Introduction to Neurobiology II
Todays Learning Objectives…
1. Review basic principles of gene expression and atlases for studying the cellular
and molecular organization of the nervous system.
2. Define important structural and functional features of neurons/synapses that
contribute to diversity in neuronal subtypes/circuits.
3. Be able to explain the basic contributions of different glial subtypes to nervous
system function.
Organizational Principles in the Nervous System
100-105 per mRNA
1011 neurons
1011 – 1012 non-neuronal
~20,000 genes
5-100 mRNA’s per gene
>1014 synapses
SPAUN - 2.5-million neuron
model of visual function
>106 miles of myelin
Schwanhausser et al 2011, Azevedo et al., 2009, Eliasmith et al, 2012
Genetic Organization of the Nervous System
Genetic Organization of the Nervous System
• Differential Gene Expression is crucial for generating cellular
diversity and maintaining cell-specific functions.
• For example:
– What genes regulate the generation of excitatory vs inhibitory
neurotransmitters?
– How does aberrant gene expression (autism, drug use, etc) disrupt
nervous system function?
The Central Dogma of Biology
Gene expression is
controlled in the cell at
multiple levels including:
• Transcription
• mRNA processing
• Translation
Review of Eukaryotic Gene Structure
• Here is the gene model for Ptpn11 adapted from NCBI
(https://www.ncbi.nlm.nih.gov/gene/19247)...
Studying gene expression via RNA: In situ hybridization (ISH)
1. Must fix and permeabilize cells
3. Add antibody that
recognizes digoxigenin
conjugated to enzyme
2. Apply antisense mRNA
complementary to specific mRNA
(aka riboprobe)
4. Enzyme turns substrate blue/purple
(often amplifies signal)
http://11e.devbio.com/wt031007.html
What about Proteins?
Western Blotting
•
Quantitative technique to analyse protein levels in lysates of tissues.
Immunohistochemistry (IHC)
•
•
Uses antibodies to detect a protein of interest in a preserved tissue sample.
A wide range of detection/amplification chemistries are available
Images from Newbern Lab
Gene Expression Atlases based on ISH
•
Allen Brain Atlas (http://mouse.brain-map.org/) contains mouse brain/spinal
cord cross-sections stained for ~20,000 genes using DIG-labeled
riboprobes (generates blue/purple product).
Well-Stained
Cell Bodies
Background
Non-specific
Labeling
See technical white paper here for detailed information…
http://help.brain-map.org/display/mousebrain/Documentation?preview=/2818169/3276841/ABADataProductionProcesses.pdf
In-class Exercise
1. Go to the Allen Brain Inst. Mouse Brain Atlas http://mouse.brain-map.org/
2. Run a search for the gene NeuroD6
3. Open a high resolution viewer of the coronal expression atlas
4. Find a section that has the cortex, hippocampus, hypothalamus, and
striatum present.
• Helpful Hint: Click here to open a handy reference atlas next to the
image…
What two anatomical structures exhibit the
highest expression of NeuroD6?
http://www.nlm.nih.gov/exhibition/hooke/images/
Forefathers of Cell Theory:
Theodor Schwann, Matthias Schleiden, Rudolph Virchow, Robert Hooke, others…
The cell is a fundamental unit of structure, function, and
organization in all living organisms.
Cellular Organization of the Nervous System
Neurons
1. Highly polarized and
compartmentalized
•
•
•
Cell Body (aka Soma or
Perikaryon)
Axon
Dendrite
2. Transmit electrical
signals
•
•
Propagates action potentials
Specialized machinery for
synaptic communication
3. Post-mitotic
~100 billion neurons in human
nervous system
The term “neuron” was coined by Heinrich Wilhelm Gottfried von Waldeyer-Hartz in the 1890‘s.
Reticular vs. Neuron Theory
Modern neuroscience has a long history in the study of neuronal morphology
• Hot Question circa 1850-early
1900s: Is the nervous system
continuous (reticular theory)
or does it possess individual
units (neuron theory)?
Fundamental Neuroscience 3rd Ed. Fig 2.2
Camillo Golgi
• In 1873, Golgi stained brain tissue (in a converted kitchen) with silver nitrate and
potassium dichromate to yield silver dichromate (silver impregnation).
• Surprisingly, he found that only a few random cells were stained black, providing an
unprecedented technique to study neuronal morphology with impressive detail (still
utilized to this day).
• Concluded nervous system is continuous (reticular theory).
Santiago Ramon y Cajal
•
Customized the Golgi stain and
meticulously traced and studied a
variety of neurons.
•
Concluded neurons were individual
separable cells (neuron theory).
