Neurochemical effects of long-term ingestion of ethanol by Sinclair (S-1) swine

Pharmacoloe~ Bu~chemt~tr3& Behavior, Vol 18, pp 363--367, 1983 ~ Copyright Ankho International Inc Printed m the U S A

Neurochemical Effects of Long-Term Ingestion of Ethanol by Sinclair (S- 1) Swine R M

A HARRIS,* D FENNER,t D FELLER,I" G SIECKMAN,* S LLOYD'~ MITCHELL,t J D DEXTER,$ M E TUMBELSON§ AND D B BYLUNDt

*Truman Veterans Hospital and t D e p a r t m e n t o f Pharmacology, SDepartment o f Neurology, §Department o f Veterinary A n a t o m y and Phys:ology ~.§Smclalr Comparative Medic me R e s e a r c h Farm, Umverslty o f Missouri-Columbia, Columbia, M O 65212 R e c e i v e d 21 J u n e 1982 HARRIS, R A , D FENNER, D FELLER, G SIECKMAN, S LLOYD, M MITCHELL, J D DEXTER, M E TUMBLESON AND D B BYLUND Neuro~hemt~al effec t~ of long-term mgestton of ethanol by Sinclair (S-I) s~*me PHARMACOL BIOCHEM BEHAV 18(3) 363-367, 1983 ----SmclaJr (S-I) mmmture swine were given access to a mixture of ethanol and beer for three years Control swine were fed an ~socalorJc d~et w~th corn starch substituted for ethanol Both groups had free access to tap water The alcohol group consumed about 4 g ethanol/kg/day (about 50% of their caloric retake) resulting in plasma ethanol concentrations of about 100 mg/dl Brain membranes were prepared for analysis of neurotransmitter receptor binding, membrane hpld composmon and physical properties Receptor studies demonstrated an increase m the binding of 3H-GABA to cortical and cerebellar membranes from the alcohohc pigs as compared to control Binding of 3H-hgands to muscanne chohnerglc,/3-adrenerglc, a-adrenerglc, dopamme and benzodmzepme receptors was not changed by chronic ethanol mgesuon These results are slmdar to those obtained m studzes of human alcohohcs by other investigators The cholesterol content of myehn and synaptlc plasma membranes was not altered by ethanol consumpt~on The fluorescence polarization of d~phenythexatnene, a measure of membrane order, d~d not detect any d~fferences m the membranes from control or alcohol-treated swine either before or after tn vgtro exposure to ethanol These results are d~fferent from those reported for rodents after short-term ethanol treatments and emphasize the importance of evaluating different models of experimental alcohohsm Alcohohsm

Neurotransm~tter receptors

GABA

Membrane flmdlty

T H E n e u r o c h e m l c a l effects of chronic alcohol c o n s u m p t i o n have been the subject of many recent investigations T w o of the mam areas of interest are neurotransm~tter r e c e p t o r binding, and m e m b r a n e composition and physical properties Administration o f ethanol to m~ce or rats for one or two w e e k s Increases m u s c a n m c cholinerg~c r e c e p t o r s m brain [15,22], although this change is not seen after four days of treatment [10] T r e a t m e n t with ethanol for four to nineteen days does not alter rodent brain a-adrenerglc, benzodlazepine, 3,-amlnobutyrlc acid (GABA), fl-adrenerglc or dopamine receptor binding [7, 10, 12, 15, 21, 23] H o w e v e r , fl-adrenergic r e c e p t o r binding is d e c r e a s e d In rat brain and heart after c o n s u m p t i o n o f ethanol for 60 days [I] and m o u s e brain b e n z o d l a z e p l n e receptor binding IS d e c r e a s e d after 7 months of alcohol ingestion [7] In addition, increased G A B A r e c e p t o r binding is o b s e r v e d in brams of human aicohohcs [24] T h e s e results suggest that prolonged ethanol c o n s u m p t i o n may produce changes in neurotransmltter rec e p t o r bmdlng which are not o b s e r v e d after short-term treatment S o m e changes in rodent m e m b r a n e physical properties and c o m p o s i t i o n have been reported C o n s u m p t i o n of ethanol increases synaptic plasma m e m b r a n e (SPM) choles-

