Lecture 2: The

• The neuron is the structural and functional unit responsible for transfer of information via electrical (ionic movement) and chemical communication. • are excitable cells à capable of transmitting electrical events (action potentials) • A neuron is an excitable cell that transmits signals to other excitable cells, in one direction, via specialized junctions (). • The performs three overlapping functions: • Sensory Input à Integration (occurs in CNS) à Motor Output (effectors) • Unidirectional Signaling: • Sensory Systems (afferent - towards) direction of signal is from the periphery (e.g., skin, joints) towards brain. • The trigger may be from outside the body (e.g. visual world) or internal (e.g. via visceral receptors) • Transduced by a special sense organ/structure (e.g. pressure receptor in skin, photoreceptor in retina, chemoreceptors) • Transmitted to the for interpretation/integration • Motor Systems (efferent - away) direction of signal is toward the periphery. • Voluntary (to skeletal muscle) and involuntary signals (to smooth muscle contraction, glandular action) are transmitted. • Direction of transmission: • à Cell body à

• Parts of the cell: • Cytoskeleton: • Composed of , microtubules and actin filaments (microfilaments). • Provide structural support, movement of proteins and organelles, contractile properties. • Form a basket around the axon hillock to keep the proteins in the right compartment • Neuronal : • A nucleus containing chromosomal DNA and RNA in the nucleolus • A prominent Golgi apparatus - for processing of proteins • An elaborate endoplasmic reticulum (ER) & abundant free ribosomes • Abundant mitochondria – the sites of oxidative metabolism • Neurons – have massive protein production and very high metabolic activity (recall oxygen and blood flow stats). • Rough Endoplasmic Reticulum: • An abundant organelle in neurons • Reflects the very high level of protein synthesis • Nissl bodies (same as a Nissl substance) are composed of granular endoplasmic reticulum and ribosomes. • Made up of membrane stacks dotted with ribosomes • Ribosomes contain mRNA & are sites of protein synthesis (protein translation) • Axon Hillock: • The axon hillock plays an essential role in the integration & transmission of signals. • Plasma membrane at the axon hillock forms trans membrane protein barriers with actin filaments to block free diffusion of proteins from soma to axon. • Has no synapses • Region of summation of excitatory & inhibitory activity • The usually generated in the axon hillock • Axon: • Cytoplasmic extension of neuron • Very thin (only a few µm in diameter at most in humans) • May be very long (can be > 1m in humans) • Carries information from the soma to another neuron or effector cell • : • Specialized junctions between neurons • Sites where neurons communicate through chemical messengers – • The Neuronal Membrane: • Composition: phospholipid bilayer & special embedded proteins • Ion channels and carrier proteins (‘pumps’) • Function - preserves the highly-controlled internal environment & intercellular contact necessary for excitation • Regulates permeability • Excitation, neuronal communication, sensation, movement, integration, behaviour and thought

• How does the neuronal membrane regulate permeability & ionic segregation? • Na+/K+ pumps maintain (negative inside cell) & Ca2+ pumps keep Ca2+ concentration very low inside cell • 1. Passively - lipid solubility

• O, N, CO2 & alcohols are lipid soluble freely diffuse through the cell membrane and, even though H2O is lipid insoluble, H2O diffuses through (it’s small & has high kinetic energy). • Ions do not diffuse through. • Neurons maintain a charge potential difference across the neuronal membrane. • 2. Carrier proteins - active ‘ pumps ’ • Require energy - ATP - for transport of ions against the gradient; that is, from low to high concentration (ionically uphill) • 3. Ion channels - passive • Require no energy for ions to pass through • Selective for particular ions (charge/size) • Passive non-gated ion channels • Gated ion channels à Voltage-gated and Ligand-gated • Channels do require energy for production, removal maintenance and insertion into the membrane • Gated Ion Channels: • Gated channels are not exclusive to the nervous system or to neurons, but neurons use these in a very specific manner. • Voltage-gating - conformational change with change in voltage • Ligand-gating - conformational change with binding of effector molecule • Voltage-gated Na+ and K+ Channels: • Open & close depending on the voltage (charge difference) across the membrane around the channel. • Voltage-gated ion channels mediate changes in membrane permeability to propagate the action potential (AP) within the neuron. • Difference: • Inactivating voltage-gated Na+ channel • Delayed (rectifying) voltage-gated K+ channel • Sodium Channels: • Closed at -80mV to -65mV (resting) • Begin opening between -65mV & -40mV • Open Na+ channels allow Na+ to flow into cell • Cell becomes more positive with Na+ influx • Depolarises & more channels open • Fully open at -15mV At about +30mV, Na+ channels close Na+ influx ceases • Can’t reopen immediately (refractory period) • The highest density of sodium channels is at the axon hillock - allows generation of AP down the axon. • Potassium Channels: • Closed at -65mV (resting) • Na+ influx causes cell to ‘go positive’ • K+ channels open at +30 to 35mV • That is, after Na+ channels open • K+ flows OUT of cell • Re-establishes resting charge potential behind Na+ flux • Reclose at resting (-65mV) potential • How does the microanatomy of the neuron support the unidirectional signal? • Different regions of the neuronal cell have different densities of voltage- and ligand-gated channels • High density of ligand-gated channels at the ‘input’ end of the cell () • These channels respond to ligands - chemical input (neurotransmitters). • At the axon hillock & along the axon there is a high density of voltage-gated ion channels. • These generate and conduct action potentials. • Membranes of dendrites & soma à Higher density of ligand-gated channels (open with binding of ) • Membranes of axon hillock & axon à Very high density of voltage-gated ion channels. • Glial cells influence the conduction speed and segregation of ions across the neuronal membrane. • ( membrane) insulates the axon à Reduces ion leakage and allows faster conduction of AP.

