Physiology and Behavior. Vol. 6, pp. 191-198. Per gamon Press, 1971.

Pr i med i n Gr eat Bri t ai n

Physical Dependence on and Tolerance to
Alcohol in the Rat'
T. J. CI CERO, S. R. S NI DE R, V. J. P E R E Z AND L. W. S WANS ON
Laboratory of Neuropsychology, Department of Psychiatry, Washington University School of Medicine,
St. Louis, Missouri 63110, U.S.A.
( Re c e i ve d 7 Au g u s t 1970)
CICERO, T. J., S. R. SNIDER) V. J. PEKEZ AND L. W. SWANSON. Physical dependence on and tolerance to alcohol in the rat.
PHYSIOL. BEHAV. 6(2) 191--198, 1971.--Twenty-four rats of the Holtzman strain were maintained on ad lib food and either
a forced-intake regimen of 7 per cent alcohol (v/v) or tap water only from weaning (21 days) until 154 days of age. Upon
withdrawal of alcohol most of the alcohol rats were extremely hyperactive and appeared to be engaged in frantic, highly
disorganized, exploratory behavior in an open field. The behavior of the water rats was in marked contrast to these data.
Alcohol rats were also much less responsive to an injected dose of alcohol then water animals, even though there was no
difference in the rate of disappearance of alcohol from the blood. These dat a suggest the development of a cellular tolerance
to alcohol after a chronic exposure period. The intake of alcohol, which appeared to exceed the rats' ability to metabolize it,
increased progressively throughout the initial exposure and remained unchanged when water or even a thirdchoice (saccharin)
was simultaneously offered. These dat a would thus seem to suggest that a model for alcohol addiction is possible in the
rat which satisfies, for the first time, the classical pharmacologic criteria of addition, i.e. physical dependence,
tolerance, etc.
Alcohol addiction Physical dependence Tolerance Alcohol drinking Animal model Drug addiction
TH~ use of ani mal model s shoul d do much t o el uci dat e t he
bi ol ogi cal - bi ochemi cal f act or s i nvol ved i n human addi ct i on
t o al cohol . The i ni t i al obser vat i ons of Ri cht er in 1926 [28]
cl ear l y est abl i shed t hat a r at wi l l vol i t i onal l y consume var i ous
concent r at i ons of al cohol in a choi ce si t uat i on wi t h wat er.
Numer ous i nvest i gat or s si nce t hat t i me have concl uded t hat a
r at ' s sel f-sel ect i on of al cohol can be i nfl uenced by a var i et y of
envi r onment al , physi ol ogi cal and psychol ogi cal f act or s
[I, 6, 11, 13, 15-17, 21-23]. Al t hough t he r at ' s i nt ake of
al cohol under a var i et y of condi t i ons has of t en been i nt er -
pr et ed as exper i ment al al cohol i sm or addi ct i on i t appear s
t hat t he cl assi cal cr i t er i a of addi ct i on t o al cohol (i.e. physi cal
dependence, t ol erance, etc. ) have not been satisfied in any
of t hese experi ment s.
The pr esent paper r epor t s t he resul t s of exper i ment s under -
t aken i n our l a bor a t or y i n an at t empt t o f or mul at e a model of
addi ct i on t o al cohol i n t he r at . Recent l y, a number of i nvest i -
gat or s have r epor t ed t he devel opment of one aspect of addi c-
t i on, i.e. physi cal dependence i n monkeys [8], mi ce [10],
and dogs [9] fol l owi ng t he admi ni s t r at i on of al cohol . However ,
none of t hese appr oaches has r el i ed on t he nor mal or al i nt ake
of al cohol , and, i n a number of cases, t he possi bi l i t y of
i nt er - cur r ent di sease and ot her physi ol ogi c compl i cat i ons
dur i ng wi t hdr awal , unr el at ed t o t he effects of al cohol , has not
been excl uded.
I n t he pr esent exper i ment , r at s were mai nt ai ned on a
7% al cohol (v/v) sol ut i on f r om weani ng (21 days) unt i l 154
days of age in an at t empt t o pr oduce, vi a t he nor mal r out e o f
admi ni st r at i on, a si t uat i on anal ogous t o addi ct i on t o al cohol
whi ch woul d sat i sfy t he maj or phar macol ogi c cr i t er i a of
addi ct i on, i.e. physi cal dependence, t ol er ance and a need f or
al cohol . The r at i onal e f or t hi s a ppr oa c h was t hr eef ol d:
(1) To al l ow cont i nuous exposur e t o an al cohol sol ut i on
whi ch is r eadi l y i ngest ed i n l arge vol umes by most r at s [4, 23,
24]; (2) Si nce t he per i od f r om 21 days t o 60 days of l i fe i n t he
r at is cri t i cal in t he devel opment of t he cent r al ner vous
syst em ( CNS) , par t i cul ar l y i n i t s chemi cal mat ur at i on [5, 26,
32], al cohol coul d have a par t i cul ar l y si gni fi cant i mpact on
t hese syst ems dur i ng t hi s per i od. Si nce CNS mechani sms
undoubt edl y pl ay an i mpor t ant r ol e i n t he addi ct i ve process
[18-20, 22], ear l y exposur e t o al cohol coul d wel l be an
i mpor t ant f act or i n t he devel opment of addi ct i on t o al cohol ;
(3) The use of rat s, r at her t han anot her species, woul d seem
t o be par t i cul ar l y advant ageous i n devel opi ng a model of
al cohol addi ct i on si nce ( apar t f r om t he obvi ous advant ages
of economy, housi ng, etc.) a gr eat deal is al r eady known about
t he f act or s gover ni ng t he r at ' s i nt ake of al cohol .
