## Anatomical connectivity * C. elegans neuronal network (279 out of 302 total neurons) * Determined through tracing and electron microscopy * red: sensory neurons, blue: interneurons, green: motor neurons * 6393 chemical synapses, 890 electrical junctions, and 1410 neuromuscular junctions

Principles of Neurobiology, Garland Science Fig. 13-2, White et al, Phil Trans R Soc Lond B 1986 [http://www.wormatlas.org/neuronalwiring.html](http://www.wormatlas.org/neuronalwiring.html)

Note: Wiring diagram of C. elegans (302 neurons total, 279 neurons shown here. 20 pharyngeal (of or relating to the pharynx) nervous system neurons not shown as well as 3 that do not make synapses with other neurons) red: sensory neurons blue: interneurons green: motor neurons number of possible node pairs is N*(N-1)/2 for c. elegans: > (302*301)/2 [1] 45451 > (279*278)/2 [1] 38781 6393 chemical synapses, 890 electrical junctions, and 1410 neuromuscular junctions horizontal axis represents closeness of connectivity (spring embedded graph layout) vertical aix represents signal flow from top to bottom from D. Chklovskii. White et al, Phil Trans R Soc Lond B 1986 --- ## Investigating gene function

Principles of Neurobiology, Garland Science Fig. 13-4

Note: Forward and reverse genetics. forward genetics, researchers start by observing an altered trait (phenotype) to id. the gene responsible for causing the phenotype of interest. in reverse genetics, researchers start with a gene of interest and disrupt the gene function, examing the phenotypic consequences. --- ## Making mammalian genetic models

Principles of Neurobiology, Garland Science Fig. 13-6

Note: Making fancy mice. --- ## Targeted editing of the genome * CRISPR/Cas9 system for faster genetic engineering

Principles of Neurobiology, Garland Science Fig. 13-8

Note: Charpentier & Doudna Nature 2013, Ran et al., Nat Protocol 2013 crispr-cas9 system. crispr: clustered reguarly interspaced short palindromic repeat cas: crispr associated any eukaryotic dna that contains a PAM sequence can be a target. PAM sequence: protospacer associated motif, usually two or three nucleotides. Occurs frequently. * -canonical PAM is the sequence 5'-NGG-3' where "N" is any nucleobase followed by two guanine ("G") nucleobases * -Guide RNAs (gRNAs) can transport Cas9 to anywhere in the genome for gene editing, but no editing can occur at any site other than one at which Cas9 recognizes PAM. A guide RNA that contains sequences complementary to a piece of DNA from the target gene of interest brings the Cas9 enzyme to the target site on the chromosome through DNA-RNA base pairing (purple and red) Two nuclease domains of Cas9 create a double strand break in the genomic DNA. The double strand break can be repaired by the nonhomologous end joining system, through which small deletions or insertions may be created at the repair site. The ds break can also be repaird by the homologous recombination system with a donor DNA as a template, through which specific modifications such as insertion of a transgene can result. --- ## Timeline of genome sequencing advances

Principles of Neurobiology, Garland Science Fig. 13-7

Note: timeline of selected milestones. Graph show exponential growth of sequencing technology in 10 yrs since draft of human sequences first published. top graph from Mardis Nature 2011 --- ## Traditional anatomical methods for determining cortical areas

Principles of Neurobiology, Garland Science Fig. 13-18

Note: Nissl stain and cortical area divisions. Border of V1 and V2. Adapted from Brodmann K (1909). --- ## Confocal and light sheet fluorescence microscopy

Principles of Neurobiology, Garland Science Fig. 13-19

Note: confocal is on left. light sheet microscopy on the right. Confocal microscopy uses a small pinhole before the detector to allow emitted fluorescence from only the focal plane. In light sheet microscopy, illumination is proved from the side to produce a thin sheet of excitation light. --- ## Making brain tissue transparent for better microscopic imaging

Principles of Neurobiology, Garland Science Fig. 13-20, Chung et al., Nature 2013

Note: clarity based tissue clearing for fluorescene imaging. Intact tissue is fixed in prescence of hydrogel monomers that covalently link DNA RNA, and proteins into a mesh during subsequent polymerization. Lipids (which are a major cause of opacity for fluorescence imaging) are not covalently linked and are removed during subsequent clearing process by passive diffusion or electrophoresis in presence of detergent. Tissue is then transparent for better and deeper imaging. This example is from a Thy1-gfp transgenic mouse imaged with a confocal microscope. Shows neocortex, hippocampus, and thalamus. --- ## Injecting intracellular dyes to trace axonal projections

Principles of Neurobiology, Garland Science Fig. 13-21; Johnson et al., J Neurosci 2000

Note: Intracellular dye filling to trace axonal projections of a single neuron. Rat posterior piriform cortex pyramidal neuron. Injected in vivo followed by several days. Johnson et al., J Neurosci 2000. --- ## Super-resolution fluorescence microscopy