terol [2], alters the acyl composition o f SPM phospholiplds [13], increases the rigidity of SPM [19] and decreases the perturbation of brain m e m b r a n e s p r o d u c e d by :n v:tro exposure to ethanol [3] With the e x c e p t i o n of the study o f G A B A binding in human alcohohcs and benzodtazepine b m d m g in mice, the studies discussed a b o v e used force c o n s u m p t i o n o f ethanol for short periods of time as a model o f chronic ethanol exposure The r e l e v a n c e of these paradigms to human a l c o h o h s m remains controversial An animal m o d e l which Incorporates several aspects of human alcoholism is the Sinclair (S-l) miniature swine This species will v o l u n t a n l y c o n s u m e intoxicating quantities o f ethanol o v e r a long period of time [4,25] N e u r o c h e m i c a l correlates of alcohol d e p e n d e n c e have not, h o w e v e r , been reported for this paradigm The purpose of the present study was to investigate the effects o f long-term c o n s u m p u o n o f alcohol on neurotransmltter rec e p t o r binding, and m e m b r a n e c o m p o s i t i o n and physical properties F o r these studies, we obtained brain tissue from two groups o f swine one which had voluntarily c o n s u m e d ethanol for 3 years (alcohol group) and one (control group) w h o s e caloric, carbohydrate and protein-intake was matched to that of the alcohol group

363

H A R R I S ET AL

364 TABLE l

C O N D I T I O N S FOR BINDING OF RADIOACTIVE LIGANDS TO BRAIN M E M B R A N E S FROM MINIATURE SWINE

3H-Llgand Prazosm p-Amlnoclonldlne Yohlmbme Splropendol Qulnuchdmyl benzfiate Dlhydroalprenolol Flumtrazepam y-Amlnobutync Acid (GABA)

Buffer 40 mM Tns pH74 40 mM Tns pH74 25 mM Glygly* pH74 40 mM Tns pH 8 40 mM Tns pH 8 40 mM Tns pH 8 40 mM Tnsc~trate pH 7 2 40 mM Trl~citrate pH 7 2

Assay Vol Iml)

Protein (mg/ml)

Assay Temp {°C)

Assay Time (mln)

Conc Range LNmd (nM)

Blank (/~tM)

1

0 25

25

30

0 2 -6

I )NE (100)

1

0 12

25

~;0

0 1 -2

I-)N E (I}

1

0 08-0 12

25

30

0 1 -1

I - ) N E (10)

2

0 03~

25

60

0 05-7

(+)Butaclamol (1)

1

0 03-0 05

25

30

0 2 -3

Atropine {1)

1

0 25

25

30

0 1 -2

I-)Propranolol l0 3)

05

0 25-0 5

4

15

0 3 -10

Dlazepam I I0)

05

0 25-0 5

4

15

8 -64

GABA (I00)

*Glycylglycme

METHOD

Ammals Male a n d f e m a l e Sinclair (S-l) m l m a t u r e swine (9 m o n t h s o f age at the b e g i n n i n g o f the study) were h o u s e d individually T h e lifespan o f t h e s e animals is a b o u t 12 y e a r s Initial b o d y weights a v e r a g e d 36 kg for the c o n t r o l g r o u p a n d 31 kg for the alcohol g r o u p Final b o d y weights were 70 kg icontrol) and 51 kg (alcohol) T h e e t h a n o l g r o u p was given free a c c e s s to 10c~ w/v e t h a n o l m b e e r B e e r was delivered by Lixlt spouts w h i c h r e d u c e d spillage to a b o u t 10% of the total c o n s u m p t i o n Spillage was collected m a pan u n d e r the spout a n d m e a s u r e d daily B o t h ethanol a n d control g r o u p s were allowed free a c c e s s to t a p wate~ and were given 53 g o f p r o t e i n / d a y in a d d i t i o n to 500 g/day pig c h o w To c o m p e n sate for the calories derived from e t h a n o l and beer, the control g r o u p was g i v e n 450 g/day o f c o r n s t a r c h A n i m a l s were m a i n t a i n e d on t h e i r r e s p e c t i v e r e g i m e n s for 35-37 m o n t h s T h e y c o n s u m e d a b o u t 4 g of e t h a n o l / k g / d a y w h i c h was e q u i v a l e n t to a b o u t 50% o f their caloric retake P l a s m a e t h a n o l c o n c e n t r a t i o n s were d e t e r m i n e d weekly (2-4 p m ) a n d a v e r a g e d a b o u t 140 mg/dl for males a n d a b o u t 100 mg/dl for females for the last 12 w e e k s of the s t u d y T h e animals c o n s u m e d a b o u t 50% of t h e i r daffy e t h a n o l b e t w e e n 8 a m a n d 2 p m a n d blood ethanol levels were near maximal at the tune of m e a s u r e m e n t T h e s e data will be p r e s e n t e d in detail e l s e w h e r e ( T u m b l e s o n and D e x t e r , in p r e p a r a t i o n ) Animals w e r e not w i t h d r a w n from ethanol and the b l o o d e t h a n o l conc e n t r a t i o n s at the t~me o f d e a t h (9-11 a m ) was 50 to I00 mg% T h e a n i m a l s s h o w e d n o signs o f w i t h d r a w a l T h e s w i n e were given 12 mg/kg k e t a m i n e HC1 0 088 mg/kg atropine sulfate a n d sufficient t h i a m y t a l s o d i u m to p r o d u c e light a n e s t h e s i a T h e y were killed by e x s a n g u i n a t l o n and dec a p i t a t e d T h e b r a i n was r e m o v e d , p l a c e d on ice a n d dissected T h e b r a i n regions were w e i g h e d and h o m o g e n i z e d for m e m b r a n e p r e p a r a t i o n as d e s c r i b e d b e l o w

