through active channels in the whole cell by inserting the pipette into the cytoplasm of the cell. It is good for looking at cell currents in response to drugs added from the outside, or the regulation of channels by the cell itself.

Cell attached configuration allows the current through a few active channels to be recorded at the cell surface by sucking the pipette onto the surface over the cell over a certain type of channel. This focuses the attention on single channel currents in response to the regulation of the whole cell.

When we don’t want any interference of the cell on the functioning of the channel, a piece of membrane can be ripped off and analysed away from the cell. Voltage gated channels always have an intracellular face, which binds cytoplasmic regulators and enzymes, and an extracellular face, which binds drugs and toxins. The same is true for ligand binding. Solutions in patch pipettes cannot be easily changed, but the bath solution can.

Inside-out configurations are used to record currents though a channel away from the cell by removing the membrane and placing it in a bath, the environment of which can be changed. This is good for looking at agents that modulate the channel at its intracellular face. Agents can be added to the bath solution to change the cell environment. Outside-in configurations use the same technique, but using agents that modulate the channel at its extracellular face.

LECTURE 10 – GROSS ORGANISATION OF THE NERVOUS SYSTEMThe nervous system is divided up into the central and peripheral nervous systems. The peripheral nervous system is further divided into the somatic PNS; which innervates and collects information from the skin, muscles and joints, and the visceral PNS; which innervates smooth muscle in blood vessels and glands.

The telencephalon (cerebrum) has two hemispheres and consists of the cortex (the outermost layer of the brain), and the olfactory bulb.

The diencephalon is located at the midline of the brain, above the mesencephalon (midbrain) of the brain stem and contains the thalamus and the hypothalamus.

The mesencephalon contains the tectum (dorsal midbrain) and tegmentum (ventral midbrain). The midbrain controls movement and sensory input.

The rhombencephalon (hindbrain) contains the pons, which connects the cerebellum to the cortex, the medulla (sensory functions), and cerebellum.

The spinal cord is protected by the spinal column and is the primary channel for messages from skin, joints and muscles to and from the brain. Dorsal roots bring information into the spinal cord and ventral roots send information from the spinal cord. The cerebrum is the largest part of the brain, which contains two hemispheres, separated by the sagittal fissure running down the centre of the brain. The right hemisphere controls the left side of the body, whilst the left hemisphere controls the right side (decussation). The cortex controls voluntary actions, cognition and perception and the number of neurons is related to intelligence. Because the skull is a confined area, this needs to be kept to a minimum area, and so the cortex is kept thin and folded; peaks are called gyri and troughs are called sulci. The cerebellum is the old part of the brain that co-ordinates movement and contains extensive connections to the cerebrum and spinal cord. Diseases of the cerebellum include ataxias, which result in coordination problems. Babies have floppy heads because their cerebellum has not yet fully developed. The brain stem is the oldest part of the brain that controls vital functions such as breathing.

In acute schizophrenic disease patients are unable to separate real from unreal experiences. Patients with disorganised schizophrenia may be difficult to understand as their speech may be incomprehensible and their behaviour is often inappropriate or bizarre. Patients with paranoid schizophrenia experience hallucinations and delusions.

David Berkowitz, a paranoid schizophrenic, killed 6 people in NY, 1977 and believed he was possessed with demonic power.

Brain activity can be measured through the use of electroencephalograms; measuring brain waves in response to activity. This is fast and cheap to do, but produces a poor resolution and is hard to interpret. Functional Imaging is safe and produces high resolution results, but is expensive. An fMRI (Functional Magnetic

Resonance Imaging) scan detects differences in the way hydrogen nuclei of water behave in different situations and PET (positron emission tomography) scans detect the positrons emitted after injection of a labelled drug. A positron is the

antiparticle counterpart of the electron with a charge of +1. Both methods detect changes in blood flow and metabolism within the brain. The image to the left shows a panic disorder

patient (right) with a significant global reduction in binding sites (too few GABA receptors), mainly in the orbitofrontal and temporal areas which indicate anxiety.