MATERIAL AND METHODS
Animals
Twent y- f our r at s of t he Hol t z ma n st r ai n were r ear ed under
s t andar d l abor at or y condi t i ons on a 12 hr l i ght - da r k cycle.
At weani ng (21 days) t he r at s were s epar at ed f r om t hei r
mot her s and i ndi vi dual l y housed in s t a nda r d wi re mesh cages.
1This research was supported in part by USPHS grants MH-09247 and MH-07081.
191
192 CICERO, SN1DER, PEREZ AND SWANSON
Apparatus
Fo r t he t est i ng per i od t he r at s were housed in 12 12 36 in.
chamber s whi ch have been mor e ful l y descr i bed el sewhere
[22], Thr ee hol es were dr i l l ed on t he f r ont wal l of t hese
chamber s, 1 in. apar t , t o hol d t hr ee 100 ml Ki max dr i nki ng
t ubes; each chamber was fi t t ed wi t h a speci al met al feedi ng
cup t o per mi t accur at e measur ement of f ood i nt ake. Fo r
open field t est i ng an encl osure, 4 f t 4 f t 18 in. was con-
st r uct ed of in. pl ywood and pai nt ed flat gr ay; t he base was
equal l y di vi ded i nt o 8 in. squares.
Procedure
Initial period (Phases A and B). The pr ocedur e empl oyed
in t hi s exper i ment is out l i ned in Tabl e 1. At weani ng (21
days), t he r at s were di vi ded i nt o t wo gr oups of 12 r at s, one
desi gnat ed t he al cohol gr oup (7 mal es and 5 femal es) and one
t he wat er gr oup (6 of each sex), and were i ndi vi duaUy
housed in mesh l abor at or y cages. The al cohol gr oup was
mai nt ai ned on 7 % al cohol , vol umet r i cal l y pr epar ed wi t h t a p
wat er a nd 95~o al cohol [3] f or t he next 133 days, whereas t he
wat er gr oup was gi ven t a p wat er onl y dur i ng t hi s per i od
( Tabl e 1, Phase B). The 7 % al cohol sol ut i on was freshl y
pr epar ed each day t hr oughout t hi s per i od. Body weights
were r ecor ded every ot her day and fl ui d i nt akes were r ecor ded
dai l y. Fo r t he l ast 24 days of t he i ni t i al per i od, t he r at s were
moved t o t he t est chamber s (see above) , a nd were i dent i cal l y
mai nt ai ned, i.e. al cohol r at s recei ved 7% al cohol as t hei r
sol e fluid, whereas wat er r at s were given wat er onl y ( Tabl e 1,
Phase B). Dur i ng t hi s phase f ood i nt akes, as wel l as fluid
i nt akes and body weights, were al so r ecor ded.
Open fieM testing (Phase C). The r at s were wi t hdr awn f r om
al cohol af t er t he 133 day exposur e per i od and for t he next
4 days, dur i ng whi ch wat er onl y was avai l abl e, open field
behavi or was r ecor ded ( Tabl e 1, Phase C). Each r at was
t aken f r om t he home chamber a nd pl aced in a c ome r of t he
open fi el d and r est r ai ned t her e by ~ s of a bar r i er f or t he
next 15 sec. The r at s were t hen al l owed t o expl or e t he open
field for t he next 3 mi n and t he squares crossed and number
of feces el i mi nat ed were r ecor ded [7]. I n addi t i on, t he r at s
were r at ed on a behavi or al checkl i st coveri ng such i t ems as
expl or at or y behavi or , curi osi t y, gr oomi ng, sniffing, reari ng,
i nvol unt ar y mot or act i vi t y, etc. Thi s por t i on of t he wor k was
conduct ed doubl e- bl i nd, i.e. t wo peopl e i ndependent l y
obser ved and r ecor ded each r at whi ch was br ought i nt o t he
t est r oom in r a ndom or der by a t hi r d person. The classifica-
t i on of each r at (al cohol or wat er) was known onl y by t he
per son bri ngi ng t he r at s i nt o t he r oom.
Self-selection of 3, 7 and 14% alcohol (Phase D). I n an
at t empt t o assess t he st rengt h of t he r at ' s preference f or or
aver si on t o al cohol , t he r at s in bot h t he al cohol and wat er
gr oups were given one day of wat er onl y (i.e no testing) and
were t hen offered a si mul t aneous choi ce of 3, 7 and 14%
al cohol f or 2 days; t he t ubes were r andoml y r ot at ed (25)
each day (Tabl e l , Phase D). I n t hi s way, i t was possi bl e t o
det er mi ne not onl y t he absol ut e vol umes of al cohol consumed
aft er t he wi t hdr awal per i od, but al so t o assess t he rel at i ve
preference for or aver si on t o al cohol wi t hi n a single t est
session.
Tolerance (Phase E). Fol l owi ng one day of wat er onl y,
each of t he r at s in t he al cohol and wat er gr oups was i nj ect ed,
i nt r aper i t oneal l y, wi t h a dose of al cohol (2.5 g/ kg body wei gh0
in a 25 % sol ut i on, vol umet r i cal l y pr epar ed wi t h 95 % al cohol
and i sot oni c saline. The t i me r equi r ed f or l oss of t he ri ght i ng
reflex, t he t i me t o t he onset of anest hesi a, t he l at t er bei ng
defi ned by l oss of t he reflexive response t o pai n, and t he
dur at i on of anest hesi a, r ecover y bei ng defi ned as t he poi nt at
whi ch t he ri ght i ng reflex r et ur ned, were r ecor ded ( Tabl e 1,
Phase E). Each of t hese cr i t er i a was consi der ed sat i sfi ed onl y
when t he f our i ndependent observers unequi vocal l y agr eed
upon each. Bl ood sampl es were t aken by sni ppi ng t he t ai l o f
each r at and t hen filling one hepar i nl zed capi l l ar y t ube.
