Effects of chronic ethanol administration on acetylcholinesterase activity in the somatosensory cortex and basal forebrain of the rat

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Brain Research, 627 (1993) 104-112 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 19370

Effects of chronic ethanol administration on acetylcholinesterase activity in the somatosensory cortex and basal forebrain of the rat Michael W. Miller a,h,,, Richard W. Rieck

c

"Research Service (151), Veterans Affairs Medical Center, Iowa City, IA 52246-2208, USA b Departments of Psychiatry and Pharmacology, University of Iowa College of Medicine, Iowa City, IA 52242-1057, USA c Department of Anatomy, Louisiana State University, School of Medicine, New Orleans, LA 70112-1393, USA (Accepted 8 June 1993)

Key words." Alcohol; Acetylcholine; Barrel; Diagonal band of Broca; Nucleus basalis; Dementia

A chronic diet of ethanol has detrimental effects on the cholinergic system in adult humans and rats. This study examined the effects of chronic exposure to dietary ethanol on the anatomical organization of true acetylcholinesterase (ACHE) active elements in rat cerebral cortex. We focused on the somatosensory cortex because of its highly organized chemical and cellular structure. Following 42 days of exposure to an ethanol diet (6.7% v/v), there were marked changes in the cortical plexus of AChE-positive fibers. The AChE-positive plexus in ethanol-treated rats was reduced in all cortical layers, in comparison to age-matched pair-fed control and chow-fed rats. The most marked reduction was evident in layers II/III, IV, and Via. Moreover, the density of AChE-positive cell bodies was significantly reduced in the cortices of ethanol-fed rats, particularly in the deep laminae. These alterations in the chemoarchitecture of somatosensory cortex occurred in the absence of changes in the cytoarchitectonic organization of neocortex. There was no detectable ethanol-induced change in the density of Cresyl violet-stained neurons either in the horizontal limb of the diagonal band of Broca or in the nucleus basalis. The density of AChE-positive neurons in the nucleus basalis, however, was significantly lower in ethanol-fed rats than in controls. Thus, it appears that a mere 6 weeks of ethanol exposure is sufficient to alter the cholinergic innervation of the cerebral cortex. These cortical alterations occur despite the lack of an ethanol-induced death of neurons in the basal forebrain. Such changes may contribute to the memory loss associated with alcohol dementia.

INTRODUCTION One of the sequelae of long-term chronic abuse of alcohol is Korsakoff's syndrome. This syndrome is characterized by three classical clinical signs: an irreversible loss of memory, confabulation, and disorientation 39. Chronic alcohol exposure also alters the cortical content of acetylcholine 3'6 and causes the degeneration of cholinergic afferents 4'1°'38. Behavioral and biochemical deficits similar to those in humans with Korsakoff's syndrome have been modeled in rats chronically exposed to ethanol 2'3'6'7'67. Chronic exposure to ethanol selectively affects the physiology and biochemistry of the cholinergic system 22'24'30'41'54"56'60. Interestingly, the behavioral deficits induced by ethanol exposure apparently can be reversed by transplanting acetylcholine-rich brain tis-

sue (derived from the fetal basal forebrain) into the cortices of rats exposed to ethanol 2'3. The cholinergic input to cerebral cortex has a strong facilitatory role in cortical function 13'14'42'64'68. Alterations in this input have marked effects on the ability of the cerebral cortex to process other inputs. Moreover, the cholinergic afferents to cortex are essential for maintaining the plasticity of the mature nervous system, i.e., the ability of neural tissue to adapt to insults 5,59. Taken together, the data described above support the hypothesis that chronic alcohol exposure causes the degeneration or dysfunction of cholinergic afferents within the cerebral cortex 4'1°'38. We examined the effects of chronic exposure to ethanol on the structure of the cortical cholinergic system. This study shows that chronic alcohol exposure in the adult animal has a

* Corresponding author. Department of Psychiatry, University of Iowa College of Medicine, 200 Hawkins Drive #2887 JPP, Iowa City, IA 52242-1057, USA. Fax: (1) (319) 353-3003.