LECTURE 11 – CELLULAR ORGANISATION OF THE NERVOUS SYSTEMThe brain is a hierarchical structure: brain → systems → pathways → local circuits → single neurons. In 1880, new improvements in glass making allowed the advance of microscopes. Methods were developed to preserve (fix) nervous tissue and cut it into thin sections and to stain nervous tissue, such as the Golgi silver stain. Cajal examined sections from throughout the nervous system and identified that neurons exist in different forms, but most have a cell body (soma) with two types of processes: Axons, specialized for transmission of information, and dendrites, specialized for the receipt of information. He also discovered that neurons communicate indirectly by contact, rather than fusion, with synapses.

The more recent introduction of the electron microscope allows images to be resolved to 0.1nm, confirming the existence of synapses. Fluorescence microscopy, with the aid of Green Fluorescent Protein, allowed a powerful way to determine the protein distribution in cells, but the disadvantage is that this is limited due to the range of antibodies available and confocal microscopy allowed 3D images of live cells to be rendered with the use of lasers as a light source.

The two major cell types in the nervous system are glia and neurons. Glia outnumber the neurons by 10:1, their primary role is to support neurons, and they have the ability to divide. In the PNS, glia exist as Schwann cells, which myelinate the periphery neural axons. In the CNS, the glial cells exist as astrocytes, which fill the space between neurons and regulate the composition of the extracellular fluid, oligodendrocytes, which myelinate the central neural axons, and microglia, which act as the main form of active immune defence in the CNS.

Neurons are highly polarised cells, simple polarised cells include those with different apical and basolateral surfaces, and a simple non-polarised cell is just a standard cell with a regular, unchanging membrane shape.

Main features Axon DendritePhysiological Propagate information Receive informationOrganelles Synaptic vesicles ER, ribosomes, GolgiStructural Long (mm-m), branch at 900 Short, tapered, branchedSpecialisations Synapses Dendritic spinesMyelination/ nodes Often NevermRNA Never

How does the polar structure of neurons arise? The mechanisms are still unclear. Do the proteins have address markers on them and if so what reads this address? The neuronal cytoskeleton gives structural support to the shape and calibre of axons and dendrites, allows the transport of cargo from axons and dendrites and tethers components at the membrane surface. It contains Microtubules; large tubulin polymers that run longitudinally down axons and dendrites, providing structure and transport with the ability to undergo polymerization and depolymerisation. Neurofilaments are 10nm wide protein threads that provide the neuron with mechanical strength, and Microfilaments are 5nm wide actin polymers, that are tethered to the membrane and mediate shape change.

Diseases of the neuronal structure include mental retardation, which occurs due to an impoverished environment during the “critical period” of brain development, and Alzheimer’s disease, which has genetic origins and is characterised by neurofibrils (dead and dying neurons) and neurofibrillary plaques full of cytoskeletal junk. Some signs appear in 80% of 80 year olds, consisting of mental impairment; irritability and forgetfulness.

LECTURE 12 – TRANSMISSIONCurrents that pass down dendritic cells become smaller over time, to understand this better we can use the analogy of the transatlantic telegraph cable; if it were the perfect conductor with perfect insulation you would get out what you put in, but the cable core resists current flow and current also leaks through the insulator. The principle equation demonstrates an exponential decrease in current over a greater distance.

V=V O exp− xλ

λ is the length constant; the distance over which the voltage drops to 37% of the original value

when x=λ , then VV O

=0.37 (37%)

Cables with big length constants transmit further than cables with small length constants, which depends on Rm (leakiness), Ri (conductivity) and d (diameter). The ideal cable should be fat, with thick insulation and a heavy core. In neurons, the core conductivity and insulation are 106 times worse than the transatlantic cable. The typical dendrite is 1-4µm in diameter, which limits the transmission to short ranges of <1mm. Dendrites combat this problem by having lots of synapses, which help to maintain current flow.