Mumblallu Plupalalloll All steps were d o n e at 0-5°C F o r r e c e p t o r binding assays, tissue was h o m o g e n i z e d for 30 sec in 50 m M T n s HC1 IpH 7 4) with a T i s s u m l z e r ( T e k m a r Co , C i n c i n n a t i O H ) and c e n t r i f u g e d at 4 8 , 0 0 0 × g for 10 rain T h e s u p e r n a t a n t was d i s c a r d e d and the pellet was h o m o g e n i z e d again This susp e n s i o n was c e n t r i f u g e d as before and the pellet was again h o m o g e m z e d This s u s p e n s | o n was c e n t r i f u g e d as before, a n d the pellet was stored at - 7 0 ° C F o r binding studies, the m e m b r a n e s were s u s p e n d e d m the a p p r o p r i a t e buffer (Table 1) a n d used w i t h o u t f u r t h e r t r e a t m e n t , e x c e p t in the case o f G A B A r e c e p t o r binding F o r G A B A binding, m e m b r a n e s were h o m o g e m z e d with 0 01% T r i t o n X-100 a n d i n c u b a t e d at 37 ° for 30 rain [5] M e m b r a n e s were pelleted a n d resusp e n d e d in 50 m M Trls citrate (pH 7 2) buffer for the binding assay F o r analysis of m e m b r a n e hpld c o m p o s i t i o n and physical p r o p e r t i e s , brain n s s u e ( c e r e b e l l u m a n d cortex) was h o m o g e n i z e d m 0 32 M s u c r o s e with 10 m M H E P E S , p H 7 4 Myelin and s y n a p t i c p l a s m a m e m b r a n e s (SPM-2) were prepared by d i s c o n t i n u o u s g r a d i e n t centrIfugatlon as d e s c r i b e d previously [6,8]

Re~ eptol Btndm~,, T h e binding o f tritlated h g a n d s ( N e w E n g l a n d N u c l e a r , B o s t o n , M A ) to b r a i n m e m b r a n e s was m e a s u r e d by incubating the m e m b r a n e with 5-7 c o n c e n t r a t i o n s of e a c h h g a n d E a c h m e m b r a n e was a s s a y e d m d u p h c a t e or t r l p h c a t e R a d i o a c t i v i t y b o u n d to the m e m b r a n e s was d e t e r m i n e d after filtration ( W h a t m a n G F / B filters) and w a s h i n g for all h g a n d s e x c e p t G A B A , w h i c h w a s d e t e r m i n e d by centrffugatlon ( 3 0 , 0 0 0 x g for 10 mln) NonspeclfiC binding was d e t e r m i n e d b y a d d i t i o n o f an a p p r o p r i a t e e x c e s s of a c o m p e t i n g h g a n d T h e assay conditions used for e a c h hgand are given in Table 1 T h e n u m b e r o f binding sites (B .... ) a n d the affinity o f binding

