Today: Ultrastructure Axoplasmic transport Glia I want you to call me Tony. Please don't call me Professor, Professor Stretton, or Dr. Stretton. Technique that Golgi made for seeing neuroanatomy, illustrated with the picture from the book on page 6 of Chapter 1. In the picture on the lower right, you can see a picture of the section of the retina. This gives you an idea - the point that I made last time is that you can't really see what is going on here. The beauty of the Golgi technique is that it stains a minority of the neurons and it stains the entire neuron. This enabled Ramon y Cajal to create a diagram of what a neurons look like. Upper left corner picture on the same page is the summary of the way the retina is arranged. You need a technique like this to sort out the different categories of neurons. Cajal not only was a predigious worker, he also had incredible insights. On this diagram, there are arrows drawn, and this was his interpretation and insight that this was the direction of information flow. He was way ahead of his time. It was interesting that Golgi and Cajal both got the Nobel prize for their contributions to neuroanatomy, but when they were receiving the prize, one of them was gracious in his statement about the work and Golgi was swearing because he thought Cajal had stolen his technique and misinterpreted it. Dispute: whether or not the Golgi stain did in fact stain the entirety of the neuron. Golgi said no, what we've got is an example of a stain in a big cell and suddenly for some reason the stain stops at a particular point and that's why you see these endings, like in the pictures on page 7 of Chapter 1. Golgi would say the stain just ended there where the branches are, but in fact the cell continued and all the cells are connected to other cells. Golgi thought there was cytoplasmic continuity between cells. Cajal said, no. What you are seeing stained is the real end of the cell. At the time, he had no way of proving that so this was a bitter disagreement between these two very great scientists. We now know that Cajal was right and that the nervous system, each neuron is a discrete entity. The reason we know that is firstly electron microscopy - if you look at the place where the stain ends, you can see a cell membrane, so that really is the end of the cell. The second technique that was used to corroborate this involves the injection of substances into a single neuron (such an enzymes that turn an entire cell black) and it gave the same picture as the original stain that Cajal and Golgi both saw. So basically, Cajal got it right. General terms about the differences between dendrites and axons: Dendritic spines on the dendrites, and then here's the axon. One of the differences is morphological: axons tend to be uniform in diameter (easy to spot in Golgi stains) whereas dendrites tend to be tapered - they start off being large in diameter and get smaller the further out you go. When people started to do electron microscopy on neurons they started to see other differences and some of those are summarized on the handout we were given last lecture (Lecture 1). Handout: Picture of a generic neuron. Axon giving rise to axon terminals which are pre-synaptic elements: parts of the neuron that are going to make synapses onto another cell. Dendrites receive synapses. Most neuron cells do not divide, but there are some exceptions. On the surface of the axon you often find a particular classes of neurons - myelin sheath. Called a myelinated axon. Not all axons are myelinated, so some are unmyelinated, meaning they don't have a myelin sheath. The function of myelin is to increase the speed of the function of propagation of action potentials. In the nucleus of neurons, there is stuff called nissl substance. This is rough endoplasmic reticulum (RER). This has ribosomes on them and ribosomes do protein synthesis. Neurons are prodigious synthesizers of proteins. The density of the nissl substance is very, very high in neurons (we know this because of stains that work on the nucleic acids of ribosomes, which also stains nissl substance). Lots of nissl substance in the cell bodies and the proximal dendrites. This is part of the secretory part of neurons. Neurons secrete like crazy because the synaptic terminal has these synaptic vesicles and they are packed with transmitter. They release their cargo transmitter by synaptic secretion in secretory vesicles, then these vesicles fuse with the dendrites of the next neuron in the line. There is no nissl substance in the axons or the axon terminals because they do not need to make or package any proteins there. On the dendrite, there is post-synaptic density where the axon terminals of the previous cell will dump its neurotransmitters. Increased density in the pre-synaptic and post-synaptic terminal. Neurofilaments (not shown on diagram) - made up of different proteins assembled in a long, slow helix. Found in neurons, but they belong to a subset called intermediate filaments that appear in many types of cells, but in other cells they are made of different proteins and go by different names. Diameter is 100 nM. Microtubules are made up of tubulin, highly polymeric form arranged in protofilaments that are typically 30 protofilaments in a microtubule. They go on, and on, and on. Found in all sorts of cells. Diameter 25 nM. Initial segment is also very close to the region called the axon hillock. This is important because it is where the axon potential gets propagated. Area of low threshold, meaning the axon potential goes off there easiest. This area integrates all the excitatory/inhibitory signals and decides whether or not to fire an action potential. This all happens in the axon hillock (aka Initial Segment). Very few ribosomes in the cytoplasm of the axon (much lower than that in the cell body or the dendrites). In the axon slide, you