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Programmed feeding as a model of chronic alcoholism in the rat.

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Programmed Feeding as a Model
of Chronic Alcoholism in the Rat
J. R. Craig, MD, PhD, T. L. Munsat, MD, and M. Chuang, BS, MS
Programmed-feeding polydipsia results in a reliable model of chronic alcoholism in the rat. High oral ethanol
consumption and a predictable withdrawal reaction associated with audiogenic seizures are produced. The maintenance of high blood ethanol levels for three weeks in 18 male Charles River rats was associated with audiogenic
seizures after 6 to 8 hours of withdrawal. These chronic alcoholic rats had enhanced blood clearance of ethanol. T h e
cerebral cortical crude mitochondrial fraction showed a decrease in total and magnesium-dependent adenosine
triphosphatase activity in alcoholic and control (water-fed) rats compared with normal rats.
Craig JR, Munsat TL, Chuang M: Programmed feeding as a model of chronic alcoholism in the rat.
Ann Neurol 2:311-314, 1977
Investigation of the chronic effects of alcohol has been
hampered by lack of an adequate experimental small
animal model. T h e major behavioral and physiological
criteria for an experimental model of human chronic
alcoholism should include: (1) oral ingestion of high
doses of ethanol, resulting in prolonged elevation of
blood ethanol levels; (2) a separate supply of ethanol
and food, so that factors related to ethanol consumption are not directly tied to nutritional needs; (3)
voluntary ethanol consumption not associated with
extrinsic reinforcing factors; and ( 4 )demonstration of
physical dependence. The rat model utilizing programmed feeding described by Falk e t a1 [2, 31 satisfies
these criteria. In this study we present an improved
model, report the blood ethanol clearance and brain
adenosine triphosphatase (ATPase) activity, and describe withdrawal seizures [ ll.
Methods
Thirty male Charles River rats weighing 175 to 250 gm were
individually caged with food pellet dispensers. A 250 or 500
ml plastic graduated cylinder with a Richter feeding tube
was used for monitoring fluid intake, and less than 2 ml per
day was lost. The pellet dispenser (Model PD-104 or
PI)-109, Davis Scientific Company, Los Angeles, CA) released individual 4 5 mg pellets at 2-minute intervals. The
dispenser operated continuously for 1 of every 4 hours
around the clock. The food pellets (P. J. Noyes Company,
Lancastrr, NH) were composed of meat, vegetable meals,
and vitamin supplements, resulting in a total daily food
intake of 8.1 gm (36.5 Kcal).
Before they were placed in the experimental cages, the
rats were fasted to 80% of their free-feeding weight. ExFrom t h e Neuromuscular Unit, Department of Neurology, University of Southern California School of Medicine, Los Angeles,
CA .
Accepted for publication Apr 20, 1977.
perimental rats received 5% vlv ethanol in water containing 0.35% saccharin (Sigma Chemical Company, St Louis,
MO). Control racs received saccharin solution alone. The
room was kept at constant temperature (27°C) and illumination.
For ethanol clearance studies, blood ethanol was measured in tail vein samples by an ultraviolet enzymatic method
(Sigma Chemical Company, St Louis, MO). Clearance of
ethanol was determined by measuring the time for all of it to
be cleared after intraperitoneal injection at 1.5 gm per kilogram of body weight. Tail vein blood samples were obtained
at 3 , 4 , and 5 hours. Results were calculated by least-square
analysis. To determine blood ethanol levels during the feeding cycle, blood was collected within 1 hour after feeding.
Physical dependence on ethanol was tested during
ethanol withdrawal by sensitivity to audiogenic seizures. A11
rats were placed on water (with saccharin) at 8:00 AM, and
after 4 hours a ring of keys was shaken adjacent to the cage
for 5 to 30 seconds. The test was repeated at 30-minute
intervals for 4 hours or until a seizure occurred.