105

106 significant effect on the structure of the cortical cholinergic plexus. We suggest that the basalocortical affere n t s c o n t r i b u t e a t l e a s t in p a r t t o t h e f u n c t i o n a l c h a n g e s caused by chronic alcohol exposure. MATERIALS AND METHODS The subjects were twelve 3-month-old Long-Evans rats; 6 male and 6 female, The diets of the rats were carefully monitored for 42 days. The feeding protocols were similar to those used in previous studies conducted in our laboratory 47'49. The rats were separated into 4 triads of sex- and weight-matched cohorts. One rat from each triad was fed ad libitum a high protein, nutritionally balanced liquid diet containing 6.7% (v/v) ethanol (Et; BioServ, Frenchtown, NJ). A second rat was fed an isocaloric, nutritionally matched liquid control diet (Ct; BioServ). The daily allotment of this diet was regulated so that the Ct-fed rat received the same amount of food as the matched Et-fed rat. The third rat in each triad was fed chow and water ad libitum. The weights of all rats were monitored every other day. Samples of the blood from the tail veins of the rats were taken on the 10th and 30th days of the regimen and the blood ethanol concentrations were determined using a Sigma diagnostics kit (UV no. 332). After being fed their particular diets for 42 days, all rats were sacrificed. Rats were given a lethal dose of pentobarbital and perfused transcardially with 25 ml of 0.10 M phosphate-buffered saline (pH 7.4) followed by 250-350 ml of 4.0% paraformaldehyde in 0.10 M phosphate buffer. The brains were removed from the skull and post-fixed for at least 2 days in buffered fixative with 30% sucrose. The brains were frozen and cut into a complete series of 52 /zm sections. Every other section in the series was stained with Cresyl violet and used for cytoarchitectural analyses. We studied the primary somatosensory cortex in the present study because of its highly defined structural and functional organization 14Jsa°'aS'sm7°'71. The identity of this cortex and of its constituent laminae was determined using established cytoarchitectonic criteria 1s'48'52. In specific, the quantitative analyses focused on the posteromedial barrel subfield, i.e., the area containing the representation of the mystacial vibrissae t5'2°'7°'73. The structure of segments of the basal forebrain was also examined. We directed these analyses to the horizontal limb of the diagonal band of Broca and the nucleus basalis, two segments of the Ch4 sector of the cholinergic system u'33'34'46. Neurons in the Ch4 sector project topographically to cortex. Changes in the density of AChE-positive neurons may result from decreases in the total cell number or in reductions of AChE content or activity. This issue was partially addressed by determining the

numbers of Cresyl violet-stained neurons in a 500 tzm wide column of the somatosensory cortex centered within the posteromedial barrel fields. Counts were made from each of 5 consecutive sections per rat. These data were used to estimate neuronal densities with a modification of the formulae described by Floderus m'61. Such formulae were used to correct for overestimation due to counting cell fragments and whole cells equally. A full description of these procedures is provided in previous publications 5°'sl. All statistical differences were assessed using t-tests. The alternate series of sections was processed histochemically for AChE activity 31's5. The first step in this staining procedure was to deplete the endogenous butyrylcholinesterase activity. Sections were incubated in 0.10 mM tetraisopropyl-pyrophosphoramide for 20 min at 37°C, Subsequently, the tissue was reacted with a solution (pH 5.85) of 0.50% acetylthiocholine iodide, 0.50 mM ferricyanide, 3.0 mM copper sulfate, 5.0 mM sodium citrate, and 63 mM sodium acetate for 2-3 hr at 37°C. The processed sections were dehydrated, cleared, and coverslipped. Sections from the Ch-, Ct-, and Et-fed rats were processed in parallel. This procedure minimized variations due to the vagaries of the histochemical reactions, and thereby strengthened the interpretation of the data. The densities of AChE-positive neurons in primary somatosensory cortex and in the basal forebrain were determined. For cortex, the numbers of intensely and weakly AChE-positive neurons in each layer within a 500 tzm width of the posteromedial barrel field. For the basal forebrain, the numbers of neurons in a 1.00 mm 2 area of the horizontal limb of the diagonal band and the nucleus basalis were counted. Counts and calculations were performed by procedures used to determine the number of Cresyl violet-stained neurons. RESULTS The weight of the rats did not change significantly while the Et was administered

