CSB332 Lecture 6 Notes

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Department
Cell and Systems Biology
Course
CSB332H1
Professor
Francis Bambico
Semester
Winter

Description
CSB332 Lecture 6 Slide 10 - You can use a tool to measure the membrane potential in neurons. The reference electrode is in the extracellular environment, and the other electrode is sticking into the neuron, and you connect them to a voltmeter that measures the membrane potential. - Membrane potential is a potential energy, which is stored energy. Energy is the ability to do work. Membrane potential is the work that can be invested because of the presence of the potential energy. Work is force times distance. If there is work done, then there is a force that displaces the object. An electrical potential may refer the work done, or the force (electromotive force), or the potential energy. o You can measure the energy that is stored in nervous tissue using a voltmeter. Voltage is equivalent to one coulomb of positive charge (e.g., one coulomb per change in voltage). - Neurons are able to store energy indefinitely in the form of chemical energy. The movement of ions can only be possible based on the chemical composition of the neurons. The chemical composition (e.g., concentration of ions inside and outside of the neuron) is key to be able to store energy. You would be able to convert the stored chemical energy into kinetic electrical flow depending on the behaviour or the property of the lipid bilayer. The neuron acts like a battery. Slide 11 - The specimens in a petri dish are placed under a stereomicroscope. There are recording electrodes (e.g., record the neuron) and stimulating electrodes (e.g., apply current to artificially stimulate the neuron). You can stimulate many components of the neuron at the same time. The glass micropipette or electrode is connected to the hydraulic microdrive that controls the lowering of the electrodes. You need an anti-vibration table. A faraday cage prevents electrical interference. - The signal that you pick up is in the form of bioelectricity based on movements of ions within the neural tissue, but it has to be measured with electronic instrumentation. How can the movement of the ions within the neuron be translated into something that can be picked up by electronic instruments? The flow of ions would have to be converted into current in the form of electrons to be picked up by the measuring instruments. In physics, electricity is the flow of electrons. In neurophysiology, electricity in a neural tissue is the flow of positive ions or charges. o Protons do not flow. - The flow of current will be relayed to an amplifier because the signals that are picked up are very small (e.g., in orders of hundreds of mV). The amplifier allows you to visualize the spikes. The stimulus device controls the amount of current that you pass onto the stimulating electrodes. Stimulating electrodes introduce electrons into the neural tissue. You see the changes in membrane potential on an oscilloscope (old) or monitor (new). You can hear the firing of neurons or the movement of ions in and out of the membrane with an audio output. Slide 12 - Changes in membrane potential appear in different forms. The membrane potential of neurons change and transition to different forms. They transition from the resting membrane potential to an action potential. Some neurons do not fire action potentials. Some neurons would make use of their graded or electrotonic potentials to convey their information to neighbouring neurons. o The axons projecting from the ganglion cell bodies are what form your optic nerves. - If you stick a recording electrode into different cell bodies, only ganglion cells would fire action potentials. Bipolar receptor and receptor cells would not fire action potentials. You would be able to record changes in electrotonic or graded potentials. - Electrotonic or graded potentials are a type of change in membrane that is characterized by a slow rise in the amplitude of the membrane potential followed by a slow decrease in the action potential (e.g., bell-shaped signal). o The size depends on the intensity of the stimulus (e.g., light). The greater the intensity of the stimulus, then the greater the amplitude. o In contrast to action potentials, electrotonic potentials are not self-sustaining and they rapidly dissipate. If they propagate along a medium (e.g., along axons or cell bodies), they rapidly die down and they eventually dissipate. Slide 13 - Action potentials are self-sustaining, regenerative, and can travel far distances. - Ganglion cells have graded potentials. When a ganglion cell reaches a certain level of membrane potential, called the threshold of excitation, then it generates an action potential. Many other types of neurons in the brain convey action potentials. - Some neurons do fire action potentials. Some neurons don’t fire action potentials. Some neurons fire one action potential at a time when it reaches threshold. Some neurons fire many action potentials in response to achieving a threshold of excitation. Some neurons can be invoked to fire an action potential in response to an input. Some neurons are spontaneously