After the withdrawal seizure test indicated physical dependence, an additional week of programmed feeding was
instituted before sacrifice. Cerebral ATPase was measured
in rats killed by cervicomedullary dislocation. Preliminary
experiments confirmed that both ether and pentobarbital
anesthesia resulted in lower activity [9]. A cerebral
homogenate was prepared by adding 10 vol of 0.32 M
sucrose and 1 mM Tris, p H 7.0, to the cerebral hemispheres.
After centrifugation at 100 g at 0°C for 10 minutes, the
pellet was discarded. The supernatant was centrifuged at
12,000 g at 0°C for 20 minutes to obtain a pellet of crude
mitochondria and synaptosomes. This pellet was resuspended and centrifuged at 10,000g for 20 minutes to obtain
the crude mitochondrial fraction. Fractional centrifugation
was not performed. The supernatant was used as the crude
Address reprint requests to Dr Munsat, Department of Neurology,
Tufts-New England Medical Center, 171 Harrison Ave, Boston,
MA 02111.
311
microsomal fraction. ATPase assay conditions were: 3 mM
adenosine triphosphate, 3 mM magnesium, 1 mM ouabain,
100 mM sodium, 20 mM potassium, and 100 mM Tris
buffer at pH 7.4 [3]. Protein assay was done by the method
of Lowry et a1 [lo]. The total reaction volume was 1.0 ml,
and the assay was linear in time for the 15-minute test
period.
Results
Ethanol-exposed rats gained an average of 36 gm
during the study period, whereas control rats lost an
average of 58 gm despite food supplements. The
mean daily fluid intake was higher in control than
experimental rats (ethanol group, 35 1 ml/kg/day; controls, 453 rnllkglday). O n e experimental rat died of
uncertain cause during the second week. The control (water-fed) rats maintained body grooming
and were alert to the feeding mechanism. Following a
1-hour feeding period the alcoholic rats were lethargic, but by the time of the next feeding they were alert.
If food supplements were not provided to control
rats to maintain body weight, they had poorer grooming behavior. Mean blood ethanol within 1 hour of
feeding was 142.8 ? mg/dl (Fig 1).
Ethanol Consumption
In preliminary studies we had observed that the mean
daily ethanol consumption rose with increasing
ethanol concentrations up to 5 %; concentrations
above 596 resulted in a reduction of fluid intake. W e
therefore began with 5% v/v ethanol and were able to
achieve a rapid high ethanol intake. The effect of
adding saccharin to the ethanol solution was also
evaluated. Ethanol consumption (18 rats) over a
three-week period was 7.37 gm/kg/day; with saccharin
added the consumption increased to 11.85 gm/kg/day
(p <0.0005) in a separate three-week period. The
mean daily ethanol intake for the four-week study
F i g 1 . Tail vein ethanol levels obtained within 30 minutes of
feeding on multiple dqJ.
period was 12.9 gm/kg/day. Because ethanol (7 Kcal
per gram) provided approximately 19.6 Kcal per day
per rat, the chronic alcoholic rat was ingesting approximately 375% of his calories as ethanol.
Ethanol Metabolic Rare
After four weeks of ethanol consumption, mean
blood ethanol clearance in 16 alcoholic rats was 32 1 ?
76 mg/kg/hr. I n 8 control (water-fed) rats the clearance was 226 ? 45 mg/kg/hr @ <0.005).
Withdrawal Seizures
All 18 chronic alcoholic rats had audiogenic seizures
within 8 hours of alcohol withdrawal (Fig 2). None of
the 11 control rats had a seizure during the same
period. The seizure pattern was variable. Usually, a
brief period (seconds) of premonitory hyperactivity
and increased alertness was followed by tonic “jumping” and then clonic movements of all four extremities. Occasional animals demonstrated peculiar
mouthing and nose-rubbing behavior. At times,
asymmetrical focal seizure activity was seen. During
the clonic phase the animals were opisthotonic and
cyanotic. Seizures lasted 15 to 90 seconds, and no rat
died.
Following the seizure test, the rats were placed on
ethanol again and observed for one more week before
sacrifice. The mean daily ethanol intake in the week
before and after a seizure test was unchanged. A second seizure test was performed on 7 rats one week
following the first test. All these animals had a seizure
between 6% and 7 hours after ethanol withdrawal.