Lecture 3:

• Categories of glia: • Macroglia à and • • Macroglial cell lineage: • Astrocytes, oligodendrocytes & neurons share a common stem cell à neural stem cells • All originate from neural stem cells. • Microglial cell lineage: • Microglia are macrophages & have a monocyte (bone marrow derived) lineage. • Move from the bone marrow into the brain. • Microglia are monocytes. • Microglia remove waste from the brain, e.g. dead cells, synapses etc. • Macroglia: • In the central nervous system à Astrocytes and Oligodendrocytes • In the peripheral nervous system à Schwann cells myelinate the peripheral portions of neurons (). • Schwann cells are derived from neural crest cells à stem cells that surround the neural tube in early development. • Astrocytes: Star Shaped • Labeled with immunohistochemistry using antibodies against GFAP • Cytoskeleton confers strength and shape to cell. • GFAP is specific to astrocytes. • Astrocytes line the capillaries in the brain.

• Astrocytes fill spare volume in the brain. • The ratio, number and size of astrocytes per neuron has increased significantly in humans. • Jobs of an : • Provide structural support • Invest endothelium & induce blood brain barrier tight junctions • Provide support for synapse formation & maintenance • Guide neuronal process growth & regulate neurogenesis • Maintain biochemical balance around neurons: • - Sequester/redistribute K+ during neural transmission • - Remove glutamate & GABA at the synapse • - Synthesise glutamate & GABA precursors • - Detoxify ammonia • - Provide energy substrates (lactate) to neurons • - Maintain brain/water homeostasis • Form scars • Support neuroinflammatory system • Astrocytes form adhesion molecules between cells.

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• Astrocytic processes are joined by cell-cell contacts; increase strength • Astrocytes stabilise neuronal configurations • Astrocytic processes interweave between neuronal processes • Astrocytic endfeet à Invest >90% of brain capillary (endothelial cell) surface • Induce endothelial cells to form the blood brain barrier

• Astrocytes induce and support the blood brain barrier (BBB). • The blood brain barrier is formed of tight junctions between endothelial cells. • Astrocytes induce endothelial cells to form tight junctions. • In the absence of astrocytes, endothelial cells do not form tight junctions. • Astrocytes promote angiogenesis, the growth of new blood vessels.

• Pericytes are contractile cells that wrap around the endothelial cells of capillaries and venules throughout the body. • Activated astrocytes: • Produce abundant GFAP & extracellular matrix (such as laminin, tenascin, fibronectin & proteoglycans) • CNS repair à Astrocytes form scars after injury • Astrocytic scars ‘fill in’ around where neurons are injured or dying. • Astrocytic scars can interfere with re-growth of neuronal processes in CNS (partly accounts for poor CNS regeneration).