NEUROCHEMISTRY

OF LONG-TERM

ETHANOL

365

TABLE 2 RECEPTOR DENSITY (BM~0AND AFFINITY {KD)IN MINIATURE SWINE BRAIN AFTER CHRONIC CONSUMPTION OF ALCOHOL

KD (nM) Receptor

Llgand

Benzodlazepme

Flumtrazepam

Muscanmc Dopamme fl-Adrenerglc c~-Adrenerglc

QNB Splropendol DHA Yohlmbme PAC Prazosm

Brain Area

Control

Cortex Cerebellum Caudate Caudate Cerebellum Cortex Cortex Cortex

54 + 09 55 +03 009-+ 001 0 03 _+ 0 02 0 28 _+ 0 02 0 27 _+ 0 02 1 2 _+ 0 1 0 13 _+ 0 02

BM.x (pmol/mg Protein) Alcohol

50 56 0 ll 0 03 0 32 0 26 11 0 10

_+03 _+04 -+ 002 _+ 0 03 _+ 0 02 _+ 0 02 _+ 0 1 _+ 0 01

Control

Alcohol

2 0 _+ 0 2 l 0 _+01 1 3 -+ 0 2 0 58 _+ 0 06 0 13 _+ 0 01 0 17 _+ 0 02 008_+ 001 0 22 _+ 0 01

2 4 _+ 0 1 l 0 +_01 l 5 _+ 0 1 0 63 _+ 0 02 0 15 _+ 0 01 0 16 _+ 0 02 008_+ 001 0 23 _+ 0 02

Values represent mean _+ SEM N = 8 - 9 There are no slgmficant differences between alcohol and control

(KD) were d e t e r m i n e d b y a R o s e n t h a l [18] analysis o f the

80"

binding curves

Female

Male

C9rtex I

Female

Membrane Composttlon and Phwtc al Propertte~ Llptds w e r e e x t r a c t e d f r o m S P M a n d m y e l i n m e m b r a n e s o f c o r t e x a n d c e r e b e l l u m as d e s c r i b e d p r e w o u s l y [6] T h e c h o l e s t e r o l c o n t e n t w a s d e t e r m i n e d by gas c h r o m a t o g r a p h y [6] T h e " f l u i d i t y " o f the S P M a n d t h e i r sensitivity to m vttro e x p o s u r e to e t h a n o l w a s studied u s i n g d l p h e n y l h e x a t r l e n e ( D P H ) as a f l u o r e s c e n t p r o b e o f the m e m b r a n e core [8] F l u o r e s c e n c e p o l a r i z a t i o n o f D P H was m e a s u r e d as des c r i b e d p r e v i o u s l y [8] at 37°C using a n HH-1 s p e c t r o fluorimeter (BHL Assocmtes, Burhngame, CA)

40-

6°"1 I 20-

IE eQ

80-

Mole

Il'

"04 c o

Cerebellum

['7 Control 17 Alcohol

+l

'2D

~ E

'16

~r

'12

o

| O0

P r o t e i n was d e t e r m i n e d by the m e t h o d o f L o w r y [14] u s m g B S A as the s t a n d a r d

40

RESULTS

20

Re~ eptor Binding Chrome consumption of ethanol increased the binding of 3 H - G A B A to b r a i n m e m b r a n e s In the c o r t e x a n d c e r e b e l l u m o f female s w m e , t h e i n c r e a s e d b m d m g w a s due to a d e c r e a s e In KD with no c h a n g e I n B m a \ (Fig l) In male swine, the KD w a s d e c r e a s e d in c e r e b e l l u m w h d e Bmax was i n c r e a s e d In c o r t e x F o r t h e c o n t r o l g r o u p , t h e r e w a s a c l e a r sex differe n c e m a H - G A B A b i n d i n g , with f e m a l e s displaying twice as m a n y b i n d i n g sites as m a l e s for b o t h c o r t e x a n d c e r e b e l l u m T h e b i n d i n g affinity did n o t differ b e t w e e n m a l e s a n d f e m a l e s (Fig l) W i t h the c o n c e n t r a t i o n s o f a H - G A B A u s e d in the p r e s e n t study, we d e t e c t e d o n l y 1 p o p u l a t i o n o f b i n d i n g sites In c o n t r a s t to the c h a n g e s in 3 H - G A B A b m d l n g , c h r o m e alcohol c o n s u m p t i o n did n o t a l t e r the binding o f h g a n d s for benzodmzeplne, muscanmc chohnergic, dopamlne, /3-adrenerglc or a - a d r e n e r g l c r e c e p t o r s (Table 2) T h e data In T a b l e 2 are the a v e r a g e o f values o b t a i n e d f r o m b o t h male a n d f e m a l e s w i n e W h e n a n a l y z e d s e p a r a t e l y , t h e r e w a s no significant d i f f e r e n c e b e t w e e n m a l e s a n d f e m a l e s