f o r t h e 4 2 days. I n f a c t ,

t h e m e a n w e i g h t g a i n f o r t h e E t - f e d r a t s w a s 2.1 + 1 . 5 % . This was

similar

to the

1.3 + 2 . 1 %

and

3.2 + 2 . 7 %

weight gains for the Ct- and Ch-fed rats, respectively. The mean

blood ethanol concentration

r a t s w a s 142 + 13 m g / d l

of the Et-fed

on the 10th day of the regi-

m e n o f t h e E t d i e t a n d 149 + 11 m g / d l

on the 30th

day. Chronic ethanol

exposure had no apparent

effect

on the cytoarchitectonic organization of somatosensory c o r t e x (Fig. 1). N o t e t h a t n o q u a l i t a t i v e o r q u a n t i t a t i v e differences between the sexes were detected. Thus, the

600

d a t a w e r e p o o l e d f o r t h i s a n a l y s i s a n d as w e l l as f o r

mira Chow

other Ethon01

a n a l y s e s in t h i s r e p o r t . T h e

primary

450

somatosensory

cortex was similar in Ch-fed

( 1 7 8 8 + 38 / z m ) , C t - f e d ( 1 7 7 4 + 46 fzm), a n d

I

(1860+50 300

/xm)

rats.

Moreover,

the

laminae

Et-fed were

e q u a l l y d i s t i n c t in E t - f e d a n d c o n t r o l rats. T h e t h i c k -

E--

ness of each layer of somatosensory 150

total thickness of

[ I

cortex was unaf-

f e c t e d b y t h e e t h a n o l e x p o s u r e (Fig. 2). C h a n g e s in t h e A C h E - p l e x u s

were evident through-

o u t s o m a t o s e n s o r y c o r t e x ( F i g . 3). T h e s e c h a n g e s w e r e II/111

IV

v

VIo

VIb

LAYER Fig. 2. Effect of ethanol on laminar thickness. The radial depth of the cortical layers in primary somatosensory cortex of Ch-, Ct-, and Et-fed rats are depicted. The broad bars represent the means and the T-bars signify the S.D.

particularly

marked

within

the posteromedial

barrel

field of primary somatosensory cortex and in secondary somatosensory cortex. On the other hand, the area that exhibited the least damage

w a s l o c a t e d d o r s a l l y , i.e.,

the region which corresponded

to the hindlimb repre-

107

Fig. 3. Organization of AChE-positive plexus in somatosensory cortex. In contrast to the cytoarchitecture which was not affected by consumption of ethanol for 6 weeks, chronic ethanol exposure had qualitative effects on the distribution of AChE-positive fibers. Bars = 100 ~m.

108 sentation where somatosensory and motor cortices overlap. No differences in the pattern of A C h E activity were evident between the Ch- and Ct-fed rats. The distribution of the AChE-positive axons was markedly different in all layers of the Et-fed and control animals (Fig. 3). The density of the AChE-positive plexus was reduced in all cortical layers, but layers I I / I I I , IV, and V i a exhibited the greatest reduction. Control rats had a dense supragranular plexus in which

the AChE-positive plexus consisted of radial, tangential and highly branched fibers. In contrast, the AChE-positive plexus of Et-fed rats was composed of only a few radially arranged fibers. These radial fibers often were clustered in bundles in deep layer I I / I I I and IV (Fig. 4). A less marked reduction in the density of the A C h E plexus was apparent within layer VI of the somatosensory cortex. As in the infragranular layers, the changes in layer V i a included a reduction in

Fig. 4. Radial arrays of AChE-positive fibers in layers II/III and IV. Please note the staining of the neurons. One neuron is intensely AChE-positive (solid arrow) and many of the others are weaklyAChE-positive(open arrows). Bars = 100 ~m.

109 4.0

TABLE I Chow

£~

3.0

Density of AChE-positive neurons m the basal forebrain Each value is expressed in terms of n u m b e r of neurons per 1.00 m m 2.

ono

~.° ~

Site

Stain

Ch-fed

Ct-fed

Et-fed

Mean

S.D.

Mean

S.D.

Mean

S.D.

