Alterations of Connexin43 in Alcohol Preferring Rats

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Alterations of Connexin-43 in Alcohol Preferring
Rats
Presentation by Laura Kathryn Jackson
Mentor Dr. Javier Miguel-Hidalgo
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Alterations of Cortical Synaptic Markersin
Alcohol Dependence
To identify the synaptic and glial changes that
occur as a consequence of chronic alcohol abuse.
Primary Goal
Secondary Goal
To assess the effects of neuroprotective
treatments that may alleviate or prevent the
synaptic and glial changes that occur as a result
of chronic alcohol abuse.
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What Defines Chronic Alcohol Abuse?

• Craving A strong need, or compulsion, to drink.
• Loss of control The inability to limit ones
  drinking on any given occasion.
• Physical dependence Withdrawal symptoms,
  such as nausea, sweating, shakiness, and
  anxiety, occur when alcohol use is stopped after
  a period of heavy drinking.
• Tolerance The need to drink greater amounts
  of alcohol in order to get high.

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Alcohol dependence and abuse are associated with

• Atrophic changes in prefrontal cortex.
• Alterations of the normal patterns of neural
  transmission and activity in the cerebral cortex.
• Global and regional (dorsolateral prefrontal
  cortex, anterior cingulate cortex and
  orbitofrontal cortex, hippocampus) changes in
  blood flow and glucose metabolism.

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Dorsolateral prefrontal cortex- area 9
GLIAL PACKING DENSITY
120
110
100
? 11 plt 0.003
Glial density cellsx103/mm3
90
80
70
CONTROL
ALCOHOL
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Orbitofrontal cortex- area 47
GLIAL PACKING DENSITY
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GLIAL NUCLEUS SIZE
6.0
5.5
? 3.2 plt 0.05
Diameter, µm
5.0
4.5
4.0
ALCOHOL
CONTROL
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Glial Cells

• Express a variety of receptors and transporters
  as well as ionic channels
• Receive and integrate neuronal messages
• Provide substances that activate receptors
  located on neurons and modify their
  properties, particularly their excitability and
  synaptic activity
• Occupy a privileged position in the CNS at the
  interface between capillaries and neurons
• Interact with neurons in different pathological
  situations such as neurodegenerative diseases
• Can change their morphology and phenotype
  concomitantly with behavioral changes
• Contain modifications of their membrane
  potential which have long time constants
  compatible with some behavioral modifications
  such as adaptation and learning
• Coupled by gap junction channels and form a
  syncytium allowing the intercellular spread
  of signaling molecules over hundreds of microns

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Pia mater
Astrocytes

• Uptake GLUTAMATE (GLUT) and GABA through
  specific transporters.
• Are also essential for replenishing GLUT in
  terminals by converting glutamate to
  glutamine.
• Regulate production and maintenance of
  synaptic contacts. Express functional
  receptors for most neurotransmitters.
• GLUT increases cytoplasmatic Ca and sends
  Ca waves to adjacent astrocytes through GAP
  junctions. Release NMDA receptor agonist
  homocysteic acid and GLUT after cytoplasmic
  Ca elevations.

Other astrocytes (gap junctions)
Synapses
Bloodvessels
Axons and dendrites
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Area under study Prelimbic cortex (RAT)
Human
Gray Matter
White Matter
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Rat Models
N Rats Normal ratsThese are Wistar rats that
serve as our control group. They are given ONLY
water to drink.
P Rats Alcohol-preferring rats One group of
P rats are given only water. Another group of
P rats are given both water and a 10 alcohol
solution to drink.
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Studies in a rat model of inherited risk for
alcohol-dependence

• Time Two-month continuous access to water and/or
  alcohol
• Arrangement Single-caged
• Groups
• 9 male alcohol-preferring (P) rats on alcohol
  for two months
• 9 male alcohol-naïve P rats, water only
• 6 male Wistar rats, water only
• Tissue Obtained 30 µm slices of prelimbic cortex

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Astrocyte Markers Under Study

• GFAP (Glial fibrillary acidic protein)
  Cytoskeletal protein that functions to give
  support to the cell
• Glutamine Synthetase Synthesis of glutamate to
  glutamine. Glutamine is returned to neurons to
  further synthesize Glutamate.
• Connexin 43 (Gap Junction Protein) Direct
  communication between astrocytes. Spreads Ca
  waves in response to changes of extracellular
  glutamate.

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Methods
Immunohistochemistry for Connexin 43
First Antibody Mouse Monoclonal antibody to
Connexin 43 Second Antibody Biotinylated
anti-mouse Stain Diaminobenzidine and
H2O2 Analysis Image analysis of optical density
in the prelimbic cortex
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GFAP in the rat PRELIMBIC CORTEX
Control (Wistar)
P rat on water
P rat on alcohol 10
Two months
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Connexin-43 Immunostaining
N Rats N11 , N16 Wistar Control
P Rats R11, R13 Alcohol naïve
P Rats R17 , R8 Alcohol exposed
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ANOVA F 26.616 P .0002
Plt0.001vs. Control
Plt0.001vs. Control
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Conclusions
The Connexin-43 immunoreactivity is significantly
lower in the prelimbic cortex of
alcohol-preferring rats even predating the
beginning of alcohol intake. There was no
significant difference between naïve P rats and
ethanol P rats. These results suggest that
communication between astrocytes at the level of
gap junctions might be reduced in the prelimbic
cortex. This reduction may lead to a decreased
responsivity of astrocytes to neuronal activity.
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the End.
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ASTROCYTE
Glutamine synthetase