firing action potentials without a clear input from other neurons, called pacemaker neurons (e.g., dopaminergic (VTA) and serotonergic (raphe) neurons). Slide 14 - How can action potentials be encoded? One type of information that can be encoded by action potentials is patterns (e.g., the patterns of action potentials). - When the light from an object is evenly distributed, then the object does not have any edges. - You are recording firing pattern from a cortical neuron (which is a subtype of retinal ganglion cells) in an awake cat. - (C) o If the object or the luminosity of the object is evenly distributed and strikes a group of adjacent photoreceptors on the retina, and the information is relayed to a retinal ganglion cell, then you can observe a slow action potential pattern. - (A) o If the object has boundaries so that the brightest part of the object falls on the middle of the field, then light would stimulate the central domain of the retinal ganglion cells. You would not have intense illumination in the adjacent photoreceptors. The same neuron may respond in a different way. It may respond by increasing its action potentials. - (B) o If the object has boundaries, and the brightest part of the object falls on the periphery of the field, then the bright light would stimulate the peripheral photoreceptors. The same retinal ganglion cell could respond in a different way. It could shut down its action potential firing. - Cortical neurons have an on center-off surround receptive field. The pattern of firing of action potentials depends on whether the object has edges or whether the light coming from the object is smoothly distributed across the receptive field of the retinal ganglion cell. The receptive field of a neuron is the area of the retina whose illumination influences the AP firing of the neuron. The retinal ganglion cell has an on center-off surround receptive field because if light shines on the center, then it will turn on the neuron (e.g., rapid AP firing activity). It has an off surround receptive field because if light shines on the peripheral area of the receptive field, then it will turn off the firing activity of the neuron. - The pattern of AP firing of a neuron can depend on the characteristics of an object or a stimulus in the visual space. Slide 16 - In the neocortex, there are different neurons that respond to other characteristics of an object. - This is a neuron that responds best to a vertically oriented object. If the object is in a vertically oriented position, then that could result in an increase in the firing activity. The same neuron will not respond if the object is oriented in different ways. This neuron is sensitive to a particular characteristic of an object, which is the orientation in space. - These experiments utilizes an extracellular recording because the action potentials are being recorded in live animals. The cats are anesthetized, but alive and breathing. Slide 17 - Know how action potentials are generated. - Know the ion channels involved in the generation of the action potential. Slide 18 - The changes in membrane potential are measured as a form of electrical current. - The ion channels have certain characteristics that are relevant in neurophysiology, which can be measured using electrophysiological recording techniques. o Siemens is the standard unit of conductance, but in a biological preparation, it is in the order of picosiemens. 1 S is equivalent to 1 A per unit change in voltage. o Ampere is the standard unit for current, but in a biological preparation, the movement of ions is in the order of picoamperes. o Permeability is sometimes synonymous with conductance, but they are slightly different.  If you have high permeability, then there is high conductance.  If you have a potentially high conductance, then you have very large amounts of ions, but you don’t have any ion channels open, then it wouldn’t amount to anything.  Conductance refers to the flow of current, which requires permeability.  If conductance is high, then permeability should be high at the same time. There should be a high number of ion channels that are open to conduct ion flow. Slide 19 - There are different kinds of ion channels based on the genetic and molecular structure. These are four examples of different kinds of ion channels. The differences in molecular structure would allow for differences in gating of the ion, gating of the ion channel, and selectivity of the ion channel. - (A) Ligand-gated ion channels (e.g., cholinergic receptors) o They are ion channels that are activated by ligands. They are open by chemical transmitters. They are also called ionotropic receptors, which mean that they are receptors that are ion channels at the same time.  