Bruin ATPase
The total and MG-dependent ATPase activity of t h e
crude mitochondria1 fraction was slightly but significantly increased in chronic alcoholic rats compared
with controls @ <0.025) (Fig 3). However, both valF i g 2. Time of seizzlre folloim’ng ethanol zi~ithdraioal.
No. of 1
Rats
No. Of 10-
n
Rats
8-
60
80
100
140 160
180
Blood Ethanol Conc.
120
312 Annals of Neurology Vol 2 No
5Vz
200
220
240
6
6112
7
Time of Seizure
4
October 1977
73/2
0
Normal Rats 1 1 1 )
Control Rals ( 5 )
0.300j-
EIOH (17)
z
W
I-
2a
0.200
Tolal -ATPase
Mg-ATPase
MITOCHONDRIAL
Na. K-ATPase
F i g 3. ATPase acti.uitq’ i n c-rnde mitochondrial and
sq’naptosomalfraction of rut cerebra/ hemisphere ufter three
weeks of programwiedfeeding. (EtOH = ethanol
hydroxide; Pi = inorganic phosphorus.)
0.200-
zw
&
0.150-
7
0Normal
Rats 1 1 1 )
0 Control
Rats 15)
EtOH 117)
a
n
.G 0.100 E
+-
6m
0.050-
E
3
0Total-ATPase
Mg-ATPase
Na. K-ATPase
MICROSOMAL
Fig 4. ATPase activity in microsovialfrrtction of rat cerebral
hemisphere after three wrrkJ of programmed feeding.
(Abbreziatians .ram8 as i n F i g 3.)
ues were significantly less than in normal rats subjected to neither control nor experimental conditions
Cp <0.005). Crude microsomal ATPase activity was
the same in chronic alcoholic and control rats (Fig 4 ) ,
but again, both values were less than in normal animals.
Discussion
Polydipsia induced by programmed feeding is a useful
model for the production of chronic alcoholism in rats
[ I , 31. Previous models of chronic alcoholism in small
animals have not been fully successful because of the
aversion of rodents to ethanol and a naturally rapid
metabolic rate [ 151. The programmed-feeding model
does encourage high daily ethanol intake but has not
been uniformly accepted. Some investigators have not
observed seizures even after a prolonged exposure of
three months [7, 111. The unsuccessful trials have
been attributed to inadequate ethanol consumption.
In the initial report, 8 male Holtzman rats were fasted
to 80% of their free-feeding body weight, given 1%
increments of ethanol in the drinking fluid, and maintained on 5% v/v ethanol for three months [ 3 ] . The
mean daily ethanol intake during the last ten days of
exposure was 13.1 gm per kilogram, whereas we
observed a mean daily ethanol intake of 12.9 gm per
kilogram.
The nutritional status of rats in studies of experimental alcoholism has correctly been the subject of
much controversy. In our study, the chronic alcoholic
rats increased significantly in weight over controls,
which required food supplements to maintain weight.
Falk et a1 [21 noted that the high level of ethanol intake
provided 44.8% of total calories, and our findings
were similar. Freed and Lester [41have theorized that
the programmed-feeding model results in chronic
hunger and that ethanol is consumed entirely for its
caloric value.
Increased blood ethanol clearance after chronic alcohol exposure has been reported by several investigators [13, 141. We found a 41% rise in clearance
after three weeks. A higher increase (71%) was noted
after one week of ethanol exposure by Tobon and
Mezey [13], who studied male Mistar rats given a
liquid diet containing ethanol as 36% of the total
caloric intake.
Several experimental methods have been used in
determining physical dependence. Withdrawal seizures have been observed in several models, and the
programmed-feeding model has produced erratic results. Although Falk et al [2] observed seizures in rats,
others have failed to produce withdrawal seizures in
mice [121, primates [I I], and rats [6].This failure may
be due to strain differences, the method of seizure
testing, or both. Subsequently we have found a 12-volt
house bell to be effective, and it gives more reproducible sound intensity.