In axons, the currents that pass down do not get smaller as they travel because they have active membranes where dendrites have passive membranes; axons have a high density of Na + channels to fire action potentials which enable signal transmission over long distances. To increase the conductivity of an axon, either the diameter or insulation needs to increase, an increase in diameter was employed by primitive animals such as squids, but for more complex mammals, this proved a problem as there is simply not enough space to fit these fatter neurons in our bodies. Therefore the insulation must be better and this is achieved through myelination.

There are two types of glial myelinating cells in the nervous system: oligodendrocytes in the CNS, and Schwann cells in the PNS. Most axons are wrapped 30 – 50 times by a myelin sheath, and some parts of the axon, known as Nodes of Ranvier are left unmyelinated at regular intervals along the axon. The unmyelinated regions contain high proportions of Na+ channels, which allow salutatory conduction through repetitive firing, rather than a constant signal. The appearance of white matter is due to myelination.

Multiple sclerosis is an immune mediated demyelinating disease of the CNS, where the immune system breaks down the myelin in the axons. The myelin sheath can be revealed in microscopy by staining with fluorescent antibodies to myelin basic protein, and bare axon can be revealed by staining with antibodies to the cytoskeletal filament Neurofilament H. In MS, the brain starts to repair itself by the fourth month, but eventually the processes take over, leading to an effective shrinking of the brain, or cerebral atrophy. Other important types of de-myelinating disorders include optic neuritis; the inflammation of the optic nerve that may cause a complete or partial loss of vision, and Guillain-Barré syndrome; an acute, autoimmune, polyradiculoneuropathy (deranged function and structure of peripheral motor, sensory, and autonomic neurons), affecting the peripheral nervous system, usually triggered by an acute infectious process.

LECTURE 13 – SYNAPTIC TRANSMISSIONSynapses are important for integration; convergence allows us to recognise important objects in the environment, some specific neurons (grandmother cells) allow us to recognise particular people. Divergence makes sure we behave in a certain way to a certain stimulus, such as when you tread on a pin. Synapses are also important for plasticity; learning and memory, and as targets for drug action to reduce pain.

Because the extracellular fluid has a lower resistance than the postsynaptic axon, action poentials cannot jump synapses as they follow the pathway with lower resistance through the ECF. Charles Sherringtion, in 1932, coined the term “synapse”, in 1921 Otto Loewi demonstrated chemical transmission, and between 1970 and 2000, Eric Kandel proposed a cellular/ molecular model of learning and memory; that short-term memory was linked to functional changes in existing synapses, while long-term memory was associated with a change in the number of synaptic connections.

Electrical synaptic transmission occurs at gap junctions, where pores exist for the free travel of ions. This leads to fast conduction, but the signal is weaker at the other side due to resistance from the pores. This system is used in escape pathways in invertebrates, such as in the giant motor synapse of crayfish, because the relative speed of electrical synapses also allows for many neurons to fire synchronously. Approximately 10% of synapses work in this way. Chemical synaptic transmission is slower because the signal needs to be converted from an electrical signal, to a chemical one and then back again.

LECTURE 14 – CHEMICAL SYNAPSES & NEUROTRANSMITTER RELEASEAction potentials are all or nothing, but post synaptic potentials (PSPs) can vary in strength as they can be iPSPs (inhibitory) and ePSPs (excitatory). When a chemical such as curare (a potent paralysing agent) is added to a synapse, a much smaller post synaptic potential (iPSP) is observed.

End-plate potentials (EPPs) are the postsynaptic potentials induced at the neuromuscular junction. The EPP is not an action potential, but it partially depolarizes the membrane and can initiate an action potential in the postsynaptic cell. Miniature EPPs occur spontaneously in muscle cells.

Depolarisation causes Ca2+ influx in presynaptic terminals via voltage-gated Ca2+ channels and images showing calcium levels can be produced using calcium sensitive dyes such as aequorin and FURA.

Neurotransmitters are released in vesicles called “quanta” that each contain several thousand ACh molecules. Each EPP is made up of about 200 quanta and as extracellular Ca2+ is lowered, EPP amplitude decreases in a

stepwise manner. This is the vesicle hypothesis. SNARE proteins at the plasma membrane mediate fusion of cellular transport vesicles with the cell membrane at a target compartment.

