dissertation proposal
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The Effects of Ethanol and Ethanol Metabolites on
Locomotor Activation and Ataxia: Focus on the
Substantia Nigra pars Reticulata
Maria Arizzi, M.A.Dissertation Proposal
Biphasic Effects
• Ethanol is typically classified as a sedative hypnotic
• Many studies have shown activating as well as depressant effects after ethanol administration
Biphasic Effect in Mice and Humans• Biphasic effects first shown in mice
Read et al. 1960
• Many studies since have confirmed this finding
• Recent research in humans has also shown biphasic effects of ethanol– Self-report – BAES Earleywine and colleagues
– Biological measurements Davidson et al. 1997, 2002, Grassi et al. 1989, Higgins et al. 1993, Inder et al. 1995, Rush et al. 1993
Biphasic Effects in Rats
• Only sedative effects have been seen in rats via peripheral administration (i.p. or oral) of ethanol Bass et al. 1979, Duncan et al. 2000, George et al. 1990, Gingras et al. 1996, Little et al. 1996, Moore et al. 1993, Petry et al. 1998, Pohorecky 1977
• Recent work using intraventricular administration has shown a biphasic effect of ethanol in rats
Effects of Ethanol, Acetaldehyde, and Acetate on Locomotor Activity in Rats: Open Field
• In the proposed set of experiments the focus will be on the activating effects low doses ethanol
0.0 0.7 1.4 2.8
0
5
10
15
20
25
30
35
40
45
50
55
60
ETHANOL DOSE (moles/l)
MO
TO
R A
CT
IVIT
Y (
sc
ore
s i
n 1
0 m
inu
tes
)*
** **
Ethanol infused into lateral ventricles: locomotor activity First 10 minutes stabilimeter
Ethanol Metabolism
Peripheral MetabolismEthanol
ADH, CYP 450, CAT
Acetaldehyde
ALDH
Acetate
Brain MetabolismEthanol
CAT
Acetaldehyde
ALDH
Acetate
Are the metabolites active?
Effects of Ethanol, Acetaldehyde, and Acetate on Locomotor Activity in Rats: Open Field
0 0.7 1.4 2.80
20
40
60
80
100
120
ACETALDEHYDE DOSE (mol)
****
*
MO
TO
R A
CT
IVIT
Y (
cou
nts
in 1
0 m
inu
tes)
ETHANOL DOSE (mol)
Ethanol and Acetaldehyde infused in to the lateral ventricles: First 10 minutes, stabilimeter
Working Hypotheses
• Acetaldehyde increases behavior– Increases open field and stabilimeter
activity– Increases operant behavior on low rate
schedules
• Acetate decreases behavior– Decreases open field activity– Decreased operant on high rate
schedules
Dose (mol)
0 4.4 8.8 17.6
Mea
n L
ever
Pre
sses
200
400
600
800
1000
1200
1400
1600
1800
2000
EthanolAcetaldehydeAcetate
*
*
FR5 high doses
What are the brain areas that mediate the activating effects of ethanol and
acetaldehyde?
•VTA?•NAcc?•CPU?•SNr?
SNr and Motor Behavior
• Muscle rigidity Crocker 1997
• Lever pressing Correa et al. submitted, Trevitt et al. 2001
• Tremor Finn et al. 1997, Mayorga et al. 1999
• Catalepsy Scheel-Krueger et al. 1977
• Circling Scheel-Krueger et al. 1977
• Locomotion Abraini et al. 1999, Scheel-Krueger et al. 1977, 1981, Trevitt et al. 2002
SNc
STRIATUM SNr
DA
GABA
SNr
STRIATUM
STN
SuperiorColliculus
ReticularFormation
BrainstemMotor
MotorThalamus
SNr, GABA, and Motor Behavior
• GABA agonists and antagonists have their most potent effect on locomotion when infused into the SNr
• Dopaminergic stimulation in the SNr increases locomotion and increases GABA release in the SNr
• Locomotor effect attenuated by infusion of GABAA antagonist
Trevitt et al. 2002
Ethanol and GABA
• Modulation of GABA transmission
• Facilitation of GABAA receptor function
Why SNr?• GABA manipulations in SNr effect
locomotor behavior• Ethanol works through the GABA
receptor• SNr is a part of the basal ganglia
circuitry important for the modulation of locomotion and motor function
• SN has relatively high density of catalase and is therefore a possible place for acetaldehyde action
Focus on the SNr
SNc SNr
List of ExperimentsExperiment Task Drug
Experiment 1 Locomotor Activity Ethanol
Experiment 2 Locomotor Activity Ethanol, dorsal site
Experiment 3 Locomotor Activity Acetaldehyde
Experiment 4 Locomotor Activity Acetate
Experiment 5 Locomotor Activity Ethanol, sodium azide
Experiment 6 Locomotor Activity Acetaldehyde, sodium azide
Experiment 7 Ataxia Ethanol
Experiment 8 Ataxia Acetaldehyde
Experiment 9 Ataxia Acetate
List of ExperimentsExperiment Task Drug
Experiment 1 Locomotor Activity Ethanol
Experiment 2 Locomotor Activity Ethanol, dorsal site
Experiment 3 Locomotor Activity Acetaldehyde
Experiment 4 Locomotor Activity Acetate
