Pax-3 Analysis

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Pax-3 Analysis
Oligonucleotides
inhibition of protein synthesis
Spina Bifida
Department of Pediatric Neurosurgery
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Protein expression of HNK-1
Neural Crest Cell marker
Antisense
Sense
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Protein Expression of Pax-7
Sense
Antisense
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Retinal Ganglion Cell Extraction
Wilmer Eye Institute
Glaucoma
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Retinal Ganglion Cells

• Thy-1
• - Drug testing

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Exploring the Last Frontier Structural
Functional Studies of the Brain
Robert Lewis, M.S. Graduate Program in
Anatomy Mentor Thomas Heinbockel,
Ph.D. Department of Anatomy Howard University
College of Medicine
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• Questions and Issues
• in Cellular and Systems
• Neuroscience
• What is the structural and
• functional organization of
• the olfactory limbic
• system (olfactory bulb,
• amygdala, hippocampus)?
• What are the neural
• mechanisms underlying cellular signaling in
  these systems?
• How is sensory or synaptic information
  processed and
• integrated in the brain?

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A typical vertebrate neuron
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Compartmental Organization of a Neuron
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Structure of a synapse
Martini FH, Fundamentals of Anatomy Physiology,
6th ed, 2004
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The Olfactory Bulb is the First Relay Station in
the Brain for the Processing of Olfactory
Information
(Morphonix, 2003)
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The Olfactory Pathway
(J.C. Leffingwell, 2003)
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Layered Organization and Neural Circuitry of the
Main Olfactory Bulb
(A. Puche, 2003)
Neurotransmitter mitral cells - glutamate,
granule cells - GABA
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• Questions and Issues
• Strong regulation (modulation) by local neural
  circuitry and by
• centrifugal inputs
• Functional significance of different
  neurotransmitter
• (modulator) systems in MOB and from
  centrifugal fibers,
• e.g., glutamate, dopamine, norepinephrine,
  GABA
• Role of neurotransmitters at cellular network
  level in MOB
• Neural mechanisms through which olfactory
  sensory
• information is processed, modulated, and
  shaped in the brain

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The Olfactory Bulb Slice Preparation
Paired whole-cell patch-clamp recording from
two olfactory bulb neurons
Horizontal olfactory bulb slice with electrode
placement
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Sectioning the Brain on the Vibratome
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Transfer of Brain Slices to the Recording Bath
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Evoked Responses in Olfactory Bulb Neurons
Electrical Stimulation of the Olfactory Nerve
Mimics Odor Stimulation and Evokes a Synaptic
Response in Olfactory Bulb Neurons (Mitral
Cells)
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Mitral Cells are the Main Output Neurons and Key
Integrative Elements in the Olfactory Bulb
(Layered Organization)
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Confocal micrograph of a neuron in the CNS
stained with a fluorescent dye
Biocytin-cell tracer
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Nerve Impulses Generated by a Mitral Cell
(a-methyl 4-carboxypheynlglycine)
glutamate antagonist
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The Brain Makes its own Marijuana -
Endocannabinoids

• Cannabinoid receptor CB1
• - G-protein coupled receptor

• Endocannabinoids
• - anandamide
• 2-arachidonoyl glycerol
• (2-AG)

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The Brain Makes its own Marijuana -
Endocannabinoids
Transient Input Regulation by Endocannabinoids

• Endocannabinoids
• - anandamide
• 2-arachidonoyl glycerol (2-AG)

• Cannabinoid receptor CB1
• - G-protein coupled receptor
• on GABAergic neurons
• near the synapse

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Preliminary Results
Evoked responses at the mitral cell to granule
cell synapse
Effect of CB1R antagonist AM251 on holding
current, rhythmic EPSPs, and firing of action
potentials
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• Learning Experience
• brain dissections, brain slice preparation
  (vibratome)
• patch-clamp electrophysiology
• neuropharmacological testing
• immunohistochemistry, staining techniques
• microscopy light microscopy
• differential interference contrast (DIC)
  visualization of
• cells in living slices

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• Outlook and Future Directions
• effects of cannabinoid receptor agonists and
  antagonist on different key neuronal cell types
  in the main olfactory bulb
• regulation of intrinsic neuron properties
  (membrane potential, firing pattern) by
  cannabinoids
• modulation of neuronal responses to sensory and
  synaptic input
• staining patterns of olfactory bulb neurons for
  signaling molecules and receptors
  (immunocytochemistry)

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Acknowledgements
Dr. Thomas Heinbockel Dr. Zejun Wang Dr. John
Young Grant support NIH, MBRS-SCORE Whitehall
Foundation Howard University

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• Outline
• (1) The Olfactory System
• Experimental Approach
• Recording from nerve cells
• Outlook and Future Directions

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• Outlook and Future Directions (2)
• Role of endocannabinoids in the olfactory system
• and in the amygdala
• Functional interrelationship of these two
• modulatory systems ? from postsynaptic mGluRs
  
• to presynaptic cannabinoid receptors

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