"08

7

60 . +

Other Assays

"12

I

'08 '0t

FIG 1 Effects of chrome alcohol treatment in the binding of 3HGABA to brain membranes from S-1 mtmature swme The left half represents binding affimty (Kd, nM), the right half represents the number of binding sites (Fmax,pmol/mg protein) Values m the upper panels were obtained from cortlcol membranes, values in the lower panels from cerebellar membranes Open bars represent control animals, shaded bars represent chronic alcohol Values are mean_+ SEM, n = 8--9 *indicates slgmficantly different from control, p < 0 02

m m e d (Table 3) C h r o m e alcohol ingestion did n o t a l t e r the c h o l e s t e r o l c o n t e n t o f S P M o r m y e l i n f r o m e i t h e r c o r t e x or c e r e b e l l u m In a d d i t i o n , c h r o n i c alcohol c o n s u m p t i o n did n o t c h a n g e the acyl c o m p o s i t i o n o f S P M o r myelin (not shown)

Membrane Phystcal Propernes Cholesterol Content o f Bram Membrane~ T h e c h o l e s t e r o l c o n t e n t o f S P M a n d m y e l i n was d e t e r -

T h e f l u o r e s c e n c e p r o b e m o l e c u l e D P H was u s e d to m e a s ure the o r d e r o f the h y d r o p h o b i c c o r e o f S P M F l u o r e s c e n c e

366

HARRIS ET AL TABLE 3

0

EFFECTS OF CHRONIC A L C O H O L CONSUMPTION ON T H E C H O L E S T E R O L CONTENT OF BRAIN MEMBRANES FROM M I N I A T U R E SWINE

p. Mol Cholesterol/mg Protein Membrane SPM Myehn

Brain Region Cortex Cerebellum Cortex Cerebellum

Control 0 0 0 0

52 49 69 73

+_ 0 _+ 0 + 0 + 0

04 03 04 05

Alcohol 0 0 0 0

52 51 59 66

_+ 0 03 _+ 0 05 -+ 0 04 _+ 0 05

Values are mean _+ SEM, n=8-9

"I13_

-005

,- -010 0

• Alcohol (3yrs)

' ~

N

'-E~ -015 0

n

<1 -020 p o l a r i z a t i o n values indicated that c h r o n i c alcohol e x p o s u r e did not affect t h e rigidity o f the m e m b r a n e s (control, 0 256-+0 005, alcohol. 0 259-+0 004) I n v t t l o e x p o s u r e to e t h a n o l d e c r e a s e d f l u o r e s c e n c e polarization, indicating a d i s o r d e r i n g of t h e m e m b r a n e (Fig 2) T h e p e r t u r b a t i o n p r o d u c e d m v m o b y e t h a n o l was the same for m e m b r a n e s f r o m b o t h c o n t r o l a n d alcohol swine T h e d a t a in Fig 2 repr e s e n t an a v e r a g e o f v a l u e s from male a n d female swine a n d f r o m c o r t e x a n d c e r e b e l l u m S e p a r a t e analysis o f e a c h sex a n d b r a i n region (by analysis of v a r i a n c e for r e p e a t e d m e a s ures) i n d i c a t e d no sigmficant effect o f c h r o n i c alcohol exposure

I

0

30

I

I

|

60 120 240 Ethanol, mM

I

480

FIG 2 Effects of nl ~ttto addmon of ethanol on the fluorescence polarization of DPH In synapIlc plasma membranes from S-I mmmture swine The abscissa represents the concentration (mMI of ethanol added, the ordinate represents the change m fluorescence polarization produced by each addition Open c~rcles represent membranes from control swine filled circles represent membranes from chromc alcohol swine Values represent mean+SEM n = 8-9

