Cell Biology of the Neuron and the Synapse

L1: intro to neuronal cell biology

Objective 1: Describe the cell types and the complexity of the CN   There is a hierarchy of organisation in the brain; brain regions/circuits are made up of neurons and glia which are in turn made up of axons and dendrites. neurons come in many different types (about 50) and receive and transmit impulses. glia are the connective tissue of the nervous system. They provide structural and metabolic support for neurons. white matter has a much higher lipid content than grey matter.   Reticular theory was the old theory of the brain put forward by Gerlach in 1871. It postulated that everything in the CNS was one continuous network. The theory was put to bed first of all by Cajal who used Golgi's silver-chromate staining technique to show that the nervous system is made up of independent cells. In 1891 the term "neuron" is coined, in 1897 the concept of a synapse was put forward.    Neurons feature a high degree of polarity and excitability.  Neurons may be spiny or non-spiny. Glia are not directly involved in electrical signalling. Glia cells include astrocytes, microglia and oligodendrocyctes.  Astrocyctes: most numerous, star-like shape, take up substances from synaptic cleft, involved in BBB. Microglia: arise from cells outside the CNS, they are the macrophage of the CNS, they become activated during infection, injury and seizure.  Oligodendrocytes: schwann cells in the PNS, they ensheath axons with myelin.    

Objective 2: Outline how the neuronal cytoskeleton is specialised The neuronal cytoskeleton is made up of 3 components: microtubules, neurofilaments and microfilaments.  Specific cytoskeletal isoforms provide useful markers.

Objective 3: Discuss how neurons use axonal transport to overcome the problem of their size   experiments by Weiss and Hisco demonstrated the existence of axonal transport.  The issue with transportation is that the synapse can be very far from the cell body. This is overcome by bidirectional fast and slow transport. Transport can be fast (anterograde and retrograde, moving about 200-400 mm per day) or slow (0.2-8 mm per day). Spatial and temporal control of neuronal transport is mediated by the regulation of interactions between motor proteins and their adapters. mitochondria are transported by kinesin motor proteins along microtubules. Synaptic activity causes cytoplasmic Ca2+ to increase which in turn recruits mitochondria which uncouples the mitochondria from the kinesin. modern research uses genetically encoded fluorescent proteins and fluorescence microscopy to study axonal transport.

Lecture 2: Neuronal ion channels

Objective 1: Understand the basis of the resting membrane potential and the Nernst potential.   The inside of the neuron is negatively charged compared to the extracellular matrix. This charge difference amounts to about -60 to -70 mV. There is a largely equal charge across the membrane, only a small number of ions make the difference. Nernst potential: The membrane potential at which there is no net flow of that particular ion from one side of the membrane to the other. In neurons, the lipid bilayer is virtually impermeable, ions can only cross via channels. At rest, the passive ion fluxes are balanced so the membrane potential is constant. This balancing is done by the NA/K-ATPase pump.

Objective 2: Describe the methodology and data arising from current and voltage clamp electrophysiology AND describe properties of voltage-gated ion channels.   METHODOLOGY IN ELECTOPHYSIOLOGY Current clamp: inject a known quantity of current into the cell, measure the Vm response. Voltage clamp: used to investigate the currents flowing through individual ion channels. Here, the voltage across the cell membrane is monitored while simultaneously injecting a small amount of current to clamp the transmembrane voltage at a desired level.  Analysis of this type has shown that Na+ channel activation gates have fast kinetics, are closed at resting potential and open in response to depolarization. Also, K+ channel activation gates are the same but have slow kinetics. Patch clamp: This involves placing the electrode tip on the membrane of the cell to form a high resistance seal between the wals of the tip and the membrane. This allows one to measure the activity of single channels incorportated into the membrane trapped on the tip of the electrode. Whole cell patch clamp: A pulse of suction applied to a patch clamp ruptures the membrane and allows whole cell recording. Properties of one type of channel can be studied by blocking other types with toxins/drugs. VOLTAGE-GATED ION CHANNELS: Na+ General structure: Consist of large alpha subunits that associate with proteins. The subunit has 4 repeat domains I through IV, each containing 6 membrane-spanning segments S1 through S6.  The S4 segment is highly conserved and acts as the channel's voltage sensor. The sensitivity of this channel is due to positive amino acids located at every third position. When stimulated by a change in transmembrane voltage, this segment moves toward the extra cellular side, allowing the channel to become more permeable to ions.  There are 3 main conformational states: closed, open and inactivated. Before an action potential occurs, the channels are in their deactivated state, blocked on the extracellular side by their activation gates.  When an action potential occurs, the activation gates open and Na+ ions flow into the neuron, causing depolarization of the membrane.  At the peak of an action potential, the channels inactivate themselves by closing their inactivation gates, which can be thought of as a plug tethered to domains III and IV.  Data from experiments: Activation gate: fast kinetics, closed at resting, opens in response to depolarization. Inactivation gate: slow kinetics, open at resting potential, closes in response to depolarization. VOLTAGE GATED ION CHANNELS: K+   ACTION POTENTIAL MECHANISM Hodgkin and Huxley used the giant squid axon to make early recordings of action potential and show the role of Na+ and K+. The action potential is choreographed by the activation threshold, ion selectivity and inactivation of Na+ and K+ voltage-dependent ion channels. molecular mechanisms of an action potential: https://www.coursera.org/learn/medical-neuroscience/lecture/HLwlx/molecular-mechanisms-of-action-potential-generation-part-1

Objective 3: Understand the neurophysiological aspects of ion channel function.   toxins such as TTX are used to explore ion channel function. hereditary mutations in ion channels that cause disease alter ion channel properties. 

L3: Pre-synaptic events

Objective 1: Ca2+ dependent neurotransmitter release   Action potential arrives at synapse, the membrane depolarises and voltage-gated Ca2+ channels open.  The increase in [Ca2+] triggers synaptic vesicles to fuse with the plasma membrane. The released neurotransmitter diffuses across the synaptic cleft and interacts with specific postsynaptic receptors. Neurotransmitter can also activate presynaptic autoreceptors. Neurotransmitter can 'spillover' into adjacent synapses. Neurotransmitter is either degraded or taken up by glia or presynaptic transporters. Synaptic vesicle membrane is retrieved for recycling by endocytosis. Neuropeptides are released from dense cored granules. neurotransmitter release is couples to Ca2+ influx in the presynaptic terminal. In the late 1960s Katz and Miledi showed that membrane depolarization-induced NT release did not require Na+ influx/K+ efflux.   THE CA2+ VOLTAGE GATED CHANNEL Ca2+ alpha subunits are monomeric like Na+ channels. resting [Ca2+] in the presynapse is0.1uM. [ca2+] required to trigger synaptic vesicle exocytosis is 5-20uM. release sites are clustered around Ca2+ channels.    Neurotransmitter release is quantal. This was showed by Katz who discovered miniature endplate potentials, and subsequently concliuded that these were due to spontaneous release of NT. 

Objective 2: The synaptic vesicle cycle   in reality synaptic vesciles have numerous molecules protruding from their surface. These include SNAP 25, synaptobrevin and synaptophysin.   

Objective 3: Regulation of transmitter release