• Astrocytes - provide support for synapse formation & maintenance • Astrocytes isolate neurons à Ensure that receptive neuronal surfaces are protected from non-specific influences • Support synaptic microanatomy à Envelope neuronal terminals • Astrocyte processes are especially dense in areas of intense synaptic activity • Synaptic remodelling à Remove degenerating synapses • Astrocytes maintain brain water balance via aquaporin channels. • Astrocytic swelling can change synaptic distance, thus efficiency. • Astrocytes - guide brain development: • Radial astroglial processes provide tracks along which neurons migrate during development. • Glutamate: • Astrocytes provide neurochemical support for neurons by taking up excess glutamate, preventing neurotoxicity. • Astrocytes supply glutamine (a glutamate precursor) to the neuron. • The neuron converts this glutamine to glutamate and fires the post-synaptic neuron. • The astrocyte then clears glutamate from the synaptic cleft. • Astrocyte neuron lactate shuttle hypothesis: • Astrocytes - relay lactate (energy supply), buffer K+ • Astrocytes take up glucose and turn it into lactate. • Lactate is given to the neuron, to produce ATP that fuels the sodium-potassium pump and the conversion of glutamate + ammonia to glutamine. • Astrocytes – communicate through gap junctions: • Astrocytes propagate Ca+ waves and these waves constrict blood vessels. • The propagation timescale is slow. Consider seconds (astrocytes) vs.milliseconds (neurons) • Several potential functions in vivo • May prime activity over large distances • Spread glial activation (such as in inflammation) • Regulate blood flow in local areas. • Produce vasoconstrictive/vasodilatory factors: e.g. arachidonic acid, NO, prostaglandins Dilate or constrict blood vessels • Oligodendrocytes: • Wrap a membrane sheath around the axons of neurons. • Oligodendrocyte process spirals around axon. • Cytoplasm is extruded until the opposite membranes meet, forming a multi-layered lipoprotein coat à a myelin sheath • Myelin sheath = specialised cell membrane à a living part of the cell

• Myelin: • Myelin (oligodendrocyte membrane) insulates the axon and increases resistance across the axonal membrane. • Reduces leakage of ions. • Faster conduction, fewer ions move, less energy required to segregate the ions. • Increases the speed of conduction of impulse • Faster conducting axons have thickest myelin sheath & longest internodes • Action potential jumps from one to the next à saltatory conduction. • Node of Ranvier: • Na+ channels (responsible for supporting the AP) are concentrated at node • Very little current leakage at internodes • Microglia: • Age-dependent entry to the brain (few microglia enter after birth). • The BBB prevents monocytes from entering the brain in large numbers in adults but exchange with blood circulation is possible. • The evidence for this comes from animal studies and inter-gender human bone marrow transplants. • Microglia morphology changes with activation state. • Glia are key players in disease: • Astrocyte dysfunction: • Increased glu production in presynaptic neuron • Decreased glu uptake from synapse • Decreased energy stores, Ca+ balance • Most neurotransmitters are able to be taken up by astrocytes • Astrocyte dysfunction is implicated in epilepsy, Schizophrenia • Oligodendrocytes & Schwann cells dysfunction: • Degenerate – e.g. Multiple Sclerosis resulting in slowed conductance • Microglia activation: • Produce neurotoxins (NO) • Produce excitotoxins: glutamate, quinolinic acid • Diseases characterised by neuroinflammation Multiple sclerosis, Alzheimer’s disease, stroke, HIV, etc.