Principles of Neurobiology, Garland Science Fig. 13-25; Dani et al., Neuron 2010

Note: Mapping synaptic protein organization with super res fluorescence microscopy. double labeling with basson (presynaptic scaffold protein, red) and postsynaptic scaffold protein (homer1, green) in mouse olfactory bulb glomerular layer. Imaged using stochastic optical reconstruction microscopy (STORM), a super resolution technique. Gets you beyond the diffraction limit for light microscopy, typically 100-150 nm with the highest resolution objectives and shortest wavelengths of visible light. Third image is higher magnification view. Right shows distribution of different antibody, STORM imaging resolved proteins around the synaptic cleft. --- ## Diffusion tensor imaging

Principles of Neurobiology, Garland Science Fig. 13-26; [www.humanconnectomeproject.org](http://www.humanconnectomeproject.org)

Note: Diffusion tensor imaging. sagittal view of human brain. Axon bundles running along medial-lateral axis are colored red. Those along anterior-posterior axis colored green. Axons running through brainstem colored blue. [www.humanconnectomeproject.org](http://www.humanconnectomeproject.org) --- ## Tracing of long distance connections in the mouse brain

Principles of Neurobiology, Garland Science Fig. 13-27; left, H. Dong [www.mouseconnectome.org](http://www.mouseconnectome.org); Zingg et al., Cell 2014; Oh et al., Nature 2014; right, H. Zeng, [connectivity.brain-map.org](http://connectivity.brain-map.org)

Note: Mix of phytohemagglutnin (PHA-L, green) an anterograde trace and cholera toxin subunit b (CTb, magenta) a retrograde tracer injected into right insular cortex of a mouse brain. Stained in blue with fluorescent nissl stain. Projections from and to insular cortex. On right show adeno-assoc viruses expressing Cre-dependent GFP. Injected into motor cortex of mice expressing Cre recombinase in layer 6 or 2/3. Notice different projection patterns of neurons from these two layers in the rendered 3D views. --- ## Reconstruction of wiring diagram using serial EM

Principles of Neurobiology, Garland Science Fig. 13-29; Takemura et al., Nature 2013

Note: serial electron microscopy to construct wiring diagrams. left is EM micrograph of drosophila medulla in optic lobe. High mag view shown at bottom showing presynaptic terminal with 4 contacts. Colored view showing segmented cellular elements in this image. Neurites reconstructed by registering and linking thousands of consecutive brain sections… from Takemura et al, Nature 2013 --- ## Using modified rabies virus to do monosynaptic tracing

Principles of Neurobiology, Garland Science Fig. 13-30

Note: Gene encoding rabies virus glycoprotein is essential for viral recognition of host cells and for viral spread is replace with GFP in the rabies genome. This mutant rabies virus can no longer recognize and transduce normal mammalian neurons. Mutant rabies assembled in a cell line that helps assemble the virus with a coat protein from a different virus, the EnvA coat protein (blue). This makes it able to transfect mammalian cells that express the EnvA receptor TVA (cyan) from a transgene. A transgene that supplies the rabies glycoprotein is alos expressed in the starter cells. *Need ref* --- ## Principal methods for electrophysiology

Principles of Neurobiology, Garland Science Fig. 13-31

Note: left is photo uncaging right is optogenetics parse out signal flow in the brain. --- ## Multielectrode arrays

Principles of Neurobiology, Garland Science Fig. 13-33; Campbell et al., IEEE Trans Biomed Eng 1991

Note: multielectrode arrays (like this 10x10 silicon based prototype) are widely used now for recordings from multiple cortical neurons simultaneously and for usages with neural prosthetics. from Campbell et al., IEEE Trans Biomed Eng 1991 --- ## Laser scanning two-photon microscopy: imaging neurons in living mice

Principles of Neurobiology, Garland Science Fig. 13-39; Svoboda and Yasuda, Neuron 2006, Denk, Strickler, Webb Science 1990

Note: two photon microscopy is non-linear magic. --- ## Chemical and genetically encoded calcium indicators

Note: chemical and genetically encoded calcium indicators. fura2 is a fluorophore with a calcium chelating site from calcium buffer EGTA. With low calcium, excitation at 380nm produces stronger fluorescence emission than excitation at 350 nm. Ratiometric imaging at 350/380 gives sensitive measure of [Ca2+] first genetic calcium reporter based on FRET (fluorescene resonance energy transfer). Gcamp. permutated gfp is restored to its native 3D structure with an associated increase in fluorescene after calcium triggered binding of the calmodulin binding peptide M13 to calmodulin. Fluorescent intensity thus gives readout of Ca2+. Single APs reliably induce fluorescent intensity changes in GCaMP6 in mouse visual cortical neurons in vivo. left, Grienberger and Konnerth Neuron 2011; Grynkiewicz et al., J Biol Chem 1985; Miyawaki et al., Nature 1997 right, Chen et al., Nature 2013; Nakai et al., Nat Biotech 2001 --- ## Recording neuronal activity in awake behaving mice