Bl ood was t aken at 15, 30, 60, 120 and 240 mi n i nt er val s
f ol l owi ng t he i nj ect i on of al cohol . Bl ood al cohol levels were
det er mi ned in 2 ~l al i quot s of whol e bl ood by gas- l i qui d
chr omat ogr aphy ( GLC) as descr i bed el sewhere [27].
TABLE 1
THE PROCEDURE EMPLOYED IN THE EXPERIME~rr FOR THE ALCOHOL A N D WATER GROUPS
Phase Number of Days Alcohol Water
A 21 Home cages Home cages
B 133 7 ~o Et OH sole fluid HsO sole fluid
C 4 Open field testing HzO sole Open field testing H~O sole
fluid fluid
D 2 Selection of 3, 7 and 14~ Selection of 3, 7 and 14~
Et OH EtOH
E 1 Tolerance testing, H, O Tolerance testing, H20
sole fluid sole fluid
F 7 Preference-aversion Preferenco-aversion
testing 3- 30 ~o Et OH and testing 3- 30 ~o Et OH and
Hl O Hz0
G 7 Prefeacnce-aversion P r ~ c r a t c e - - a v e r s i o n
testing 3--30~0 EtOH, testing 3-30~0 EtOH,
H~0 and S a c c h a r i n Ht 0 and S a c c h a r i n
PHYSICAL DEPENDENCE AND ALCOHOL TOLERANCE 193
Fol l owi ng one day of wat er onl y five r at s i n t he wat er and
al cohol gr oups were ki l l ed f or a var i et y of hi st ol ogi cal and
chemi cal anal yses. The r eason f or sacri fi ci ng t he ani mal s at
t hi s poi nt was t o avoi d a ver y l ong per i od of wi t hdr awal
pr i or t o anal yses si nce i t was uncer t ai n as t o t he effect of t hi s
pr ocedur e on subsequent hi s t ol ogi cal - bi ochemi cal anal ysi s
[18, 19].
Alcohol self-selection (Phases F and G). Af t er one day of
wat er onl y, each of t he survi vi ng r at s was offered a choi ce
bet ween wat er and an al cohol sol ut i on 3, 5, 7, 10, 15, 20 and
30 %, each offered f or one day ( Tabl e 1, Phase F) . Af t er t hi s
i ni t i al sel f-sel ect i on series a second det er mi nat i on of pr ef er ence-
aver si on f unct i ons was made except i n t hi s case a t hi r d choi ce,
0.005 Yo (w/v) sacchar i n, was offered i n addi t i on t o wat er and
t he a ppr opr i a t e al cohol sol ut i on ( Tabl e 1, Phase (3). Thi s
concent r at i on of sacchar i n was sel ect ed on t he basi s of previ ous
wor k in our l a bor a t or y [3, 12] i n whi ch i t was f ound t hat t hi s
sol ut i on was i ngest ed in vol umes si mi l ar t o t hat of 7 %
al cohol i n a choi ce si t uat i on wi t h wat er.
Af t er t he fi nal day of t he al cohol sel f-sel ect i on series, t wo
al cohol and t wo wat er r at s were kept , whereas t he r emai nder
of t he r at s were sacri fi ced f or chemi cal anal yses. The sur-
vi vi ng al cohol ani mal s were t hen pl aced on a solo-fluid
r egi men of 15 % al cohol and t he wat er r at s were gi ven onl y
wat er. The dat a f or t hese ani mal s wi l l not be r epor t ed her e
si nce t hey wi l l be mai nt ai ned on 15% al cohol , a nd mor e
concent r at ed sol ut i ons, f or a pr ol onged per i od i n an at t empt
t o ext end t he fi ndi ngs r epor t ed i n t hi s paper .
RESULTS
Initial exposure period (Phases A and B). The r at s i n bot h
gr oups grew at appr oxi mat el y t he s ame r at e t hr oughout t he
exper i ment . Fi gur e 1 shows t he gr owt h curves f or mal e and
f emal e al cohol and wat er r at s f or every f our t h day dur i ng t he
i ni t i al 133 day exposur e per i od. As shown i n Fi g. 1, mal e
and femal e ani mal s were appr oxi mat el y 5 per cent l i ght er
4 0 0 , M A L E . ~ ' ' ~ " ~ "
........... "S=:',----,--':
/.,-'/.,-.
3 0 0 .
. , . . : . . ; : ' > "
p
2o0,
I - , : : /
3 - 1 0 0 , / ~ f A L C O H O L
( " ~ / - . . . . . WA T E R
LIJ
0 . . . . . . . r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . r n T r ~
> . . 3 0 0 .
t ' ~ F E MA L E
o ............ r = : : = : : = : = = ~ . ~
2 0 0 . . . , ~ , ~ . .
4: : )'"
1 0 0 . /.~,.
./'
0
o i o do 6'o 8"0 16o 1~o 11o
DAYS
FI G. 1. Mean body weights (g) for every fourth day during initial
133 day exposure period for male (top) and female (bottom)
alcohol and water rat s.
t han wat er cont r ol s but none of t hese di fferences were
st at i st i cal l y significant.
Fi gur e 2 shows t he mean fl ui d i nt akes of al cohol and wat er
mal es a nd femal es f or five day bl ocks t hr oughout t he first
109 days of t he exper i ment . The dat a f or t he r emai ni ng
days (24 days) dur i ng t he i ni t i al per i od wi l l be eval uat ed in
gr eat er det ai l i n a l at er sect i on. As can be seen, t he fl ui d
i nt akes of wat er and al cohol mal es cl osel y par al l el ed one
anot her t hr oughout t he cour se of t he exper i ment a nd t her e
was no st at i st i cal l y si gni fi cant difference bet ween t he t wo
groups. However , as shown i n Fi g. 2, al cohol femal es dr ank
appr eci abl y less t ot al fluids t han wat er femal es. An anal ysi s
of var i ance of t he l ast 30 days of t he i ni t i al per i od, when
i nt akes were at asympt ot e, r eveal ed a st at i st i cal l y si gni fi cant
difference bet ween t he al cohol and wat er femal es ( F = 19.33,
1/30 df, p < 0 . 0 1 ) .