SNARE proteins can be classed as v-SNAREs (vesicle), which are incorporated into the membranes of transport vesicles during budding, and t-SNARE (target), which are located in the target membranes. Synaptotagmin is a Ca2+ sensor that triggers synaptic vesicle fusion with the presynaptic membrane.

LECTURE 15 – CHEMICAL SYNAPSES & PSP INDUCTION2ACh molecules bind to the alpha subunits on nicotinic acetylcholine receptor channels at neuromuscular junctions and cause them to open, allowing Na+ and K+ to flow down their electrochemical gradient leading to the depolarisation of the membrane (EPP/ePSP). The nicotinic acetylcholine receptor is made up of five subunits: 2α, β and γ, arranged to form a central pore. There are a great number of neurotransmitters, and neurons can contain or release more than one type of neurotransmitter, this is called co-localisation.

Amino acids Glutamate, glycine, GABAAmines Acetylcholine, dopamine, noradrenaline, serotonin

Peptides Enkephalin, substance P, neuropeptide Y

Serotonin is responsible for arousal, sleep and appetite, Glycine and GABA are widespread inhibitors and peptide neurotransmitters are neuromodulators. Enkephalin is a natural painkiller in the brain that regulates pain and nociception, dopamine depletion leads to Parkinson’s disease.

Amino acids and amines are made in the presynaptic terminal by synthetic enzymes and transported in vesicles, neuropeptides are made in the cell body, synthesised in the rough ER, cleaved in the Golgi apparatus and transported in secretory granules. The action of a neurotransmitter depends on the receptor present; nicotinic ACh receptors excitatory at skeletal muscle, but muscarinic Ach receptors are inhibitory on the heart.

Repolarisation is due to the removal of a chemical stimulus. This is done by acetylcholinesterase action at the synapse, not all ACh actually reaches the post synaptic membrane since acetylcholinesterase is always present. Nerve toxins that attach cholinergic synapses include

Botulinum toxin From Clostridium botulinum Destroys SNARE proteins, prevents ACh releaseBlack widow spider venom From black widow spiders Induces massive ACh release and depletionα- Bungarotoxin From cobra venom Irreversibly binds to ACh receptorsOrganophosphates In insecticides and nerve gas Irreversibly inhibits acetylcholinesterase

Myasthenia gravis, muscle weakness, is caused by a reduction in the number of ACh receptors on skeletal muscle, an autoimmune response targets antibodies to the α subunit of the nicotinic ACh receptor, causing a reduction EPPs and mEPPs. Edrophonium is an acetylcholinesterase inhibitor. In people with myasthenia gravis involving the eye muscles, administration of this drug will briefly relieve weakness.

LECTURE 16 – INTEGRATION

The primary motor cortex initiates voluntary movement, while the motor association cortex (premotor area) coordinates complex movements. The prefrontal cortex deals with planning, emotion and judgement.

Synaptic integration involves summation of ePSPs and iPSPs on the postsynaptic membrane. PSPs travel to the axon hillock in a passive, detrimental form, where decision making occurs. The axon hillock has a high density of Na+ channels, while the postsynaptic neuron filters and integrates the signals. The average neuron forms about 1,000 synapses and receives up to 10,000 connections, most PSPs are only a few mV and do not take the membrane to threshold.

Summation refers to the addition of a number of impulses, the cell has to “decide” what to do depending on the inputs; spatial summation is due to multiple pathways, and threshold is reached when the impulses from the presynaptic neurons fire at the same time (the theory behind event association). Temporal summation works according to the rate at which impulses are fired along one pathway, firing at a higher frequency causes the action potentials to reach threshold as they do not have time to fully repolarise before the next signal arrives. In effect, the signal just builds up gradually to reach threshold.

Primary motor cortex

Sensory association area

Visual association area

WB

Motor association cortex

Prefrontal cortex

Auditory association

Primary Somatosensory area

In hyperekplexia (literally “excessive startle”), an affected adult will startle easily at a sudden sound or unexpected touch or bump and may fall and be injured. This is usually inherited as an autosomal dominant trait and due to a single amino acid mutation in the glycine receptor chlorine ion channel, which reduces inhibition of the central nervous system.