Principles of Neurobiology, Garland Science Fig. 13-40 left, Komiyama et al., Nature 2010; middle, Harvey et al., Nature 2009; right, Ziv et al., Nat Neurosci 2013

Note: Head fixed preparation on left during 2P imaging. The thirsty mouse can be trained to extend its tongue only when odor A, but not odor B is presented in order to receive a water reward. Motor cortical area controling tongue extension can be imaged during the learning process. Virtual reality preparation to test the neuronal correlates of memory dependent spatial navigation. Floating styrofoam ball that the mouse is trained to run on, head fixed with electrodes or 2P imaging. Screens providing continuous first person VR experience— in fact this was done by coding a VR environment based on the quake 2 game engine (if any of you have played the classic quake first person shooter games by id software). The right shows a miniaturized microscope weighing just over a gram that an adult mouse can carry on its head to image brain activity as it freely moves and navigates. --- ## Optogenetics for precise control of neuronal activity * Channelrhodpsin-2 (ChR2) from green algae is a cation channel gated by blue light * Allows more Na+ influx than K+ efflux causing depolarization * [http://www.youtube.com/watch?v=I64X7vHSHOE](http://www.youtube.com/watch?v=I64X7vHSHOE)

Principles of Neurobiology, Garland Science Fig. 13-45 left, Boyden et al., Nat Neurosci 2005; middle, Zhang et al., Nature 2007; right, Zhang et al., Nat Rev Neurosci 2007;

Note: channelrhodopsin from green algae. cation channel gated by blue light. causes neuronal depolarization. bottom is voltage traces in hippocampal neuron culture from 10ms blue light pulses. halorhodopsin NpHR from archaean species. Cl- pump activated by yellow light pulses, causes hyperpolarization (reducing AP frequency) in cultured neurons. right is optogenetics in vivo. cannula implanted surgically in brain. Viral vectors carrying opsin transgenes introduced through cannula. Light induced ChR2 depolarization mediated behavior can then be observed. [http://www.youtube.com/watch?v=I64X7vHSHOE](http://www.youtube.com/watch?v=I64X7vHSHOE) --- ## Mapping connectivity by combining optical and electrophysiological methods * Combining methods for read/write control— of the brain!

Principles of Neurobiology, Garland Science Fig. 13-47

Note: TODO: img src refs needed left is photo uncaging, light induced stimulation of caged glutamate right is optogenetics. parse out signal flow in the brain. --- ## "I didn’t have time to think" * Multimodal sensory activation, focuses attention, object tracking and stereoscopic vision/depth perception, salience of object, defensive/self preservation, limbic loop, decision making, movement selection, proximal and distal motor pool recruitment, reflex activation— but all from a previously unexperienced activity! 2016-03-10 09:48:33

Note: --- ## Response latency in macaque visual system * 30 ms before reaching LGN. 60 ms to primary visual cortex. Color information slower (M cell vs P cell pathways).

Fig. 2 Schmolesky, J Neurophysiol 1998

Note: [^Schmolesky-1998]: Schmolesky, M. T., Wang, Y., Hanes, D. P., Thompson, K. G., Leutgeb, S., Schall, J. D., and Leventhal, A. G. (1998). Signal timing across the macaque visual system, J Neurophysiol, 79(6), 3272-8. PMID 9636126 --- ## Structures of a neuron * cell body (soma)– metabolic center of the cell, contains the nucleus. * dendrites– receive incoming signals from other nerve cells * axon– carries signals to other neurons * axon hillock– initiates action potentials * synapse– site at which two neurons communicate * synaptic cleft– area between pre and post-synaptic cell Note: --- ## Ways to measure neural activity * Extracellular recording– an electrode is placed near a neuron. Measures action potentials. Useful for detecting patterns of activity. * Intracellular recording– an electrode is placed inside a neuron-can measure smaller graded potential changes. Useful for isolating responses to single inputs. * Voltage clamping– can make the membrane potential of a cell at a desired point and determine the current flow across the membrane. * Patch Clamping– can measure ion flow across a single channel. * fMRI– infer activity indirectly in a living brain based on brain oxygenation patterns Note: --- ## General rules of spinal cord organization * Neurons and axons that process and relay sensory information (afferents) are in dorsal spinal cord. * Preganglionic visceral motor neurons (innervate glands) are found in the intermediate/lateral region. * Interneurons are in intermediate zone. * Motor neurons and axons are found in the ventral portion of the cord. * Distal muscles are innervated by lateral motor neurons. * Proximal muscles by medial motor neurons. Note: --- ## Compare and contrast sensory systems * What are the peripheral receptors? What is their receptive field? What neurotransmitters are used? * How is the information translated into changes in cell potential? * What are the circuits, how do they get to the cortex? * What types of perception defects are associated with damage to different components of the system? Note: ---