The biochemical basis of withdrawal seizures has
been studied by several investigators, and alteration in
ion flux has been suggested as a mechanism. Ion flux is
regulated in part by membrane-bound ATPase, which
may be inhibited by ethanol [91. Although acute exposure to ethanol produces competitive inhibition of
Na,K-dependent ATPase activity in vitro, chronic exposure causes increased ATPase activity in brain
homogenates and partially purified preparations 191.
In another study, alcoholic male Wistar rats were
given ethanol in their drinking fluid, supplemented
with ethanol through a gastric tube (4.5 gm/kg/day)
for two weeks. Total and Na,K-dependent ATPase
of whole-brain homogenates was increased, but the
activity of Mg-dependent ATPase was reduced [81.
Our results with cerebral ATPase in the rat concur
in part with those of Israel and Kuriyama in the mouse
151. A slight increase in mitochondria1 %-dependent
ATPase was observed in alcoholic rats Compared with
Craig, Munsat, and Chuang: Rat Chronic Alcoholism Model 313
control (water-fed) rats. The total enzyme activity was significantly decreased compared with normal
rats. Our studies were restricted to a crude mitochondrial fraction containing both mitochondria and
synaptosomes. The varying published results suggest
that the activity of ATPase after ethanol exposure is
influenced by the method of brain preparation, the
assay technique, the strain of animal or type of rodent,
the timing of assay after ethanol withdrawal, the type
of control preparation (programmed feeding vs normal feeding), and the method, amount, and frequency
of ethanol administration.
Funded by US Public Health Service Grant AA00338-03 and the
Muscular Dystrophy Association of America.
Presented in part at the 28th Annual Meeting of the American
Academy of Neurology, Toronto, Ont, Canada, April, 1976.
The technical assistance of Mr John Irvine, BS, is gratefully acknowledged. We thank Rita Chick for secretarial services.
References
I . Craig JR, Mnnsat TL, Chuang M, et al: Ethanol withdrawal
seizures in the rat. Neurology (Minneap) 26:344-345, 1976
2. Falk JL, Samson HH, Tang M: Chronic ingestion techniques
for the production of physical dependence on ethanol. Adv
Exp Med Biol 35:197-211, 1973
3. Falk JL, Samson HH, Wingcr G: Behavioral maintenance of
high concentrations of blood ethanol and physical dependence
in the rat. Science 177:811-813, 1972
4 . Freed EX, Lester D: Schedule-induced consumption of
314 Annals of Neurology Vol 2 No 4 October 1977
ethanol: calories or chemotherapy? Physiol Behav J:555-560,
1970
5. Goldstein DB, Israel Y: Effects of ethanol on mouse brain (Na
+ K)-activated adenosine triphosphatase. Life Sci 11:957963, 1972
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191:482-483, 1976
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1968
8. Israel Y ,Kalant H, LeBlanc E, etal: Changes in cation transport
and Na + K-activated adenosine rriphosphatase produced by
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174~330-336, 1970
9. Israel Y ,Salazar 1: Inhibition of brain microsomal adenosine
triphosphatases by general depressants. Arch Biochem
Biophys 122:310-317, 1967
10. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the phenol reagent. J Biol Chem 193:265-275,
1951
11. Melko NK, Mendelson JH: Evaluation of a polydipsia technique to induce alcohol consumption in monkeys. Adv Exp
Med Biol 35:225-244, 1973
12. Ogata H, Ogata F, Mendelson JH, et al: A comparison of
techniques to induce aicohol dependence and tolerance in the
mouse. J Pharmacol Exp Ther 180:216-230, 1972
13. Tobon F, Mezey E: Effect of ethanol administration on hepatic
ethanol and drug-metabolizing enzymes and on rates of ethanol
degradation. J Lab Clin Med 77:llO-121, 1971
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intoxication and withdrawal. Adv Exp Biol Med 59:279-294,
1975
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