5 0
MALE i
, " ' k ~ - ' , 4
s,e'-e- " /
/ /
3 O , , " / ' . . / '
. . . . . . WATER
t ~
I0.
t ' ~ FEMALE - ' ~ t PX
; , , . . . . : ,
4 0 i
/
/ " ' e " / / ' ~
3o . : - : . y . . : . . . . . . . ' , .
/ / % / " -
/
1C
00T 2 b 40 6"0 8"0 100 1~0
DAYS
F I G . 2. M e a n f l u i d i n t a k e s ( m l ) f o r f i v e - d a y b l o c k s d u r i n g t h e f i r s t
109 days during the initial exposure period for male (top) and female
(bottom) alcohol and water rats.
The mean f ood i nt akes f or al cohol and wat er mal es a nd
femal es ar e pr esent ed i n Fi g. 3 f or t he l ast 24 days of t he
i ni t i al per i od. As can be seen, f ood i nt akes f or mal e al cohol
r at s were si gni fi cant l y l ower ( F - 45.02, 1/45 dr, p <0. 01)
t han cor r espondi ng wat er rat s. Fo r f emal e r at s t hi s di st i nc-
t i on was not appar ent , i.e. f emal e al cohol a nd wat er r at s at e
similax amount s of f ood.
Dur i ng t he i ni t i al per i od t wo wat er r at s a nd one al cohol
r at di ed. I n addi t i on, t wo al cohol r at s ha d t o be sacri fi ced
because of t he devel opment of severe r espi r at or y pr obl ems.
I n t he r emai nder of t he Resul t s sect i on, t herefore, dat a ar e
pr esent ed f or 10 wat er r at s a nd 9 al cohol rat s.
Open field testing (Phase C). Fi gur e 4 present s a hi st ogr am
of act i vi t y, i.e. t he number of squares crossed i n a 3 rai n
per i od, i n t he open fi el d f or al cohol and wat er rat s. As
shown i n Fi g. 4, t he di st r i but i on of act i vi t y scores was
mar kedl y di fferent i n t he al cohol gr oup t han in t he wat er
194 CICERO, SNIDER, PEREZ AND SWANSON
40
MALE
3 0 .
2 0 / *' / ' * " " ' " / * ' " "
/ : \ . _ . : - ' " . . . . . . .:.
1 0 :,/
t l . l
- - - - ALCOtlt I[ ~r
0 - ] / - ~ . . . . . . . . . r . . . . . . . _ . . . . . V,:D T ~i~ . . . . . . . . .
O FEMALE
O 3 0 , /
/ ~ J /
\
2o
/ . ' , , , . . : , . . , , / \ , . / ' ,
/ " ~, - " ." .-'I ~ ~ ~ I { ,. , ',' .
,, . . . . . ,
10.
0 0 4 " / ] I i 0 I i 5 1 2 0 1 2 5 1'30
D A Y S
FI G. 3. Mean food intakes (g) for the last 24 days of the initial
exposure period for male (top) and female (bottom) alcohol and
and water rats.
4
W A T E R
- 0 . . . . . - ~ - . . . . . . - . . . . . . . . . . . . . ~ . . . . . ~ . . . . .
0 - 1 5 1 6 - 3 0 3 1 - 4 5 4 6 - 6 0 6 1 - 7 5 7 6 - 9 0 g 1 - 1 0 5
S Q U A R E S C R O S S E D
FI G. 4. A histogram showing the number o f rat s i n e a c h o f s e ve n
c l as s i f i c at i ons o f ac t i vi t y s cores , eatpressed as s quar e s c r os s e d i n a
3 mi n pe r i od, f o r wa t e r ( t op) a nd a l c o h o l ( b o t t o m) rat s.
gr oup. Si nce i t appear ed t hat t he al cohol gr oup no l onger
r epr esent ed a nor mal l y di st r i but ed popul at i on, par amet r i c
st at i st i cs were deemed i nappr opr i at e f or eval uat i ng t he
resul t s of t hi s experi ment . To eval uat e t he possi bi l i t y t hat an
exper i ment al pr ocedur e (i.e. l ong t er m al cohol i nt ake)
pr oduced an ext r eme response in t he al cohol gr oup, t he Moses
t est f or ext r eme r eact i ons was used [30]. The results of t hi s
anal ysi s ( p = 0.012) conf i r med t hat dur i ng wi t hdr awal t he
act i vi t y of al cohol r at s fell i nt o t wo ext r eme cl assi fi cat i ons,
i . e. ext r emel y hi gh or l ow activities. I n t he r emai nder of t he
resul t s sect i on, t hese t wo al cohol gr oups will be consi der ed
separ at el y and wi l l be referred t o as t he hi gh al cohol (7 rat s)
and t he l ow al cohol (2 rat s) groups.
Tabl e 2 present s t he mean act i vi t y scores f or hi gh al cohol ,
l ow al cohol and wat er r at s on each of t he f our open field t est
days. St at i st i cal eval uat i on ( Ma nn- Whi t ne y LD reveal ed t hat
hi gh al cohol r at s were significantly mor e act i ve t han wat er
rat s ( U = 0, 7/9, p < 0.001), based on t he mean f our day
act i vi t y scores. The difference bet ween hi gh al cohol and
wat er r at s was par t i cul ar l y st r i ki ng i n vi ew of t he fact t hat
t her e was no over l ap in t he di st r i but i on of act i vi t y scores;
t he most act i ve wat er r at cr ossed 62 squares in 3 mi n where
t he l east act i ve hi gh al cohol r at crossed 65 squares in 3 rain.