LECTURE 17 – NEUROMODULATIONNeuromodulation refers to setting the activity level of neural pathways by changing their threshold (due to more channels, or channels having a greater sensitivity), or altering synaptic strength (with more postsynaptic receptors or neurotransmitters). For example noradrenaline acts on hippocampal neurons to make the cell more responsive to a neurotransmitter (glutamate) by causing K+ channels to close. In the long term the neuronal membrane properties or synaptic strength may permanently change.

Classical synaptic transmission Fast, short lived - ionotropic ion channels ACh on skeletal muscleLocal neuromodulation Slower, longer lived - metabotropic receptors ACh on cardiac muscleExtrasynaptic neuromodulation Slow, long lived - metabotropic receptors Oxytocin on brain pathway

Ionotropic receptors include nicotinic ACh receptors and GABAA receptors, metabotropic receptors are G-protein coupled receptors, and include muscarinic ACh receptors (where the ion channel is directly activated) and Serotonin 5HT receptors (where a secondary messenger cascade activates the ion channel).

GABA is a neuromodulator which inhibits calcium channels when released; the blue neuron effectively acts as an on/off switch to vary the strength of the action potential of the main neuron. Serotonin acts in the opposite way to GABA, and usually leads to association of events when both neurons are stimulated at the same time.

LECTURES 18 & 18B – THE BASIS OF LEARNING AND MEMORYBehaviour changes as a result of experience; learning is the process of acquisition of knowledge, while memory is concerned with storage and retrieval. An altered stimulus → response relationship is also known as Pavlovian (or classical) conditioning. Short term memory is a reverberation of electrical activity caused by changes in synaptic activity, and long term memory is due to new or enhanced synaptic connections or new gene expression. Most animals with a nervous system can learn. Drosophila can detect certain odours from food and by pairing certain “bad” odours with a mild electric shock they learn to avoid them.

In sea hares, gills retract when the pressure is applied to protect them, this reflex action is a result of classical conditioning. When the stimulus is repeated and no form of “punishment” is administered, the response decreases, showing a habituation response. But when an electric shock is administered with the stimulus, the response quickly increases again. Synaptic efficacy is reduced with repetitive use, due to a reduction in Ca2+ influx per action potential and hence reduced neurotransmitter release.

Sensitisation of gill withdrawal involves presynaptic facilitation. A facilitating interneuron (5HT) receives impulses from the electrical stimulus in the tail, and causes cAMP to be released at the motoneuron, which leads to the closing of voltage-gated potassium channels, meaning it takes longer for the action potential to repolarise and a broader response is observed as more neurotransmitter is released. In the long term, the signalling cascade leads to changes in gene expression, K+ channels are inhibited and will not function at all.

The hippocampus is located deep in the temporal lobes of the brain and is part of the limbic system involved in memory processing. It is important for spatial learning and London taxi drivers have large hippocampi. The hippocampus is also severely affected in Alzheimer’s disease. Long-term potentiation (LTP) is the long-lasting enhancement in communication between two neurons that results from stimulating them simultaneously. Since neurons communicate via chemical synapses, and because memories are believed to be stored within these synapses, LTP and its opposing process, long-term depression, are widely considered the major cellular mechanisms that underlie learning and memory. LTP can be induced by strong tetanic stimulation of a single pathway to a synapse.

LECTURES 19 AND 20 – MUSCLESkeletal muscle is a form of striated muscle that is attached to bones via tendons. They perform voluntary actions such as running and can be up to 40% of one’s body weight. Skeletal muscle contains between 100 and 10,000 muscle fibres (cells) that are made up of myofibrils and run in parallel, stretching from millimetres to tens of centimetres long, the cells have many nuclei due to the fusion of myoblasts. The origin of striations is due to the presence of actin and myosin filaments.

Tropomyosin stops muscle contraction as it covers up actin binding sites, this can be relieved through calcium binding to troponin and causing tropomyosin to undergo a conformational change, exposing the binding sites.