can see microfilaments and microtubules, which run parallel with the course of the axon. Outside the axon, you can see all sorts of processes from other neurons (what a mess if not for Golgi!) He showed slides of neurons to highlight some of the things mentioned above. Shown: Diagram of a synapse showing the tubules and filaments which end before you come to the cytoplasm of the synapse. Vesicles are destined to release their cargo to the outside world of the synapse to connect with the dendrites of the next neuron. There is a wonderful wine called Cilterne. The water has been sucked out of it, so when people trumple on these grapes the juice is much more concentrated and so the flavor is just out of sight. I belong to a wine tasting group. A bunch of scientific faculty trying to talk about wine in discrete terms. Does it taste like blackberry or horse piss? We all put in a slug of money and one year we had a lot of money in the kitty and so one guy said why don't we buy a $500 bottle of the best Cilterne? So we all swallowed hard and said okay. I can still taste it, and that was 15 years ago. Anyway, let's get back to it. The point: vesicles that look squished contain a different neurotransmitter than those that look like big round grapes, but we'll get back to that later. Axoplasmic transport The fact that the density of ribosomes in the axon is low creates a problem for the proteins which exist in the axon. The proteins which exist in the axon are made by ribosomes obviously and these functioning proteins are a long way from the place where they are synthesized. This creates an issue: how do you replace proteins which are worn out? Proteins have a finite lifetime and they need to be replaced. And secreted proteins certainly have to be replaced. So how do you get the proteins down this long axon? Diffusion won't do it because it is too slow. The process for dealing with this is called axoplasmic transport and there is a special transport mechanism that carries stuff down the axon to the terminals. There are different components to the axoplasmic flow which go at different velocities. There's fast axoplasmic flow: 240 mm/day, Intermediate: 30-60 mm/day, Slow: 2-8 mm/day, Very slow: ~1 mm/day. How do you find this out? You inject radioactive amino acids into a place (favorite place is into the eyeball) and then you let the proteins get synthesized and then at intervals you sample what is going down the axon. The fast stuff is plasma membrane proteins (proteins for external membrane of cell) and synaptic vesicle proteins and transmitters Intermediate is mitochondria Slow is bulk cytoplasmic enzymes Very slow is the cytoskeleton The mechanism for this axoplasmic flow has been well studied in the case of fast and intermediate. Special apparatus which uses the microtubules. So what you have (picture later on in the book. Figure 13.16). Microtubule, and on it, there are motor proteins (transport proteins). In neurons, there are two types of these proteins: Kinesin: moves down the microtubule toward the plus end, from subunit to subunit using ATP hydrolysis; transport goes from the cell body to out. This carries synaptic vesicles, including plasma membrane protein which will be inserted into the terminal membrane. Dynein: moves down the microtubule toward the minus end, from subunit to subunit using ATP hydrolysis; tends to bind to vesicles which are made in the nerve terminal, called endocytic vesicles (sample of external environment is pinched off). This is called retrograde transport. They move along a microtubule in an interesting way. Microtubules are polarized (minus end and plus end). You can distinguish these ends because the + end is where the new microtubule is added and the minus end has very little turnover (+ end grows, - end doesn't). Arrangement of microtubules in cells: The disposition of the microtubules in axons and dendrites is different. (showed picture from UW-Professor paper) He showed that the microtubules in the axon were all arranged with their plus ends out and the minus ends toward the cell body. The dendrites are arranged so that the microtubules are arranged equally in either direction. This means that Kinesin is going to carry things out and Dynein carries things in in the axon. In the dendrite, this is not true. Both motor proteins will end up bringing things in and out in the dendrite because of the varied arrangement (some plus and some minus) pointing at the cell body. How did he find this out? Add monomeric proteins for making microtubules. When looking at cross sections, if you see a circle with little curvy flaps coming off in the clockwise direction then you are looking at the plus end toward you. If you see a circle with little curvy flaps going counterclockwise then you are looking at a minus end. In this way, if you see all the curvy parts going in the same direction, you are looking at an axon, but if the curvy parts are not all going the same way, you are looking at a dendrite because it has both plus and minus going in both directions, whereas axons do not. Glia: Glial cells are more numerous than neurons in the NS. There are about 10 times as many glial cells as there are neurons. Glial cells come in different categories. In the PNS, the glial cells are called Schwann cells. In the CNS, there are three major types: Oligodendrocytes, astrocytes, and microglia. The function of Schwann cells and oligodendrocytes is to form myelin. Myelin in the PNS is made by Schwann cells. Myelin in the CNS is made by oligodendrocytes. Showed picture of a peripheral nerve with a myelin wrapping. Axons with a myelinated have a faster rate of propagation of action potentials. Also showed diagram of myelinated and unmyelinated axons. Shwann cells travel around and around an axon to coat it in myelin. All the myelin between each node of Ranvier is made from one Schwann cell.