As shown i n Tabl e 2, hi gh al cohol r at s aver aged 80.3 ( l 4.35)
squares t o onl y 41.9 ( 6.9) for wat er rat s. An addi t i onal
aspect of t he dat a shown i n Tabl e 2 is t hat , unl i ke t he l ow
al cohol and wat er rat s, t he act i vi t y of hi gh al cohol r at s
i ncreased significantly (z2r = 7.92, 3 dr, p < 0.05) dur i ng t he
wi t hdr awal per i od. The peak act i vi t y scores (96.1 squares
crossed) were r ecor ded appr oxi mat el y 72 hr aft er al cohol
was wi t hdr awn.
The subj ect i ve anal ysi s, conduct ed doubl e- bl i nd and in
most cases r epr esent i ng a composi t e eval uat i on of t wo
i ndependent j udges, al so reveal ed a cl ear di st i nct i on bet ween
hi gh al cohol , l ow al cohol and wat er rat s. The r at i ng of hi gh
al cohol r at s r eveal ed, as expect ed, t hat t hese r at s were most
act i ve, r ear ed and expl or ed excessively and seemed com-
pl et el y non- f ear f ul i n t he open field, I n addi t i on, t hese r at s
were f r equent l y descr i bed in such t er ms as hyperact i ve,
frant i c, hi ghl y exci t abl e, etc. I n mar ked cont r ast t o this, t he
l ow al cohol a nd wat er groups, whi ch appear ed i dent i cal i n
open field per f or mance a nd subj ect i ve descr i pt i on, were
char act er i zed by such t er ms as i nact i vi t y, nervousness,
c r o u c h i n g , etc.
Selection of 3, 7 and 14yo alcohol (Phase D). The mean
i nt akes of t he 3, 7 a nd 14yo al cohol sol ut i ons ar e shown i n
Tabl e 3 f or t he hi gh al cohol , l ow al cohol and wat er gr oups.
The dat a i n t hi s t abl e and i n t he r emai nder of t he resul t s ar e
expr essed as g of al cohol / kg body wei ght per day, r at her
t han i n ml , si nce di fferent concent r at i ons of al cohol were
TABLE 2
M E A N A C l w n ~ S C O R m (q- S , ~ M . ) ~ EACH O F ~ Fo u r OPEN FmiJ~ Test
DAYS FOR Tim H I O H ALCOHOL, LoW AL(X)HOL AND WA~ RATS (PHAsB C, SEll TIL~r)
I 2 3 4 X
H i g h a l c o h o l 6 7 . 6 ( = E 6 . 1 ) 71.0(-I- 6 . 6 ) 9 6 . 1 ( + 5 . 8 ) 8 6 . 6 ( 4 - 1 2 . 0 ) 8 0 . 3 ( 4 - 4.4)
L o w a l c o h o l * 2 4 . 0 7.5 0 0.5 8.0
W a t e r 34.4(4- 7 . 3 ) 4 5 . 0 ( - 4 - 8 . 9 ) 4 7 . 7 ( - I - 9 . 9 ) 4 0 . 7 ( - I - 9 . 8 ) 4 1 . 9 ( 4 - 6 , 9 )
* No s t a nda r d e r r or s we r e c o mp u t e d since there we r e o n l y t wo rat s i n t hi s gr oup.
PHYSICAL DEPENDENCE AND ALCOHOL TOLERANCE 195
TABLE 3
THE MEAN (:ES.E.M.) ALCOHOL II~rAKES (gm/kg) ov THE 3, 7 AND 14~o ALCOHOL
CONCENTRATIONS (PI-]ASB D , SEE TEXT) FOR Tim HIGH ALCOHOL, LOW ALCOHOL AND WATER RA'E$*
3 7 14 TOTAL
H i g h a l c o h o l 1 . 5 4 ( - I - 0 . 3 1 ) 3 . 0 5 ( - } - 0 . 6 0 ) 2 . 2 6 ( + 0 . 6 3 ) 6 . 8 5 ( - I - 0 . 5 1 )
L o w a l c o h o l ? 2 . 2 1 1 . 9 2 0 . 1 8 4 . 3 1
Water 2.34(-4- 0.31) 0.71(-4- 0.24) 0.64(:E 0.48) 3.69(-4- 0.34)
*The classification of rats as high or low alcohol animals is based solely on their
activity scores in the open field.
? No standard errors were computed since there were only two rats in this group.
used and t he effective dose of al cohol varied accordi ng t o
differences in body weight (as, f or example, in males vs
females). I n addi t i on, it is of some i mport ance t o not e t hat
in this table and in t he remai nder o f t he results section t he
classification of rats as either hi gh or l ow al cohol animals is
based solely on their respective activity scores i n t he open field
duri ng withdrawal, as described above (Phase C). Several
features of this dat a require detailed analysis. First, it is
appar ent t hat hi gh al cohol rat s dr ank significantly mor e t ot al
al cohol t han water rat s (U-----6, 6/9, p < 0.01) on bot h test
days. Secondly, t he intakes of hi gh al cohol rats were distri-
but ed generally over t he t hree al cohol solutions, t he largest
vol umes being consumed at t he 7 per cent concent rat i on. I n
cont r ast t o these dat a, t he l ow al cohol and wat er rat s con-
sumed most of their al cohol at t he 3 ~o al cohol sol ut i on and
avoi ded t he mor e concent rat ed solutions.