GPCRs are metabotropic receptors, which are not ion channels, but their biochemical signalling is associated with ion channels. o The typical structure is composed of five subunits, hence they are called pentameric proteins. Each subunit is composed of four transmembrane spanning domains or sequences of amino acids. o The N terminus and the C terminus is located in the extracellular side. - (B) Gap junctions o Gap junctions are found in glial cells and between neurons. The cytoplasm of one glial cell is continuous with the cytoplasm of the other glial cell via gap junctions. They directly connect one cytoplasm to another cytoplasm of another neuron. o Gap junctions can be found in neuronal populations (e.g., LC, noradrenergic neurons, neuronal subpopulations in the brainstem, medulla oblongata). Gap junctions allow for efficient, fast, and rapid communication among neurons. o Gap junctions are composed of two hemichannels. Each hemichannel is composed of six subunits, called connexons. Each connexon has four membrane spanning domains, called connexins. - (C) Voltage-gated channels o These are channels that are opened or activated by changes in membrane potential. It is a monomeric protein. It is a continuous sequence of amino acids. Each chunk that is formed by the sequences is called motifs. For example, Na+ channels have foth motifth Each motif has six transmembrane domains. There is a region between the 5 and 6 transmembrane domain that sticks out into the extracellular side, which is a part of the pore forming region. You have to form a pore within these ion channels to allow the ions to pass through. o There are also chunks of amino acid residues that are important for the sensitivity to the change in the internal membrane potential. o The N terminus and the C terminus of the ion channels are all located within the cytoplasmic environment. - (D) Resting channels or leak channels o Resting channels are not gated. They are always open. They are important for the maintenance and the generation of resting membrane potential. The resting membrane potential is when the neuron is at rest. o There are two types of resting channels that are important for the maintenance of resting membrane potential of a typical neuron: Na+ resting channels and K+ resting channels. o The channel is not gated, so the structure is simpler. The K+ resting channels have two P regions, which are important for the maintenance of the resting membrane potential. Slide 20 - Two characteristics of ion channels are ion selectivity and gating. - Gating o Under resting conditions, an ion channel is closed. When it is activated by a ligand, it transitions from a closed/activatable state to an open/activated state. A segment of the intracellular domain of the channel would change its conformation, open a gate, and allow ions to pass through. After being in an activated state for a while, it is going to spontaneously transition into a closed/inactivatable state. o There are two closed states. The last state is a closed/inactivatable state because adding more ligands or changing the membrane potential won’t be able to open the ion channel until it reverts into a closed/activatable state. - Ion selectivity o Ion selectivity depends on the size of the pore and on an amino acid residue lining the interior of the pore that is charged called the selectivity filter. For example, Na+ ion can bind to the negative charge on the selectivity filter. It depends on the composition of the selectivity filter, and the size of the pore. Slide 21 - Membrane potential is always associated with current. When you change the membrane potential, then it changes the flow of current, which in turn changes the membrane potential, and so on. - Current and membrane potential can be measured by different electrophysiology recording techniques. o Electrophysiology is a relatively new field. - (A) Extracellular recording o You are using an electrode that is placed around the neuron. The electrode is outside of the neuron, but it is still able to pick up small signals or echoes of action potentials being delivered by that neuron. You would require an amplifier. The changes in membrane potential of a neuron switching from a resting membrane potential to an action potential is 100 millivolts. If you are recording from afar, the echo of action potentials will rapidly dissipate and be picked up at the order of 10-100 microvolts. You need a strong amplifier to amplify the signal. o Electrodes are in the form of glass capillaries. You have to use a micropipette puller to construct one.  You place the glass capillaries into the micropipette puller. The puller is a device with a heated coil in the middle. You place the glass capillary through the coil so that the midpoint will be heated. While it is being heated up, the machine would pull it apart. After that, you get two viable glass micropipettes.  You need less than or equal to 1 micron diameter tip.  You fill the micropipette with electrolytes, which are salt solutions (e.g., high concentrations of KCl). You have to translate changes in ion flow into the flow of electrons. The changes in ion flow within the neuron would have to allow for the inflow of ions through the tip of the micropipette. The inflow of ions would perturb the kinetics of the ions that make up the intra-micropipette solution (e.g., high concentration of KCl). Salt solutions will ionize. You will find a lot of positive and negative ions in the intra-micropipette environment. If the additional flow of ions perturbs the membrane potential of the neuron, then it will also enhance the kinetics of the electrolyte ions in the micropipette. This would create a redox reaction, which will translate into electrical flow.  