DISCUSSION

T h e s e results d e m o n s t r a t e an i n c r e a s e in G A B A r e c e p t o r binding in S-1 m i n i a t u r e swine b r a i n following l o n g - t e r m c o n s u m p t i o n o f e t h a n o l This c h a n g e was selective for G A B A r e c e p t o r s as binding o f h g a n d s to a d r e n e r g l c , c h o h n e r g l c , d o p a m l n e a n d b e n z o d l a z e p l n e r e c e p t o r s was not affected b y alcohol c o n s u m p t i o n T h e s e results are v e r y similar to t h o s e o b t a i n e d by T r a n e t a l [24] In studies of h u m a n alcoholics T h e s e i n v e s t i g a t o r s r e p o r t e d an i n c r e a s e in the n u m b e r o f G A B A binding sites In cortical tissue from male alcoholics, while c h o h n e r g l c fl-adrenerglc a n d b e n z o d l a z e p l n e binding w e r e not altered (A small d e c r e a s e in fl-adrenergic r e c e p t o r s was o b s e r v e d , but this was a t t n b u t e d to p o s t - m o r t e m c h a n g e s ) L i k e w i s e , we o b s e r v e d an a l c o h o l - i n d u c e d i n c r e a s e in the n u m b e r of G A B A binding sites in cortical tissue f r o m male swine, t h e i n c r e a s e d binding in c e r e b e l l a r tissue f r o m males a n d in cortical a n d cerebellar tissue f r o m females was due to e n h a n c e d binding affinity I n t e r p r e t a t i o n o f t h e s e results is c o m p l e x T h e G A B A r e c e p t o r - l o n o p h o r e c o n t a i n s several s u b u n l t s , including b e n z o d i a z e p l n e a n d b a r b i t u r a t e binding sites [16] A n inc r e a s e in G A B A r e c e p t o r binding might be e x p e c t e d to e n h a n c e G A B A action in wvo, b u t it is i m p o r t a n t to note t h a t it is n e c e s s a r y to r e m o v e e n d o g e n o u s lnh~bitors of G A B A r e c e p t o r binding p r i o r to the binding a s s a y If alcohol cons u m p t i o n affects the level o f t h e s e lnhlbltors, t h e n the binding o b s e r v e d in v z t r o m a y n o t reflect binding tn w v o In the range o f h g a n d c o n c e n t r a t i o n s used in this s t u d y a n d t e s t e d by T r a n e t a ! [24] t h e r e was e v i d e n c e o f only one p o p u l a t i o n o f binding sites, but it m a y b e possible to d e t e c t o t h e r sites using a w i d e r r a n g e of h g a n d c o n c e n t r a t i o n s [5] R e g a r d l e s s o f the i n t e r p r e t a t i o n , it is i m p o r t a n t to note t h a t the c h a n g e s p r o d u c e d by l o n g - t e r m alcohol c o n s u m p t i o n in m i n i a t u r e

swine are similar to t h o s e f o u n d in h u m a n alcoholics This is in c o n t r a s t to studies with rats w h e r e 21 days o f alcohol e x p o s u r e did not alter G A B A r e c e p t o r binding [17 23] L i k e w i s e , the selective effect on G A B A r e c e p t o r s is in contrast to the d e c r e a s e in fl-adrenerglc r e c e p t o r binding n o t e d after 60 d a y s o f alcohol c o n s u m p t i o n b y rats [I], the d e c r e a s e in b e n z o d l a z e p l n e binding r e p o r t e d in mice after 7 m o n t h s o f alcohol e x p o s u r e [7], a n d the i n c r e a s e in m u s c a r m l c c h o h n e r g i c r e c e p t o r binding f o u n d in mice a f t e r 7 d a y s o f alcohol c o n s u m p t i o n [22] In c o n t r a s t to the studies o f r o d e n t s a n d h u m a n s , a n i m a l s in o u r s t u d y were a n e s t h e t i z e d with a m i x t u r e of k e t a m l n e . a t r o p i n e a n d t h l a m y t a l It is unlikely t h a t t h e s e d r u g s altered t h e r e c e p t o r binding b e c a u s e m e m b r a n e s were w a s h e d ext e n s i v e l y prior to the m w t t o studies F u r t h e r m o r e addition o f t h e s e drugs directly to the a s s a y solution did not alter the binding o f ~ H - G A B A u n d e r the c o n d i t i o n s of the p r e s e n t s t u d y ([26] and u n p u b l i s h e d results) A t r o p i n e can c o m p e t e with Q N B for muscarlnlC binding sites b u t in view of the high d e n s i t y o f ~ H - Q N B sites o b s e r v e d in the p r e s e n t s t u d y it is d o u b t f u l t h a t a d m l m s t r a t l o n o f a t r o p i n e r e d u c e d r e c e p t o r binding C h a n g e s m m e m b r a n e lipid c o m p o s i t i o n a n d physical p r o p e r t i e s h a v e b e e n suggested as m e c h a n i s m s for t o l e r a n c e a n d d e p e n d e n c e [9] It is, h o w e v e r , unlikely t h a t c h a n g e s in m e m b r a n e h p l d s were r e s p o n s i b l e for the i n c r e a s e in G A B A r e c e p t o r b i n d i n g o b s e r v e d in the p r e s e n t s t u d y A n a l y s i s of the c h o l e s t e r o l c o n t e n t o f s y n a p t l c and myelin m e m b r a n e s did not d e t e c t a n y effect o f c h r o n i c alcohol c o n s u m p t i o n L i k e w i s e , c h r o n i c ingestion o f alcohol failed to c h a n g e the p h y s i c a l p r o p e r t i e s of s y n a p t i c m e m b r a n e s F l u o r e s c e n c e