Fi gure 5 summarizes t he dat a f or t he last 25 days of t he
initial exposure peri od (Phase B), t he open field testing
(Phase C) and t he selection of 3, 7 and 14 ~o al cohol (Phase D)
f or t he hi gh al cohol and l ow al cohol gr oups and t he wat er
group. Al cohol intakes pr i or t o open field (Panel A, Fig. 5)
were positively correl at ed with activity in t he open field
(panel B), f or t he hi gh and l ow al cohol rats (P= ---- Jr 0.64, 9 df,
p < 0.05). I n addition, a somewhat higher positive correl at i on
was f ound between t he intakes of 3, 7 and 1 4 ~ al cohol
(Panel C, Fig. 5) and open field behavi or (Ps = + 0.74, 9 df,
p < 0.05). There was no significant correl at i on between open
field behavi or and t he intake of t he 3, 7 and 14~o al cohol
solutions in t he wat er gr oup (see Fig. 5).
As shown in Fig. 5 (panel A), al cohol intakes (g/kg)
steadily increased over t he last 25 days of t he initial
exposure peri od f r om 5.6 g/ kg t o 7.3 g/ kg f or t he hi gh
al cohol rats. Since water rat s increased their t ot al fluid
intake onl y slightly duri ng this peri od (1.5 nd over t he 24
days), t he increase in al cohol i nt ake does not appear t o be
t he result of a nor mal increase i n fluid i nt ake duri ng this
7
<
I--
Z
_J
0-5
"r"
3
A /
H / ~ o ~
o i t~ :~ ~, g o
FIVE DAY BLOCKS
,,oo
,75
e ~f
,50 ~,~/,. _z
/ , , J
~ - 1
25 o
A
"~ 2 3 4
DAYS
C
ETOH CONCENTRATIONS (%)
FIG. 5. A snmm~ry of the data for three phases in this experiment.
Panel A shows the mean al ~hol int~k~ (g]kg) for five-day blocks over
the last 25 days of the initial exposure period high alcohol (HA) and
low alcohol 0. A) rats. Panel B shows the mean open field a c t i v i t y
(squares crossed) for each of the four test days for HA, LA and
water (W) rats. Panel C shows the mean alcohol intakes (g/kg) of
the 3, 7 and 14 per cent alcohol solutions (EtOH concentrations ~ )
for HA, LA and W rats.
196 CICERO, SNIDER, PEREZ AND SWANSON
per i od. Low al cohol r at s di d not consi st ent l y i ncrease t hei r
i nt ake of al cohol dur i ng t hi s per i od.
Tolerance (Phase E). Fol l owi ng t he i nt r aper i t oneal injec-
t i on of al cohol (2.5 g/ kg) 3 of 9 al cohol r at s became anest he-
t i zed and of t he 6 whi ch di d not become anest het i zed, most
appear ed t o be onl y mi l dl y sedat ed. I n ma r ke d cont r ast t o
t hese dat a, 6 of t he 9 wat er r at s became anest het i zed and t he
r emai ni ng 3 r at s were ver y heavi l y sedat ed and exhi bi t ed
pr onounced l et hargy. Of t he r at s in bot h gr oups whi ch
became anest het i zed, t he al cohol r at s [3] were anest het i zed
for onl y 14 mi n ( 1.00) wher e wat er r at s [6] were anest het i zed
for over t wi ce as l ong, i.e. 30 mi n ( ~6. 52) .
The bl ood al cohol levels f or t he al cohol and wat er gr oups
are shown i n Fi g. 6. Bl ood al cohol levels r ose t o a peak at
60 mi n and t her eaf t er decl i ned at a l i near r at e t hr ough 4 hr
and t her e was no difference bet ween t he t wo groups. The
maxi mum bl ood al cohol levels at t ai ned were 238 mg/100 ml
for t he al cohol gr oup and 235 mg/100 ml f or t he wat er gr oup.
Ther e was no appar ent sex di fference or di st i nct i on bet ween
hi gh al cohol and l ow al cohol r at s in ei t her t he maxi mum
bl ood al cohol l evel r eached or t he r at e of di sappear ance
of al cohol f r om t he bl ood.
240"
200'
c.
o
N m0.
0
' 120.
,,.-.,
O
S
8 0 ,
|
) . ~ / ; / %%%
%%%
% . . .
%%% %
ALCO
[ . . . . . . WATER """
! %%
1~i :30 6"0 1 2 0 2 4 0
MINUTES
FI G. 6. Mean bl ood alcohol levels (Blood-EtOH [mg/100 ml]) for
alcohol and water rats from 0 to 240 rain after the injection of
alcohol (2.5 g/kg).
Alcohol self-selection (Phase F a n d G). The resul t s pr esent ed
in t hi s sect i on ar e based on 4 r at s i n each of t he al cohol and
wat er gr oups, t he r emai ni ng ani mal s havi ng been ki l l ed f or
chemi cal anal ysi s. Si nce t he amount of al cohol consumed in
t hese choi ce si t uat i ons woul d seem t o be of gr eat er i mpor t ance
t han t he shape of t he preference--aversi on funct i ons, t he dat a
pr esent ed in Fi g. 7 ar e expressed as g of al cohol / kg body
wei ght dur i ng bot h Phase F ( al cohol - wat er choi ce) and
Phase G ( al cohol - wat er - s acchar i n choi ce) f or each concen-
t r at i on. Fi gur e 7 present s t he al cohol i nt akes (g/kg) f or t wo
rat s, a hi gh al cohol (A9) and a wat er ((31) r at f or bot h
phases. These t wo anirtmls ar e shown si nce bot h have been
subj ect ed t o f ur t her al cohol exposur e and bot h ar e r epr e-
sent at i ve of t hei r par t i cul ar groups. As shown in Fi g. 7, t he
hi gh al cohol r at dr ank ver y l arge vol umes of al cohol at each
of t he concent r at i ons empl oyed in t hi s exper i ment in t he
12. A9 (ALCOHOL) /
, . . , . i - e - - i " o _ ~
- - 0 . . . . . r - - r - - r . . . . ~ . . . . . . . -1 . . . . . . . r . . . . . . . . . . . . . . ~ . . . .
