Lecture topic 1.pdf

Neurons and Electrical Signaling (CBNS 120, Currie) Membrane passive or active active passive or active types:  Example of “endogenous slow potentials”… Endogenous Bursting Membrane potential (Vm)...

Neurons and Electrical Signaling (CBNS 120, Currie) Membrane passive or active active passive or active types:  Example of “endogenous slow potentials”… Endogenous Bursting Membrane potential (Vm) Time (s) Examples of “burster” neurons: pre-Bötzinger complex neurons in the mammalian medulla (brainstem) that drive the breathing rhythm; R15 neuroendocrine cell in the abdominal ganglion of the marine mollusk Aplysia. Santiago Ramon y Cajal, self-portrait, Rodent hippocampus - Cajal indicated the probable when he was in his thirties direction of information-flow with arrows CA1 from CA3 DG EC to EC Principle of Dynamic Polarization - Information flows in a predictable and consistent direction within a nerve cell. - Information flow begins at the receiving (input) sites on the dendrites and soma to the trigger zone at the axon hillock (initial segment). - At the axon hillock, the action potential is initiated and propagated unidirectionally along the axon to the presynaptic transmitter-release sites at the axon terminal. - Neurons differ greatly in form and function, but most adhere to this pattern of information flow. Types of Neurons (based on morphology) Multipolar turtle spinal motor neuron Unipolar crayfish swimmeret motor neurons in 3rd abdom ganglion From: Kandel et al. (1995) Essentials of Neural Science and Behavior Types of Neurons (based on morphology) Multipolar turtle spinal motor neuron Unipolar crayfish swimmeret motor neurons in 3rd abdom ganglion From: Kandel et al. (1995) Essentials of Neural Science and Behavior  Signal transduction in a typical vertebrate neuron LOCAL GRADED POTENTIAL = a graded change in membrane potential (Vm) that varies continuously in amplitude with stimulus-strength and decays exponentially over distance. ACTION POTENTIAL = A transient, all-or-none reversal of the Vm produced by a regenerative inward current in excitable membranes. Does not decay over distance. In general, the more a local graded potential depolarizes a neuron, the higher the action potential frequency evoked, up to a point (the cell’s maximum spike frequency).  Signal transduction in a vertebrate sensory neuron From: Kandel et al. (1995) Essentials of Neural Science and Behavior  axon hillock (spike initiating zone) local action pots local action pots graded graded pots pots AM code FM code AM code FM code AM = amplitude modulated FM = frequency modulated Types of Neurons (based on function & inputs/outputs) 1. Motor neurons (a.k.a. “efferents” or “effector cells”) - Synapse onto muscle or gland tissue. - In vertebrates, all motor neurons are excitatory (i.e., produce excitatory postsynaptic potentials); but in invertebrates, can be either excitatory or inhibitory. 2. Sensory neurons (a.k.a., “afferents”) - Transduce sensory information. - Examples: heat, cold, light, mechanical pressure or stretch, chemical energy 3. Neuroendocrine cells - Release neurohormones into the circulation 4. Interneurons - Are both postsynaptic and presynaptic to other neurons. From: Kandel et al. (1995) Essentials - Can be either local- or projection-type interneurons. of Neural Science and Behavior Example of a non-spiking local interneuron with no axon: starburst amacrine cell in mammalian retina 6 Example of spiking projection interneurons with long axons: reticulospinal cells in vertebrate brainstem Mauthner cells & other giant reticulospinal cells in living zebrafish brainstem, fluorescently labeled with calcium green conjugated dextran (CGD) (Joseph Fetcho).  Example of spiking projection interneurons with long axons: reticulospinal cells in vertebrate brainstem Reticulospinal cells in fixed whole-mount of zebrafish brainstem, stained with Texas Red Dextran (Don O’Malley, 2008). 