NEUROCHEMISTRY OF LONG-TERM ETHANOL

367

polarization o f D P H indicated that n e i t h e r the intrinsic o r d e r o f the m e m b r a n e s nor their sensitivity to the d i s o r d e r i n g effect o f e t h a n o l w e r e altered by c h r o n i c ethanol e x p o s u r e T h e s e results are also in c o n t r a s t to the effects o f s h o r t - t e r m e t h a n o l t r e a t m e n t in r o d e n t s , w h e r e c h a n g e s in acyl saturation, c h o l e s t e r o l c o n t e n t and e t h a n o l - i n d u c e d m e m b r a n e fluidizatton h a v e b e e n o b s e r v e d [2, 3, 11, 13, 19] A l t h o u g h several o f the studies [11,13] o f r o d e n t s have u s e d a c r u d e s y n a p t o s o m a l p r e p a r a t i o n , it is p o s s i b l e that our p r e p a r a t i o n o f s y n a p t l c m e m b r a n e s was not sufficiently pure to allow d e t e c t i o n o f small c h a n g e s in SPM Our m e m b r a n e isolation p r o c e d u r e is well suited for r o d e n t brain tissue [6], but the purity o f the m e m b r a n e s o b t a i n e d f r o m swine brain has n o t b e e n c h a r a c t e r i z e d In addition, t h e r e may be s p e c i e s differe n c e s in the r e s p o n s e s o f brain m e m b r a n e s to alcohol treatm e n t , for e x a m p l e , the c h a n g e s in lipid acyl c o m p o s i t i o n m e a s u r e d by Sun and Sun [201 in guinea pig brain differ m a r k e d l y f r o m t h o s e r e p o r t e d by Llttleton et al [13] for m o u s e brain N e u r o c h e m i c a l r e s p o n s e s to ethanol may also d e p e n d u p o n the length and level o f e x p o s u r e Studies with

r o d e n t s h a v e u s e d t e c h n i q u e s w h i c h a c h i e v e high (200-400 mg/dl) b l o o d ethanol c o n c e n t r a t i o n s for a few d a y s In contrast, the t e c h n i q u e u s e d in the p r e s e n t s t u d y m a i n t a i n e d m o d e r a t e blood e t h a n o l (50-200 mg/dl) for t h r e e y e a r s Although t h e r e is e v i d e n c e for t o l e r a n c e and d e p e n d e n c e in swine c o n s u m i n g t h e s e quantities o f ethanol [4,25], it is p o s s i b l e that the d e g r e e o f t o l e r a n c e and d e p e n d e n c e is not as great as that a c h i e v e d during s h o r t - t e r m t r e a t m e n t o f rod e n t s P e r h a p s the p r e s e n t t e c h n i q u e s can only d e t e c t m e m b r a n e c h a n g e s w h i c h a c c o m p a n y a very high d e g r e e o f t o l e r a n c e and d e p e n d e n c e R e g a r d l e s s o f the r e a s o n s for the d i f f e r e n c e s b e t w e e n the p r e s e n t results and t h o s e o b t a i n e d In o t h e r studies, our findings e m p h a s i z e the n e e d for caution in extrapolating results from s h o r t - t e r m studies in r o d e n t s to long-term c o n s u m p t i o n in o t h e r s p e c i e s , including h u m a n alcoholics ACKNOWLEDGEMENTS Supported by funds from the Veterans Administration, the U S Brewers Association and PHS grant DA-02855

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