- - J
0 C1 (WATER)
"I-
0 - - A-W CHOICE
~18 . . . . . . . A-W-S CHOICE
, <
4, e / ~ ' e ~
o 5 t, m 2"o
A L C O H O L C O N C E N T R A T I O N S (%)
FI G. 7. The alcohol intakes (g/kg) of an alcohol rat (Ag, top) and a
water rat (C1, bottom) for various concentrations of alcohol
(from 3 to 30 ~) . The solid line refers to alcohol intake when water
and an ethanol solution (A-W) were available, whereas the dotted
line refers to alcohol intake when water, an alcohol solution and
0.005 ~ saccharin (A-W-S) were available.
i ni t i al sel f-sel ect i on series (Phase F). Dur i ng t he per i od in
whi ch 0.005 ~ sacchar i n was al so avai l abl e (Phase G), this
r at ' s al cohol i nt ake was r el at i vel y unaffect ed (Fi g. 7). I n
cont r ast t o t hi s finding, t he wat er r at vi r t ual l y el i mi nat ed its
i nt ake of al cohol dur i ng t he per i od i n whi ch sacchari n
(Phase G) was avai l abl e even t hough i t had consumed at
l east some of t he al cohol sol ut i ons dur i ng t he i ni t i al self-
sel ect i on det er mi nat i on. I t is of some i nt erest t o not e t he
ext r emel y l ar ge di fference bet ween t he i ni t i al i nt akes o f t he
al cohol (A9) and wat er (C1) r at . The al cohol r at oft en
consumed at l east 5 t i mes t he amount of al cohol as t he wat er
r at ; i nt akes for A9 (Fi g. 7) aver aged 8- 9 g/ kg and ext ended
up t o 13 g/ kg at t he 15 ~o concent r at i on.
D I S C U S S I O N
The resul t s of t hi s exper i ment suggest t hat chr oni c
exposur e t o a r eadi l y consumed al cohol sol ut i on f r om weani ng
may pr oduce a si t uat i on i n t he r at whi ch satisfies a number of
t he obj ect i ve phar macol ogi c cr i t er i a of addi ct i on t o al cohol .
The hyper act i vi t y of hi gh al cohol r at s in t hi s exper i ment
appear s t o be a di r ect r esponse t o t he abr upt wi t hdr awal of
al cohol f or t hr ee i mpor t ant r easons: (1) The act i vi t y of hi gh
al cohol rat s, par t i cul ar l y in t he l at t er stages of open field
testing, was of t ea so gr eat t hat i t was ext r emel y d i t ~ u l t t o
manual l y r ecor d, occa~onal l y reachi ng 130-145 squares
cr ossed i n j us t a 3 r ai n peri od. Mor eover , t he act i vi t y o f t hese
rat s, i n mar ked cont r ast t o l ow al cohol and wat er rat s, oft en
a p p e a u ~ t o be poor l y di r ect ed and di sor gaai zed. Wher eas
wat er and l ow al cohol r at s were t ypi cal l y qui t e caut i ous in
PHYSICAL DEPENDENCE AND ALCOHOL TOLERANCE 197
t he open field and were highly systematic in their expl orat i on
(i.e. movement first ar ound t he walls, t hen occasi onal
caut i ous movement i nt o t he center of t he area), hi gh al cohol
rats on t he ot her hand began runni ng immediately, criss-
crossing t he open field, and showed no apparent fear or
organi zed activity in this novel environment. (2) The activity
scores of hi gh al cohol rats increased significantly duri ng t he
withdrawal period, reachi ng a peak f r om 48 t o 72 hr after the
wi t hdrawal of alcohol. (3) Activity in t he open field was
positively correlated with t he amount of al cohol consumed
pri or t o t he wi t hdrawal period. These three findings are
consistent wi t h t he i nt erpret at i on t hat t he hyperactivity of
hi gh al cohol rats represents, at least a partial, withdrawal
response in t he rat since each is in t he predicted direction.
That is, hyperactivity, whi ch appears t o be a maj or com-
ponent of t he wi t hdrawal syndr ome i n animals [8-10, 29],
increased as a funct i on of t he length of t he abstinence period
and appeared t o be directly pr opor t i onal t o the amount of
al cohol consumed pri or t o t he wi t hdrawal period. On t he
basis of these data, it seems reasonabl e t o suggest t hat t he
hyperactivity of hi gh al cohol rats in t he open field represents
a response t o t he abr upt wi t hdrawal of alcohol.
The wi t hdrawal response described in this paper appears t o
correspond t o the mild t o moder at e withdrawal syndrome,
bot h i n terms of intensity and t emporal development,
observed in rats whi ch have been made physically dependent
on mor phi ne and ot her compounds with significant addictive
liability [29]. I n addition, t he finding t hat activity scores
reached a maxi mum f r om 48 t o 72 hr (see Tabl e 2) after t he
withdrawal of al cohol is consistent with t he t emporal develop-
ment of t he al cohol withdrawal syndrome in a number of
species [8-10, 18, 19, 29]. Since comparabl e dat a are not
available concerni ng physical dependence on al cohol in t he
rat, t he nat ure and extent of t he withdrawal syndrome is not
known. However, it seems possible t hat t he present findings
mi ght be extended t o include t remors and convulsions duri ng
withdrawal by varyi ng t he length of t he al cohol exposure
period, t he strength of t he al cohol solutions, or perhaps t he
poi nt at which t he al cohol regimen is begun.