•
Cajal & Golgi were awarded the Nobel
prize in 1906 for defining the cellular
anatomy of the nervous system.
Fundamental Neuroscience 3rd Ed. Fig 2.18
Diversity in Neuronal Morphology
Modern Techniques for Neuroanatomical Tracing
• Antibody based IHC
• Injectable tracer molecules – HRP,
DiI, Fluorogold
CLARITY prepped Mouse Hippocampus
• Viral vectors
• Fluorescent proteins – GFP
• Transgenic animal models
Chung Deisseroth et al 2013 Nature
Neuronal Diversity
Neuronal diversity can be classified by various features
1. Anatomical Location
•
Cortical, Spinal, Cerebellar…
2. Shape or Morphology
•
Bipolar, stellate, pseudo-unipolar,
multipolar, etc.
3. Neurochemical Properties
•
•
Excitatory vs Inhibitory vs Modulatory
Glutamatergic, GABAergic,
Dopaminergic, etc.
4. Electrophysiological parameters
•
Fast spiking, adapting, tonic, etc.
5. Connectivity
•
Long range vs. Local
6. Gene Expression
•
TXN factor, Ca binding proteins, etc.
and many other criteria…
Neuroscience (2nd Ed) 2001 Fig 1.2
In-class Exercise
In groups of 2-3, pick one of the following neuronal subtypes and describe
its anatomical location, axonal and dendritic morphology, primary
neurotransmitter, and axonal targets.
Spinal motor neuron
Cortical Inhibitory interneuron
Purkinje neuron
Retinal ganglion cell
DRG sensory neuron
Medium spiny neuron
Pre-ganglionic sympathetic neuron
Layer 5 projection neuron
Mitral cell
CA3 pyramidal neuron
Please go to the whiteboard and draw the model neuron of your
choosing when you are finished.
Synaptic Organization of the Nervous System
Electrical Synapses
•
Gap junctions allow exchange of small molecules such as K+, Ca++, and ATP
Chemical Synapses
• In 1897 the physiologist Sir Charles Sherrington (Nobel Prize in 1921)
named neuronal contacts “synapses” while studying spinal reflexes.
(Contacts in the CNS=synapses, PNS=junctions)
Identify these structures:
Axon terminal
Dendrite
Post-synaptic density
Synaptic Cleft
Synaptic vesicles
Synaptic Diversity in the Simple Spinal Reflex
Synaptic Diversity in the Cerebral Cortex
•
A few neurons in this mouse cortex are transgenically labeled with green
fluorescent protein (GFP)
•
Components of synapses are labeled using immunohistochemistry (IHC) with
antibodies directed toward excitatory synapses (PSD-95, red) and inhibitory
synapses (VGAT, blue)
•
A single cortical excitatory neuron can have >10,000 synapses!!
Electrophysiological Diversity in Cerebral Cortex
Lamination in the Cerebral Cortex
•
Most regions of the human cerebral cortex contain layers.
•
Layer 1 is at the pial surface while Layer 6 is close to the ventricular surface
Multiple Morphologies Provide Diverse Circuit Architectures
Cortical Layers
• Provide anatomical substrate for multiple inputs and outputs
Cortical Columns
• Cells arranged vertically across layers = column
Neural Circuit Diversity
The 100 billion neurons + 100 trillion synapses present in the human brain
generate an extremely complex multi-level system.
• Each neuron and synapse is a complex molecular machine (Block 1-2).
• Local microcircuitry in a single brain region interact with other circuits in more
distant regions, often via long-range axonal projections (Block 2-3).
• The interactions between these levels give rise to behavior, emotion, learning,
and cognition (Block 3-4).
Nervous System Glia
Glia in the nervous system
Glial cells (aka neuroglia)
•
The term neuroglia ‘nerve glue’ was coined by Rudolph Virchow
(1859), who conceived of glia as inactive ‘connective tissue’ holding
neurons together in the CNS.
•
We now know these non-neuronal cells are present throughout the
nervous system and perform many vital functions.
1.
2.
3.
4.
5.
6.
7.
Structural support
Insulation – limit spread of neural activity
Myelinate
Modulate neurotransmitter levels
Regulate metabolic components and ion levels
Immune functions – destroy and clear debris, form scars
Some populations act as stem cells
Myelination
electron micrograph of a transverse section through
part of a myelinated axon from the rat sciatic nerve
Myelination = white matter
Myelin
• a lipid enriched coating surrounding axons
• important for fast transmission of nerve
impulses, “saltatory conduction”.
•
An oligodendrocyte myelinates many
axons in the CNS.
•
A myelinating Schwann cell
myelinates a single axon in the PNS.