8  Some basic electrophysiology terms defined: MEMBRANE POTENTIAL (EM) = voltage or electrical potential (mV) across cell membranes, arising from a separation of charge. A typical EM in neurons is -60 to -70 mV (inside negative). DEPOLARIZATION = decrease in EM (decreased inside negativity), or movement of EM in a positive direction. HYPERPOLARIZATION = increase in EM (increased inside negativity), or movement of EM in a negative direction. SYNAPTIC POTENTIALS - 3 major types: chemical excitatory, chemical inhibitory, electrical ELECTRICAL (ELECTROTONIC) PSPs (post-synaptic potentials) are mediated by gap junction channels. CHEMICAL PSPs can be mediated by transmitter-gated ion channels (ionotropic receptors), or by G-protein-linked receptors (metabotropic receptors) that affect ion channels indirectly. - Binding of transmitters to inotropic and metabotropic receptors can produce either excitatory (E), or inhibitory (I) PSPs. - Binding of transmitter to metabotropic receptors can also produce neuromodulatory effects in post-synaptic cells (affecting action potential shape, spontaneous discharge, and synaptic strength (efficacy). Recording electrical activity in the nervous system 1. Extracellular recording - “Differential” extracellular recording from axons and whole nerves Crayfish motor nerve recording 1. Extracellular recording (cont.) 2. Intracellular recording - “Single-ended” extracellular recording - sharp electrode and whole-cell patch from neuronal cell bodies 15  3. Optical recording Homma R et al. Phil. Trans. R. Soc. B 2009;364:2453-2467 Squid giant axon Turtle olfactory bulb in vitro Voltage-sensitive dyes have very fast responses (𝜏 = 10 µs) Electr. stim of olfactory nerve. Odorant Mouse olfactory bulb stim in vivo (a) Changes in transmitted light intensity (absorption; dots) of a squid giant axon Comparisons of voltage (fast), stained with a merocyanine dye, XVII, calcium (medium), and intrinsic during a membrane action potential (smooth trace) recorded simultaneously (slowest) optical imaging signals. with an intracellular electrode. Calcium imaging in living zebrafish larva brainstem during startle response (Mauthner neuron) From Joe Fetcho’s Laboratory at Cornell Univ. Mauthner cell calcium responses to tail taps  Whole-brain functional imaging at cellular resolution in intact zebrafish embryos, using genetically encoded Ca2+ indicators. Misha Ahrens et al. (2013) Nature Methods 10: 413-420. This work utilizes the genetically encoded Ca2+ indicator GCaMP to report the activity of more than 80% of the neurons in the whole brain of intact zebrafish larvae at single-cell resolution. Note that video playback is sped up 21-fold from real time. Most large-scale imaging of neural activity, especially in human studies, relies on the “hemodynamic response” (neurovascular coupling, or the “Roy-Sherrington principle.”) “..the brain possesses an intrinsic mechanism by which its vascular supply can be varied locally in correspondence with local variations of functional activity.” ~ Roy and Sherrington (1890) ⬆ neural activity = ⬆ local blood flow, and ⬆ local blood oxygenation FMRI (functional magnetic resonance imaging) detects the increased ratio of O2/ de-O2 hemoglobin during the “hemodynamic response” to increased neural activity. Visualizing neuron anatomy Great effort has gone into the development of techniques for making nerve cells and their processes visible and for tracing nerve fiber pathways and synaptic connections between cells. Many of the classic anatomical methods were developed around the turn of the 20th century, especially by the great Spanish neuroanatomist Santiago Ramón y Cajal. Golgi Stain. The Golgi silver stain, invented by Camillo Golgi and perfected by Ramón y Cajal, has the mysterious (but fortunate) quality of staining only an occasional neuron - about 5% - in a slice of tissue. Because so few cells are stained, the Golgi method can be used to see entire nerve cells in very thick sections of brain tissue. The cells that are stained, are stained darkly throughout even the finest of their dendrites and axonal processes. When this method was used by Ramón y Cajal, it was the first time that individual nerve cells had been visualized - providing the first strong evidence for the Neuron Doctrine, or cellular hypothesis of neurons.  