Experiment 5 Locomotor Activity Ethanol, sodium azide
Experiment 6 Locomotor Activity Acetaldehyde, sodium azide
Experiment 7 Ataxia Ethanol
Experiment 8 Ataxia Acetaldehyde
Experiment 9 Ataxia Acetate
Preliminary data
Ethanol infused in SNr: Locomotor ActivitySecond 5 minutes (bin 6-10)
Dose (mol)
0 0.175 0.35 0.7 1.4 2.8
Mea
n ac
tivity
cou
nts
(bin
6-1
0)
10
15
20
25
30
35
40
Ethanol
*
* denotes significant difference p<.01 Preliminary data
Preliminary data
List of ExperimentsExperiment Task Drug
Experiment 1 Locomotor Activity Ethanol
Experiment 2 Locomotor Activity Ethanol, dorsal site
Experiment 3 Locomotor Activity Acetaldehyde
Experiment 4 Locomotor Activity Acetate
Experiment 5 Locomotor Activity Ethanol, sodium azide
Experiment 6 Locomotor Activity Acetaldehyde, sodium azide
Experiment 7 Ataxia Ethanol
Experiment 8 Ataxia Acetaldehyde
Experiment 9 Ataxia Acetate
Sedation Scale• 4 – awake, active
– Engaged in locomotion, rearing, or head movements
• 3 – awake, inactive– Eyes fully open, head up, no locomotion or rearing, normal
posture
• 2 – moderate sedation– Eyes partly closed, head somewhat down
• 1 – heavy sedation– Eyes mostly closed, head mostly or completely down,
flattened posture, lack of normal limb placement
• 0 – asleep– Eyes fully closed, body relaxed, loss of righting reflexSalamone et al. 1996
Hypotheses• Ethanol infused into the SNr will have
an activating effect on locomotor behavior at low doses
• Acetaldehyde infused into the SNr will also produce an activating effect, and will be more potent than ethanol
• Acetate infused into the SNr will not increase motor activity and may produce a suppressive effect on activity
Hypotheses (continued)
• The behavioral activating effect of ethanol infused in the SNr can be blocked with peripheral administration of a catalase inhibitor. The catalase inhibitor will have no effect on acetaldehyde-induced behavioral activation
Hypotheses (continued)
• Low doses of ethanol and acetaldehyde infused into the SNr will not produce ataxia
• Of all three compounds studied, acetate is the most likely produce ataxia in the dose range tested
• Locomotor activity and ataxia are dissociable and are being produced through 2 different mechanisms
The results of the proposed experiments will further elucidate the brain mechanisms and biochemical pathways through which ethanol exerts some of its behavioral effects.
Effects on neurotransmission
GABA. Like benzodiazepines, barbiturates and anesthetics, acute ethanol enhances GABA’s influence on GABAA receptor thus facilitating Cl- influx. (10-50 mM). This process seems to be mediated by the phosphorylation induced by PKC (protein kinase C). Chronic ethanol administration produces the opposite effects on GABAA receptors and reduces the synthesis of one of protein subunits of this receptor.
Glutamate. Acute administration inhibits NMDA receptor/cation channel complex (20-50 mM). This inhibition interferes with excitotoxicity in cortex, and with LTP in the hippocampus. Chronic administration increases NMDA receptor numbers.
DA. Acute and Chronic alcohol administration induces
dopamine release in the N. Accumbens.
5-HT. Acute ethanol induces the release of serotonin
and facilitates the cation-5-HT3-gated channel (10-50 mM). Chronic alcohol increases the number of 5-HT2.
Effects on neurotransmission
Endogenous Opioids. Acute alcohol
increases endorphin and enkephalin expression and
release. Chronic alcohol produces the opposite
effects.
Adenosine. Acute alcohol increases extracellular
adenosine by inhibiting adenosine uptake, and
increases synthesis of adenosine. The activation of
the A2 receptor increases intracellular cAMP. Chronic
alcohol produces the opposite effects.
Effects on neurotransmission
Evidence of central ethanol metabolism by Catalase.
Catalase is widespread in brain, and also is localized in several discrete brain nuclei.
In vivo and in vitro administration of catalase inhibitors blocks acetaldehyde formation in different brain nuclei. In contrast, when H2O2 concentration is increased, acetaldehyde fomation also increases.
Acatalasemic mice mutants have less ethanol-induced locomotor activity, more ethanol-induced sleep, and higher ethanol consumption.
The pharmacological inhibition or potentiation of catalase modifies some acute ethanol-induced effects as well as the ingestion of ethanol.
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