1.(1

Diagonal band

AChE Nissl %

72.8 430.6 16.9

17.9 50.1 2.5

76.8 433.0 17.7

18.6 52.1 2.1

68.6 399.0 17.2

21.0 45.4 2.2

o

Nucleus basalis

AChE Nissl %

41.2 130.1 31.7

8.9 17.9 5.1

39.5 * 127.3 31.0 *

7.5 17.6 4.8

27.4 123.2 22.2

4.6 21.8 4.2

N I

II/111

IV

V

VIcI

VIb

LAYER Fig. 5. Effect of ethanol on the density of intensely stained neurons. Notations as in Fig. 2.

the number of tangential and oblique fibers. These Et-induced changes in the AChE plexus in layers I I / III, IV and Via gave the cortex of the Et-fed rat a more radially organized appearance. A small number of intensely AChE-positive cells were identified. The overall density of these neurons in primary somatosensory cortex was 7.3 + 3.0 n e u r o n s / mm 2 for Ch-fed rats, 6.8 + 1.8 n e u r o n s / m m 2 for Ct-fed rats, and 7.8 + 2.5 n e u r o n s / m m 2 for Et-fed rats. Not only was the total number similar in all 3 treatment groups, but the laminar distribution of these intenselystained cells was not significantly different in the Et-fed rats and controls (Fig. 5). In contrast, a large number of neurons exhibited weak AChE activity. The density of these neurons in Ch- and Ct-fed rats was 183 + 29 and 188 + 35 n e u r o n s / m m 2, respectively. Primary somatosensory cortex of Et-fed rats contained significantly ( P < 0.01) fewer weakly stained neurons (117 + 30 n e u r o n s / m m 2 ) . Most of this difference derived

60 m Chow ~E~ Control

U~

z o

Ethaoo~ 45

~.2

N Z

15

0 I

ii i ll/Ul

IV

v

Via

VIb

LAYER

Fig. 6. Effect of ethanol on the density of weakly stained neurons. Notations as in Fig. 2. * Statistically significant differences ( P < 0.01) between the data from the Ct- and Et-fed rats.

* Statistically significant difference: P < 0.05.

from a greater than halving of the density of weakly AChE-positive neurons in layers IV, V, and Via of the Et-fed rats (Fig. 6). The densities of intensely AChE-positive neurons in the horizontal limb of the diagonal band and the nucleus basalis were determined. Ethanol had no effect upon the density in the diagonal band, but it did significantly ( P < 0.05) reduce the density of ACHEpositive neurons in the nucleus basalis (Table I). In addition, the density of Cresyl violet-stained neurons in the nucleus basalis in Et-fed rats was not significantly different from that counted in Ct-fed rats. The net result was a significant ( P < 0.05) reduction in the ratio of neurons that were AChE-positive to the total population. DISCUSSION Chronic exposure of adult rats to ethanol has a dramatic effect on the anatomical organization of the AChE-positive system within the somatosensory cortex. The density and distribution of the AChE-plexus is reduced in all cortical laminae, and most noticeably in layers I I / I I I and IV. Moreover, the density of weakly AChE-positive neurons (i.e., putative cholinoceptive neurons 36) was also reduced by ethanol exposure. Evidence from various studies shows a marked alteration in other cholinergic markers within the cerebral cortex following chronic administration of ethanol 22'24'30'41'44'54'56'60. These data indicate that the change in the AChE-positive plexus in the present study is due to ethanol-induced alterations of the cholinergic input to the cerebral cortex. The AChE-positive plexus in the rat is derived from 2 sources. One source of cholinergic cortical axons is the intrinsic network of cortical local circuit neurons 9'21'23. These neurons contain choline acetyltransferase (CHAT), the enzyme which catalyzes the produc-