The micropipette also has to have a metal wire within it to propagate the flow of electrons and deliver the electron flow signal to the electron measuring device. o The micropipette is the electrode with electrolytes in it. The micropipette would measure changes in membrane potential in comparison to a reference electrode in another area. o These recordings allow you to measure action potentials, the effects of drugs, and other experimental manipulations on action potentials (e.g., diseases). o You can record from many different subpopulations of neurons. o The advantage of extracellular recording is that you can record for a long period of time without damaging the neuron. - (B) Intracellular recording o This involves impaling the neuron. The electrode is inserted within the neuron, so that you have easy access to the electrical property of the neuron. You don’t need a powerful amplifier. It records more information than an extracellular recording. You can record changes in action potentials, changes in membrane potential, and changes in the total current going in and out of the neuron. The electrode is within the neuron and very sensitive to any changes that occur inside the neuron. o The disadvantage of intracellular recording is that you can only record for a short period of time because impaling the neuron with the electrode will impair the neuron, so it will eventually die. o There is a lot of noise that is recorded. It is difficult to distinguish between the actual signal and the noise. - (C) Whole-cell patch recording o Patch clamp recording is a family of recording techniques because it has different configurations. o In a patch clamp recording, the ring of the electrode tip is large. The ring of the electrode tip and the membrane forms a tight seal. If it is sealed, then no current will flow out of the sides of the electrode. If there is an extra outflow of current from the periphery of the electrode tip, then you would get a lot of noise. - In vivo recordings done on anesthetized animals are simpler because there is no movement. It is more difficult to produce single unit recordings. You can’t use one electrode because there is high probability that you will lose neurons. In awake animals, you may use several electrodes (e.g., tetrode is an aggregate of four metallic electrodes) that will be able to pick up many signals from many neurons. Slide 23 - How do you translate the flow of ions into electrical signals that can be measured by a recording electrode? You need an interface reaction that would be able to transduce the ionic signals into the flow of electrons. - Electrode wires are immersed in the electrolytic environment within the glass micropipette. Most electrode wires are coated with Ag and AgCl. Some electrode wires are coated with platinum. - AgCl junction produces a redox reaction. If Ag reacts with Cl- ions, then it will form AgCl and release electrons. The electrons would flow into the wire and into the measuring device. - If you neuron is hyperpolarized, Cl- ions in the neuron will diffuse into the interior of the glass micropipette that is filled with high concentrations of K+ and Cl- ions. If the Cl- ions get into the interior of the glass micropipette, then it would increase the kinetics of Cl- ions that are already present. The increase in the movement of Cl- ions would increase the probability of Cl- ions bumping onto the Ag or AgCl junction. Then Cl- would react with Ag, forming AgCl, liberating electrons, and electrons would flow onto the wire into the recording electrode. - If you want to use the recording electrode as a stimulating electrode, then the current generator (which is integrated into the amplifier, so the amplifier also functions as a current generator) will produce a flow of electrons. The flow of electrons will propagate onto the wire, and will lead to the decomposition of AgCl. The electrons will associate with the Cl- ions, the Cl- ions will be liberated, and the Cl- ions will go to the KCl electrolytic solution. The increase in Cl- concentration allows for the flow of Cl- ions into the neuron, which would change the membrane potential of the neuron. - Redox reactions are important for stimulating and recording electrodes. Slide 25 - Patch clamp recording are always done in vitro (e.g., brain slices or dissociated neurons). - When the electrode reaches a neuron, you would apply a suction to minimize the noise. You want to increase the tightness of the seal between the rim of the electrode and the membrane of the neuron. This forms a tight seal, called a gigaohm seal. This eliminates current from escaping out of the sides of the micropipette. - Cell-attached patch clamp recording o The electrode is able to capture one ion channel. You can record ionic flow from the single ion channel. o There is a tight seal between the rim of the electrode and the membrane. You are able to pick up one ion channel. You can apply a current to change the membrane potential, and so on. - Inside-out patch clamp recording o From a cell attached-patch configuration,
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