Peripheral Nervous System Glia
Myelinating
Schwann Cells
• ‘Myelinate’ a single axon
in peripheral nerves
Non-Myelinating
Schwann cells
• ‘Ensheath’ multiple axons (Remak bundle)
Satellite cells
• Surround neurons in peripheral ganglia
Enteric glia
• enteric ganglia
Nave and Schwab (2005) Nature Neuroscience
Central Nervous System Glia
Myelinating
Oligodendrocytes
Non-Myelinating
Astrocytes
Microglia
Radial Glia
Ependymal Cells
Endothelial Cells
Allen N. and Barres B. (2009) Nature
Central Nervous System Glia
Astrocytes
•
Provide a supportive environment
for neurons.
•
‘Endfeet’ often on blood vessels,
pia, neurons, or ependyma.
•
After CNS injury, astrocytes become
phagocytically active, proliferate,
and form a glial scar. (reactive
astrocytosis or gliosis)
Image of the cortex from a mouse that has been
transgenically modified to express GFP under the
control of the astrocyte specific, Aldh1l1 gene
promoter
GENSAT
Astrocytes
Astrocytes regulate…
•
•
•
•
Ion Homeostasis
Metabolic Support
Blood flow
Synaptic Transmission
Fundamental Neuroscience (3rd Edition) 2008. Fig 3.11
Allen N. and Barres B. (2009) Nature
Microglia
•
Mediate immune responses in nervous system
•
Actively probe and “sense” the environment
(see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2652634/bin/NIHMS71683supplement-Movie_S1.mov)
•
Phagocytose and clear debris following injury
resting
activated
(phagocytic)
G W Kreutzberg, TINS 19:312-319
Ventricular System/Cerebrospinal Fluid (CSF)
•
•
•
•
Ventricles contain cerebrospinal fluid
(CSF), the specialized fluid that encases the
brain and spinal cord.
CSF produced by choroid plexus, a
specialized tissue in the ventricles.
CSF circulates from ventricles to cerebral
aqueduct, central canal and into the
subarachnoid space.
Absorbed into dural sinuses.
Ependymal Cell line the Ventricular System
•
Ependymal cells line the ventricular
surfaces of the brain.
•
The microvilli and cilia on the apical
surface ‘beat’ and assist in
cerebrospinal fluid (CSF) flow.
•
They lack tight junctions and provide a
permeable barrier for CSF to
equilibrate with the underlying brain
tissue.
Tissir et al, Nature Neuroscience 13, 700–707 (20
Blood Brain Barrier
•
The brain must carefully regulate its ionic milieu, and avoid toxic molecules (or certain drugs)
in the vascular space derived from ingestion, infection, or other means.
•
~15% of cardiac output (near 1L/min) travels through ~400 miles of vasculature in the human
forebrain.
Zlokovic and Apuzzo (1998) Neurosurgery
Blood vessels in human brain. A plastic emulsion was injected
into the brain vessels, and brain parenchymal tissue was
dissolved.
Endothelial Cells
Endothelial Cells make up the
Blood Brain Barrier (BBB)
•
Physically separate blood from
neurons, tight junctions fill gap
between endothelial cells.
•
Regulates transport of cellular,
subcellular, and ionic components
into brain.
•
Implicated in a range of pathological
states (AD, ischemia, ALS,
Parkinson, MS, etc).
Non-neuronal cells are involved in many disease states
•
•
•
•
•
•
Multiple Sclerosis
Guillian-Barre Syndrome
Charcot Marie Tooth disease
Leukodystrophy
Traumatic Brain injury
Stroke/Ischemia
Hemmer et al, Nature Reviews Neuroscience 3, 291-30
2002)
http://www.youtube.com/watch?v=K8R5N7ZMlNk
Gene Expression Atlases
GEN-SAT Database
•
•
Atlas of gene expression in mouse
brain using transgenic mice where a
promoter for a gene driving the
expression of EGFP (enhanced
green fluorescent protein) is inserted
into the genome.
Any cell expressing the gene will be
fluorescently labeled in its entirety
http://www.gensat.org/index.html
Go to the GENSAT website and identify what kind of cells are primarily
labeled in the following links?
1. http://www.gensat.org/imagenavigator.jsp?imageID=2351
2. http://www.gensat.org/imagenavigator.jsp?imageID=38255
3. http://www.gensat.org/imagenavigator.jsp?imageID=66370
4. http://www.gensat.org/imagenavigator.jsp?imageID=60174
5. http://www.gensat.org/imagenavigator.jsp?imageID=26746
6. http://www.gensat.org/imagenavigator.jsp?imageID=23247
7. http://www.gensat.org/imagenavigator.jsp?imageID=34699