110 tion of acetylcholine. Although the ChAT-immunoreactive neurons do not appear to co-localize with the intense AChE reaction product 37, the number of intensely AChE-positive neurons is indicative of the number of cholinergic neurons. The density of the cell bodies of the intensely AChE-positive neurons was not affected by ethanol exposure. Interestingly, these local circuit neurons co-localize G A B A 21'29'45. Chronic exposure to ethanol does not reduce the GABA content of cortex 62'63'66, hence, ethanol may not affect the network of local circuit neurons. Most cortical AChE-positive axons arise from the large cholinergic n e u r o n s in the basal foreb r a i n 8'16'25'26'32-35'40'46'57'68'74. Specifically, the neurons in the Ch4 sector of the basal forebrain (the horizontal limb of the diagonal band of Broca and the nucleus basalis) project topographically to somatosensory cortex. The neurons in the basal forebrain are considered to be cholinergic for besides exhibiting intense AChE activity, they are also immunoreactive with antibodies against ChAT 36. The density of intensely AChE-positive neurons in the horizontal limb of the diagonal band is not affected by exposure to ethanol. The density of AChE-positive neurons in the nucleus basalis, however, is reduced by ethanol exposure. The differential effect on components of the Ch4 sector may relate to the strength of the cortical projection; the nucleus basalis projects to dorsal cortex more strongly than does the horizontal limb of the diagonal band 8'33'34'4°'46. Note that the loss of AChE-labeling in the nucleus basalis apparently occurs in the absence of a loss of neurons. This implies that 6 weeks of ethanol exposure produces a reduction in AChE content or activity in the neurons of the nucleus basalis. Thus, the reduction in the density of the AChE-positive plexus in cortex suggests that a relatively short (6 week) exposure to ethanoL-leads to the degeneration of cortical axons (or possibly the loss of axonal AChE activity) without killing basal forebrain neurons. The loss of the AChE-positive axons is most noticeable in the supragranular cortex. In contrast, the greatest reduction in the number of weakly AChE-positive cell bodies is most evident in deep cortex. This incongruity likely results from changes in the structure of cortical neurons. As the cholinergic axons enter cortex, they pass through the white matter and the deep layers of cortex before reaching supragranular cortex 4°'58. Therefore, the ethanol-induced alterations of the AChE-positive axons may begin by the degeneration of the distal ends of the axons in the supragranular layers. Most AChE-positive axons and ChAT-immunoreactive axons from the basal forebrain form asymmetric syn-

apses L2'23 and the most common post-synaptic elements in such cortical synapses are dendritic spines 17'73. Chronic ethanol exposure reduces the density of dendritic spines not only in the neocortex, but also in the cerebellum and hippocampus 27'18'43'65. Dendritic spines in neocortex are characteristic of pyramidal neurons. Many of the spines in the neuropil of layers I I / I I I and IV are on the apical dendritic trees of pyramidal neurons with cell bodies in layers IV-VIa. Thus, the depletion of the AChE plexus in layers I I / I I I and IV would be expected to impact on cholinoceptive neurons in deep cortex. The degeneration of the AChE-positive axons presages the death of large neurons in the basal forebrain. The sequence of retrograde (basalopetal) degeneration apparently underlies the time-dependent effects of ethanol exposure on memory loss. An 8 week exposure to ethanol leads to memory deficits that are reversible following a withdrawal period, however, the memory deficits are not reversible following a 28 week exposure 1,2. The permanence of this memory loss may relate to the degeneration of the cholinergic system. That is, there is a conspicuous lack of death of projection neurons in the basal forebrain following only 6 weeks of ethanol exposure, whereas longer ethanol exposure (e.g., 14 weeks) leads to the permanent loss of basal forebrain neurons 4. Thus, so long as the cell bodies of the cholinergic basal forebrain neurons survive, the memory deficits can be overcome. Cholinergic systems are crucial for maintaining neuronal plasticity in the mature nervous system 5'28'59'69. Lesion of these afferents leads to permanent physiological and anatomical changes in cerebral cortex. Chronic exposure to ethanol leads to a loss or impairment of cortical plasticity and reactive synaptogenesis 53'72. Intriguing results from Arendt and colleagues 1'2 show that ethanol-induced learning deficits can be ameliorated by transplanting cholinergic-rich fetal tissue (from the basal forebrain) into the cortex of mature rats chronically exposed to ethanol. The anatomical alterations resulting from this manipulation are still being worked out. Nevertheless, the present and published data strongly support the conclusion that the ethanolinduced loss of plasticity results from the degeneration in the cholinergic cortical afferents arising in the basal forebrain. Acknowledgements. This research was supported by AA 06916, AA 07568, and DE 07734.