The results of this experiment clearly indicate t hat t he
behavioral or physiological response t o al cohol may be
at t enuat ed after a l ong peri od of al cohol exposure. The fact
t hat t he in rive rat e of di sappearance of al cohol f r om t he
bl ood was t he same f or al cohol and water rats woul d suggest
t hat t he tolerance observed in this experiment is a cellular
t ol erance medi at ed by one of a number of possible mechanisms
[2, 14, 18, 19]. These dat a woul d seem t o indicate t hat t he
report ed changes in t he in vitro activity of al cohol dehydro-
genase ( ADH) in animals, exposed t o some peri od (often
unspecified) of al cohol intake, may be of questionable value
in explaining t he mar ked tolerance t o al cohol observed i n
this experiment. Al t hough t he present results do not exclude
t he possibility of alterations in t he activity of ADH, since
direct measurements were not made, it seems clear t hat t he
tolerance observed in this experiment was unrel at ed t o t he
ability of rats t o eliminate al cohol (see Fig. 6). Hence, a
plausible expl anat i on of these results woul d seem t o be t hat
t he observed tolerance is due to, as yet unspecified, changes
at a cellular level within t he CNS.
The amount s of al cohol consumed by hi gh al cohol rats,
bot h before and after open field testing, averaged 7- 8 g/ kg
body weight, with individual rats dri nki ng as much as
15 g/ kg. Since it is welllestablished, and we have confirmed,
t hat rats mai nt ai ned on a 12 hr l i ght - dar k cycle dri nk 90-95
per cent of their t ot al fluids at night, it can be assumed t hat
an average of 700-800 rag of al cohol / kg/ hr was consumed
over this period, assumi ng an even distribution of drinking.
Since rats frequently dri nk in bursts t he effective dose of
al cohol at any given time coul d have been considerably in
excess of these figures. Fr om t he dat a in this experiment
(Fig. 6) it appears t hat t he rat can metabolize al cohol at a
rat e of 250-300 g/kg/hr, which is in good agreement with
ot her studies [31], and t hus it seems clear t hat t he ingested
al cohol coul d not have been fully degraded during t he
dri nki ng period. These dat a therefore suggest t hat al cohol
was exerting a pharmacol ogi c effect in this experiment.
An i mpor t ant aspect of t he present dat a woul d seem t o be
t hat very large amount s of al cohol were consumed by hi gh
al cohol rats, not onl y during t he initial forced-exposure period,
but also in t he free-choice situations. These findings, t aken
in conj unct i on with t he fact high al cohol rats t ended t o
increase their al cohol intakes with increasing concent rat i ons
of al cohol and preferred al cohol t o a saccharin solution,
suggest t hat t he need f or al cohol was appreciable in these
rats. Moreover, t he fact t hat virtually t he same amount s of
al cohol were consumed i n choice situations as in t he forced
exposure peri od suggests t hat it may be possible t o formul at e
a model of al cohol addi ct i on in the rat by relying, at least t o
some extent, on a free choice situation rat her t han solely on
a forced-i nt ake regimen.
The essential features of t he addictive model described in
this experiment can be summari zed as fol l ows: rats may
develop physical dependence on and cellular tolerance t o
al cohol when mai nt ai ned f r om weani ng (21 days) until 154
days of age on a forced-i nt ake regimen of a readily consumed
al cohol solution. Moreover, large amount s of al cohol were
consumed, which appeared t o exceed t he met abol i c rate, when
a free choice regimen was instituted or even when a palatable
third choice was offered.
The results of this experiment suggest t hat a large pr opor -
t i on of rats may meet objective pharmacol ogi c criteria of
addi ct i on t o alcohol. I n fact, onl y t wo al cohol rats in the
present experiment did not meet these criteria, i.e. t he low
al cohol group. These animals consistently performed mor e
like t he water rats t han the high al cohol gr oup with one
not abl e exception. There was no distinction between hi gh
and low al cohol rats in t he degree of tolerance developed.
These dat a woul d seem t o indicate t hat t he amount of
al cohol necessary t o pr oduce tolerance may be somewhat less
t han t hat needed t o produce addiction. This concl usi on
appears t o be support ed by t he findings t hat low al cohol rats
dr ank considerably less al cohol t han high al cohol rats duri ng
t he initial exposure peri od (e.g. Fig. 5, Panel A).
The mai n question raised by t he present results is whet her
the efficacy of this procedure, in cont rast t o previous negative
results, is primarily due t o the poi nt at which t he rats were
forced t o drink al cohol (weaning) or j ust t he durat i on of t he
initial al cohol exposure period. Al t hough the latter possi-
bility seems somewhat unlikely, it shoul d be not ed t hat in
most studies of t he l ong t erm effects of al cohol 90-120 day ol d
animals are used. To obt ai n an exposure peri od similar t o
t he one used in this experiment, i.e. approxi mat el y 85 per cent
of t he rat s' lifetime f r om 21 days t o t he t ermi nat i on of t he
experiment (154 days), these rats woul d have t o be main-
tained on al cohol f or 2-3 years, a procedure which has not
yet been employed. A possible explanation of t he effects
report ed i n this experiment may be t hat al cohol was intro-
duced at a particularly critical poi nt in t he devel opment of
t he CNS of t he rat (days 21-60). Since CNS mechanisms
undoubt edl y pl ay a significant role in t he addictive process
198 CICERO, SNIDER, PEREZ AND SWANSON
[18-20, 22], it seems likely that the effect of alcohol on these
systems would be particularly significant during the develop-
mental period. Although a solution to this problem is not
provided in the present study, experiments designed to assess
the effects of exposing rats to alcohol for equivalent periods,
but at different times, from weaning to adulthood could well
answer the question of whether a critical period exists during
which the addictive liability of alcohol is most significant.
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