Levels of a1 Subunit of GABAA Receptor in

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Levels of a1 Subunit of GABAA Receptor in
µ-opioid Receptor Knockout Mice

• Vignesh Shetter
• Mentor Dr. Tangeng Ma

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Labs Project

• The labs project is to determine the role of the
  m-opioid receptor in methamphetamine addiction
• It is known that opioid receptor antagonists can
  prevent amphetamine-induced increase in dopamine
  and behavioral hyperactivity.

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Effects of Methamphetamine

• Loss of dopamine neurons similar to patterns seen
  in Parkinsons disease
• Competitively inhibits re-uptake of dopamine
• Increases release of dopamine

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The mesolimbic dopamine pathway originates from
the midbrain ventral tegmental area and
terminates in the nucleus accumbens and the
medial prefrontal cortex.
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VTA
Dopamine neuron
GABA (decrease)
GABA receptor
NAc
GABA neuron
m-opioid receptor
Stimulation
Dopamine (increase)
Dopamine receptor
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VTA
Dopamine neuron
GABA
GABA neuron
GABA receptor
NAc
m-opioid receptor
Stimulation
Dopamine
Dopamine receptor
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Scientific Question

• In comparison to wild-type mice, do m-opioid
  knockout mice have significantly different levels
  of GABAA ?1 subunit?
• Specifically in the Hippocampus
• Used as a predictor of the level of GABAA
  receptors

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m-opioid receptor knockout
Wild-type
Loh et al. (1998) Mol. Brain Res. 54321-326.
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m-Opioid Receptor (MOR)
Structure G-protein-coupled receptor
398 amino acids/seven hydrophobic
domains Functions Autonomic
nervous system, pain perception, behavioral
reinforcement,
Extracellular
Cell membrane
Intracellular
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?-Aminobutyric acid (GABA)

• The major inhibitory neurotransmitter in the
  central nervous system.
• Regulate neuronal excitability
• 20-50 of all neuronal synapses use GABA as a
  transmitter.
• Most of the physiological actions of GABA are
  produced via GABAA receptors.

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GABAA receptor

• GABAA receptor is a member of a superfamily of
  ligand-gated ion channels.
• Cl- ion
• GABAA receptors are composed of five subunits.

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?
?
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GABAA receptor
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Multiplicity of GABAA receptor subunits
 
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GABAA ?1 Subunit

• ?1 is the major subunit in the brain
• The most common GABAA receptor in the CNS is
  comprised of two ?1, two ?2, and one ?2
• 43 presence of above combination
• Use of ?1 subunit provides more substantial
  evidence of change in level of GABAA due to its
  high abundance

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Western Blot

• Western blot is a method used to detect a certain
  protein in a sample by using an antibody specific
  to that protein
• Gel electrophoresis is the main technique used in
  the Western Blot
• Proteins are separated according to weight (kD)
• The proteins are transferred to a nitrocellulose
  membrane and then exposed to primary and
  secondary antibodies
• Secondary antibodies are exposed to
  chemi-luminescent reagents that can be detected
  by film
• In this experiment the ?1 subunit is the targeted
  protein
• Previously identified as the largest most visible
  subunit on the film

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Western Blot (continued)

• The levels of the ?1 subunit appear as bands on a
  film
• The thickness of the band and/or the clarity of
  the band can be used as an indicator for relative
  specific protein level
• The band cannot be used alone to determine the
  protein level therefore, an internal control
  (GAPDH) was used as a reference
• The internal control/housekeeping protein can
  either validate or disprove if the thickness of a
  band is significant
• Image quant

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Results - ?1 Curve
20 ug
40 ug
80 ug
10 ug
5 ug
2.5 ug
1.25 ug
132 kDa
90 kDa
55 kDa
43 kDa
34 kDa
23 kDa
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Results - Graph
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Results

• No significant difference between ?1 subunit
  protein levels in wild type mice and m-opioid
  knockout mice

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Conclusions

• ?

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Acknowledgements

• Dr. Tangeng Ma
• Dr. Lu-Tai Tien
• CPN Neuroscience Scholars Summer Program
• Dr. Ian Paul
• NSSP scholars