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Methods Lecture 3:
Molecular Biological
techniques
Dr Bill Phillips, Dept of Physiology
Rm N348 Anderson Stuart Bldg
Uses of molecular biology
techniques in neuroscience:
• Study connections among neurons in the brain and
spinal cord
• Investigate the role of particular genes in the
function of the nervous system
• Analyse the molecular structure and function of
neurons and glia
• Identify changes in gene expression that occur in
developmental, adaptive or disease states
Tracers for tract tracing- Inject a “tracer” into one brain
nucleus or ganglion, it travels along the nerve fibres to label
full extent of the nerves
• Retrograde tracers: eg HRP (active transport),
rhodamine (diffusion)
• Anterograde tracers eg radioactive amino acids
• Functional pathway tracing- activation of
immediate early genes (c-fos) when a
nucleus is repetitively activated.
• Recombinant viruses- transported retrogradely,
trans-synaptically
Poly-synaptic tract tracing with
Herpes virus
• Some strains of Herpes
virus are transported
anterogradely (fig) others
retrogradely
• Cross synapses to label
2nd and 3rd order neurons
(pontine nucleus &
cerebellum here)
• Visualise on section by
staining for antigen or by
histochemical enzyme
Kandel,Schwartz and Jessel 2000
Imaging individual living
neurons in the brain
• During development and during
remodelling of synapses neurons change
shape, grow and withdraw dendritic spines
• Too many neurons packed together to be
able to study individuals
• Solution? Selectively label just a few using
a recombinant virus and Green Fluorescent
Protein (GFP) from jellyfish
GFP Labelling studies combine
molecular techniques with:
• Confocal microscopy
• High sensitivity low-light fluorescent
imaging so cells are not damaged
• Developmental and/or behavioural studies
Investigate the role of particular genes in
the function of the nervous system
Genetics
Identify Phenotype
Reverse genetics
Clone suspect gene
(say poor motor control)
Study Inheritance
Inactivate gene by
homologous
recombination
Clone gene
responsible and try
to relate its function
to the pheno type
Study Phenotype and
try to relate known
function of protein
to the pheno type
Genetic approaches to understanding the role of
genes and proteins in the nervous system
• identify neurological phenotypes (trait):
behavioural, electrophysiological,
neuroanatomical
• clone the gene based upon the pattern of
inheritance of the trait
• sequence the gene, identify the protein
• infer from the phenotype the function of this
protein
Examples of forward genetic
studies in nervous system
• human startle disease- identified as a mutation to the
glycine receptor a-subunit
• low IQ in boys with Duchenne Muscular Dystrophyhow does lack of dystrophin leads to this phenotype?
• Spontaneous neurological mutants in mice such as
stargazin. Stargazin mice are ataxic and prone to epilepsy.
Stargazin protein expressed in cell lines showed that it is
important for delivering and clustering AMPA receptors in
the postsynaptic membrane
• Chemically induced mutants in Drosophilla and C.
elegans
Reverse Genetic approaches: clone the gene
for the receptor or other synaptic protein then:
• Express the gene in cultured cells- to
study the function of the protein in neuronal
neuronal-like (PC12) or non-neuronal cells
• Inactivate candidate gene
(“gene knockout” in fly, worm or mouse)
• ‘Overexpress’ the gene
(transgenic mice, flies, worms etc)
Studies of neuronal proteins in
cultured cells
• Introduce the cDNA encoding the protein
into Xenopus oocyte (injection) or “cell
line” (transfection)
• Study its ion channel properties, protein
interactions (adaptor proteins) or transporter
function (eg amino acid transporters)
• Infer the likely role of the protein in neurons
Structure/function studies of ion
channels with patch-clamp
•
•
•
•
Clone channel
Make specific mutations to particular amino acids
Express wild-type cf mutant in Xenopus oocytes
Measure changes in gating, conductance etc with
patch clamp recordings of membrane currents
Example: Properties of P2X
receptor sub-types
• New family of channels is cloned ~7
members• what are their channel properties?
• how might their presence on a particular
neuron modify its excitability
• Can we find new drugs that will be selective
• Channel permeability, agonist sensitivity,
gating kinetics, inactivation
Gene knockout in mice
• Isolate (clone) “candidate gene” DNA
• Mutate gene DNA so that it can’t function
• Replace natural gene in chromosomes of
embryonic stem cells by homologous
recombination in cell culture
• Derive mice by injecting modified stem cells into
blastocyst stage of embryo
• Mate progeny so that mutation becomes
homozygous
• Examine abnormalities in structure and function of
nervous system and infer the normal role of the
protein
Gene knockout- recent examples
• Synaptotagmin I - testing the hypothesis
that synaptotagmin is the Ca2+ sensing
trigger
• Clarifying the role of P2X receptor subtypes
• Role of GABAA receptor subunits (a6-, dsubunits) in cerebellar granule cellsinhibition.
‘Overexpress’ the gene in transgenic mice
eg NMDA receptor NR2B
• NMDA receptor subunit
expressed in developing
but not adult mice
• Might NR2B help make
developing brain more
adaptable than adult
brain?
• Overexpressed NR2B
using a ‘strong’ promoter
• LTP response AND
learning ability improved
Tang et al. Nature 401, 63 - 69 (1999)
© Macmillan Publishers Ltd.
Basic molecular methods widely
used in neuroscience:
• Recombining DNA: PCR (amplify, modify),
restriction enzymes (cut) and ligase (paste) DNA
• Cloning DNA: E coli, lambda bacteriophage, yeast
• Studying where and when mRNA is expressed: in
situ hybridization, RT-PCR, Northern blots, Gene
arrays
• Transgenic mice
• Homologous recombination
Gene Arrays- flavour of the
month
• Genome sequencing projects have identified and
sequenced most human genes
• These sequences are like fingerprints and are easy to match
to on Web databases (NCBI USA, ANGIS Australia etc)
• Gene arrays/chips allow one to determine changes in
expression of mRNA for a wide range of genes
simultaneously.
• IF properly controlled, may give clues to how expression
of certain genes makes neurons behave differently in
health and disease. Great care needed with controls.