Genetic muscular diseases include:

Muscular dystrophy Progressive skeletal muscle destruction and weakness

Myotonia Sustained contraction followed by slowed relaxation due to a mutation in protein kinase

Channelopathy Mutations in ion channelsMcArdle disease Lack of phosphorylase so glycogen cannot be broken down

Malignant hyperthermia

General anaesthetics can make SR Ca2+ channels open, leading to an uncontrolled increase in skeletal muscle oxidative metabolism which overwhelms the body's capacity to supply oxygen, remove carbon dioxide, and regulate body temperature, eventually leading to circulatory collapse and death if not treated quickly.

Calcium accumulates into the sarcoplasmic reticulum by the calcium ATPase pump and is released into the cytoplasm through specialised release channels (Ryanodine receptors). The opening of these channels is caused by a surface membrane action potential that spreads down t-tubules and makes charged particles in t-tubule membranes move; these are connected to the SR release channel and cause it to open.

A nerve (motor neuron) action potential releases ACh at the neuromuscular junction and opens the channels at end plates, depolarising the muscle membrane and initiating the action potential.

The action potential travels down to the t-tubule, where a charged particle opens the Ryanodine receptors, causing calcium to be released from the sarcoplasmic reticulum.

The calcium binds to troponin, causing the tropomyosin to undergo a conformational change and lever off the actin filaments allowing the myosin heads to bind and leading to muscle contraction by the shortening of the sarcomeres.

On relaxation, SERCA (Sarco/Endoplasmic Reticulum Ca2+-ATPase) takes calcium back into the SR.

A muscle action potential lasts for a similar length of time to a nerve action potential, but the muscle contraction force lasts much longer. Muscle impulses can take many forms:

Single twitch Short contractions, blink of an eyeSummation Impulse increases gradually

Unfused tetanus Muscle contracts and relaxes quicklyFused tetanus Muscle remains contracted, maintained movement

Smaller motor neurons have lower thresholds and activate smaller motor units, allowing for a small muscle contraction of a certain area when greater contraction is not required, fast fibres allow for rapid shortening, but at a high energy cost as ATP is hydrolysed quickly, slow fibres are used for posture and contain myoglobin as an oxygen store with many mitochondria. Fatigue results in a state of decreased performance due to lactic acid buildup in the short term, and glycogen depletion in the long term. Ageing results in the loss of motor neurons and a decreased number of muscle fibres (which can be partially restored with exercise), whilst damage to muscle cells results in them being replaced with satellite cells, leading to muscle growth. Other factors such as disease also have different effects on muscle (see table at top for examples)

Cardiac muscle is only found in the heart, it is striated and the component cells are joined by intercalated discs which provide low resistance and allow the spread of an action potential. Cardiac muscle effectively acts as one big cell, the mechanism of calcium release is different to that of skeletal muscle since cardiac muscle has no need for nerve excitation, it is myogenic. Because the heart beats continuously it cannot rely on anaerobic glycolysis and so requires its own blood supply for aerobic respiration.

Smooth muscle is not striated and consists of small spindle shaped cells with varicosities; swollen regions of the autonomic neuron containing neurotransmitter vesicles that are released when action potentials arise. Smooth muscle is found in hollow organs such as blood vessels and bronchi to regulate flow, and in the gut and uterus in order to propel contents. As with striated muscle, smooth muscle is regulated by calcium, but this is not just from the sarcoplasmic reticulum, it also comes from the extracellular fluid. Calcium is released from the SR via both Ryanodine receptors (RyRs) and Inosital Triphosphate Receptors (IP3Rs)

t-tubule

Caldesmon is a calmodulin binding protein, and Calponin is a calcium binding protein, they both inhibit smooth muscle contraction until they are phosphorylated in the presence of agonists. Some smooth muscles have pacemaker activity, such as those in the gut and uterus. Vascular smooth muscle controls blood flow. The endothelium of blood vessels releases factors to control smooth muscle and noradrenaline helps to raise the intracellular calcium concentration.