Lecture 4: Looking at neurons

• Cells of the nervous system: • Neurons. • Supporting Cells. Schwann+, Satellite+, Perineural cells+, Astroglia*, Oligodendroglia*, Microglia*. • Other Cells: *, *(Pia, Arachnoid, Dura). • Plus blood vessels, including capillaries, which are not unique to the nervous system. • * Found in the CNS. + Found in the PNS. • Schwann Cells. Schwann cells are found surrounding some peripheral cell processes where they form the axonal myelin sheath, which improves the conduction velocity of impulses. They probably also have other functions. They also have unmyelinated fibres embedded in them. • Satellite Cells. Satellite cells surround nerve cell bodies located in the ganglia of the peripheral nervous system and have functions similar to astroglia. • Perineural Cells. Perineural cells are found in peripheral where they form the . The perineurium defines the fascicular substructure of peripheral nerves. • Astrocytes. Astrocytes, prominent in the (protoplasmic) but are also found in (fibrous). Their processes are found near blood vessels, pia, ependyma and neurons including near synapses. Function, mechanical support, metabolic, maintains homeostasis of extracellular fluid, form scar tissue. • Oligodendrocytes. Oligodendrocytes, prominent in white matter, processes surround axons. Function myelination. • Microglia. Microglia, enter and leave the CNS, widely distributed. Function immunological, phagocytic. • Ependyma. Ependyma, line the ventricles and along with the pia and capillaries form the choroid plexus, which secretes cerebrospinal fluid (CSF). • Meninges. The meninges (pia, arachnoid, dura) are membranes, which surround the brain and protect it. They are composed of varying proportions of flattened epithelial like cells and . • Structure: • The Structure of a Typical Neuronal Cell. Is comprised of a body (soma, perikaryon) containing the nucleus (karyon) and processes (neurites). The processes are of two types, many branching dendrites and a single branching axon. The proximal part of the axon is called the axon hillock. • Schematic Neurons. Particularly when drawing circuits neurons can represented in a schematic way by a circle (cell body and dendritic tree) a line of varying length (axon) with a small V at the end (the synapse). This notation can be varied to indicate various neuronal shapes and ways of connecting with other neurons. • Neurons can be described according to their processes: • A neuron with one process is a , with two processes is a and with more than two processes is multipolar. • Projection neurons project over long distances have long axons à Golgi Type 1 • project over short distances and have short axons à Golgi Type 2 • About 9 interneurons to 1 projection neuron. • Specific location: • Exact location of the neuron. • Purkinje cells are found in the middle layer of the cerebral cortex. • Grey and White Matter: • Grey matter is distributed in several ways. In lamina (e.g. cerebral cortex, cerebellar cortex) as nuclei (e.g. amygdala, mammillary nuclei, inferior olive) or as irregular collections (e.g. grey matter). • Each of these levels may have further internal organization such as the layers of the cortices. • Sometimes nuclei are given the name nucleus other times just a particular name. Cell bodies of neurons in the peripheral system are located in ganglia, or specialized tissue such as the olfactory mucosa. • White matter is given a wide range of names, depending on its location, such as a tract, peduncle, fasciculus, , commissure, funiculis, capsule, central or medullary white matter or just a particular name. • Grey matter in general is defined by the presence of nerve cell bodies and further characterized by the presence of the dendrites of projection neurons and small interneurons, axons of interneurons, proximal axons of projection neurons, distal axons of projection neurons from elsewhere, projection neuron axons in transit or passing through, synapses and astrocytes (protoplasmic). • Grey matter also has a good blood supply and contains many capillaries. It is a site of information processing. The organization of neuronal processes and their connections within the grey matter is termed the microcirciutry. • White matter is in general characterized by the axons of projection neurons, which may be myelinated or unmyelinated. • White matter also contains oligodendrocytes, which provide the myelination. It is a site of information transfer. Connections between several groups of grey matter, with some functional link is termed the macrocircuitry. • Staining Techniques: • Haematoxylin and Eosin (H & E). • Stains nuclei, including in nerve cells, a dark blue to black colour. • Eosin is an anionic dye and is related to fluorosceneeosin. It is sometimes referred to as a counterstain. • As well as providing a background contrast (pink) for cytoplasm and extracellular structures it can produce some contrast in the cell membrane and hence may show indistinctly some nerve cell processes. • Hence by staining cell nuclei the size, numbers, density and distribution of neurons can be approximated and seen. • The extent of grey matter can be defined but not of the connections. Glial and other cells are also stained. However glia in particular cannot be readily differentiated.

• Nissl stains • Stain particularly the nucleus, nucleolus and ribosomes, including those found in the endoplasmic reticulum, and the membrane to some extent. • Endoplasmic reticulum is well developed in metabolically active cells such as neurons; it is present in the cell body and dendrites but not the axon. • This stain shows the grey matter and its organization such as layers, cell numbers, density and body size and the beginnings of larger processes, but like H & E cell connections are not shown. It is a relatively simple and reliable stain to use. It can also be used as a counter stain with stains that mainly stain fibres. • Glial cells may be stained to some extent, such as their nuclei, also capillary endothelial cell nuclei and red blood cells.

• Reduced Silver Stains: • Stains all or most neurons. Cell bodies and neurites are clearly shown hence both grey and white matter are shown. • The extent to which axons can be traced is limited by the section thickness and process density. • Because all neurons are stained the number of processes obscures the detail in the dendritic tree.

• Golgi Silver Stains: • Stains only a small number of neurons in particular their dendritic trees, which are not obscured by having many cells stained. • Enables thick sections to be looked at and gives a clear picture of the cell body shape but not the intracellular structures, it particularly shows the dendritic tree and sometimes individual cell connections. • It also can show synaptic spines. It also shows glial cells. • Fibres Stains: • Fibre stains are useful for indicating fibre tracts and white matter in general but not cell bodies. • They can be used in association with cell body stains such as Nissl stains (counter staining). In serial sections large bundles of fibres can be traced but not the course of individual fibres or some groups of fibres.