Techniques have also been developed that permit the selective visualization of single neurons or groups of neurons that share common properties; for example....... Intracellular dye injection. Since the development of the sharp intracellular microelectrode by Ling and Gerard in 1949, it has been possible to first impale and characterize the electrical activity of a single neuron and then inject the same cell with an intracellular dye such as horseradish peroxidase (HRP), cobalt, biocytin, or Lucifer Yellow. This has enabled researchers to study both the electrophysiology and morphology of single neurons as well as pairs or small ensembles of synaptically connected cells. Rat cortical pyramidal neuron, intracellularly stained with Lucifer Yellow Exposure of Lucifer Yellow to blue/UV light produces free radicals that rapidly kill the cell. This is called “photoinactivation”, and has been used to delete cells from invertebrate neural circuits. Focused UV lasers can be used to “clip off” individual dendrites or axon branches with this technique.  Techniques have also been developed that permit the selective visualization of single neurons or groups of neurons that share common properties; for example....... Intracellular dye injection. Since the development of the sharp intracellular microelectrode by Ling and Gerard in 1949, it has been possible to first impale and characterize the electrical activity of a single neuron and then inject the same cell with an intracellular dye such as horseradish peroxidase (HRP), cobalt, biocytin, or Lucifer Yellow. This has enabled researchers to study both the electrophysiology and morphology of single neurons as well as pairs or small ensembles of synaptically connected cells. Rat cortical pyramidal neuron, intracellularly stained with Lucifer Yellow Exposure of Lucifer Yellow to blue/UV light produces free radicals that rapidly kill the cell. This is called “photoinactivation”, and has been used to delete cells from invertebrate neural circuits. Focused UV lasers can be used to “clip off” individual dendrites or axon branches with this technique.  Techniques have also been developed that permit the selective visualization of single neurons or groups of neurons that share common properties; for example....... Intracellular dye injection. Since the development of the sharp intracellular microelectrode by Ling and Gerard in 1949, it has been possible to first impale and characterize the electrical activity of a single neuron and then inject the same cell with an intracellular dye such as horseradish peroxidase (HRP), cobalt, biocytin, or Lucifer Yellow. This has enabled researchers to study both the electrophysiology and morphology of single neurons as well as pairs or small ensembles of synaptically connected cells. John P. Miller - Montana State Univ. Rat cortical pyramidal neuron, intracellularly stained with Lucifer Yellow Exposure of Lucifer Yellow to blue/UV light produces free radicals that rapidly kill the cell. This is called “photoinactivation”, and has been used to delete cells from invertebrate neural circuits. Focused UV lasers can be used to “clip off” individual dendrites or axon branches with this technique. Optogenetics The use of light to excite or inhibit cells, usually neurons, that have been genetically modified to express light-sensitive ion channels or pumps. Channelrhodopsins (Ch1 and Ch2) are light-gated cation channels obtained from the motile, single-cell green algae, Chlamydomonas. Ch2 is activated by blue light and causes cell depolarization & excitation. Chlamydomonas Halorhodopsin (e.g., Halo-3) is a light-activated chloride pump that drives Cl- into cells, and Archaerhodopsin (Arch) is a light-activated proton pump that drives H+ out of cells. Both are obtained from “halobacteria” (Achaea), and when activated by green/yellow light, cause cell hyperpolarization & inhibition. Halobacteria Optogenetic control of genetically-targeted pyramidal neuron activity in prefrontal cortex. Barratta et al. (2012) Nature Precedings. doi:10.1038/npre.2012.7102.1 Optogenetic control of genetically-targeted pyramidal neuron activity in prefrontal cortex. Barratta et al. (2012) Nature Precedings. doi:10.1038/npre.2012.7102.1 Optogenetic control of genetically-targeted pyramidal neuron activity in prefrontal cortex. Barratta et al. (2012) Nature Proceedings. doi:10.1038/npre.2012.7102.1 Optogenetic control of genetically-targeted pyramidal neuron activity in prefrontal cortex. Barratta et al. (2012) Nature Precedings. doi:10.1038/npre.2012.7102.1