REFERENCES 1 Arendt, T., Allen, Y., Sinden, J., Schugens, M.M., Marchbanks, R.M., Lantos, P.L. and Gray, J.A., Cholinergic-richbrain transplants reverse alcohol-induced memory deficits, Nature, 332 (1988) 448-450.

111 2 Arendt, T., Allen, Y., Marchbanks, R.M., Schugens, M.M., Sinden, J., Lantos, P.L. and Gray, J.A., The cholinergic system and memory in the rat: effects of chronic ethanol, embryonic basal forebrain transplants and excitotoxic lesions of the cholinergic basal forebrain system, Neuroscience, 33 (1989) 435-462. 3 Arendt, T., Bigl, V., Arendt, A. and Tennstedt, A., Loss of neurons in the nucleus basalis of Meynert in Alzheimer's disease, paralysis agitans, and Korsakoff's Disease, Acta NeuropathoL, 61 (1983) 101-108. 4 Arendt, T., Henning, D., Gray, J.A. and Marchbanks, R., Loss of neurons in the rat basal forebrain cholinergic projection system after prolonged intake of ethanol, Brain Res. Bull., 21 (1988) 563-569. 5 Bear, M.F. and Singer, W., Modulation of visual cortical plasticity by acetylcholine and noradrenaline, Nature, 320 (1986) 172-176. 6 Beracochea, D., Durkin, T.P. and Jaffard, R., On the involvement of the central cholinergic system in memory deficits induced by long-term ethanol consumption in mice, Pharmacol. Biochem. Behav., 24 (1986) 519-524. 7 Beracochea, D. and Jaffard, R., Memory deficits subsequent to chronic consumption of alcohol in mice- an analysis based on spontaneous alternation behavior, Behav. Brain Res., 15 (1985) 15-25. 8 Bigl, V., Woolf, N.J. and Butcher, L.L., Cholinergic projections from the basal forebrain to frontal, parietal, temporal, occipital, and cingulate cortices: a combined fluorescent tracer and acetylcholinesterase analysis, Brain Res. Bull., 8 (1982) 727-749. 9 Brady, D.R. and Vaughn, J.E., A comparison of the localization of choline acetyltransferase and glutamate decarboxylase im, munoreactivity in rat cerebral cortex, Neuroscience, 24 (1988) 1009-1026. 10 Butters, N. and Cermack, L.S., Alcoholic Korsakoff's Syndrome:" An Information-Processing Approach to Amnesia, Academic Press, New York, 1980. 11 Carey, R.G. and Rieck, R.W., Topographic projections to the visual cortex from the basal forebrain in the rat, Brain Res., 424 (1987) 205-215. 12 Carson, K.A. and Burd, G.D., Localization of acetylcholinesterase in the main and accessory olfactory bulbs of the mouse by light and electron microscopic histochemistry, J. Comp. Neurol., 191 (1980) 353-371. 13 Donoghue, J.P., Cholinergic modulation of sensory responses in cerebral cortex, Natl. Inst. Drug. Abuse. Res. Monogr. Ser., 78 (1987) 169-188. 14 Donoghue, J.P., Kerman, K.L. and Ebner, F.F., Evidence for two organizational plans within the somatic sensory-motor cortex of the rat, J. Comp. Neurol., 183 (1979) 647-664. 15 Donoghue, J.P. and Wise, S.P., The motor cortex of the rat: Cytoarchitecture and microstimulation mapping, J. Comp. Neurol., 212 (1982) 76-78. 16 Eckenstein, F. and Thoenen, H., Cholinergic neurons in the rat cerebral cortex demonstrated by immunohistochemical localization of choline acetyltransferase, Neurosci. Lett., 36 (1983) 211215. 17 Feldman, M.L., Morphology of the neocortical pyramidal neuron. In A. Peters and E.G. Jones (Eds.), Cerebral Cortex, Vol. L Cellular Components of the Cerebral Cortex, Plenum, New York, 1984. 18 Ferrer, I., Fabregues, I., Rairiz, J. and Galofre, E., Decreased numbers of dendritic spines on cortical pyramidal neurons in human chronic alcoholism, Neurosci. Lett., 69 (1986) 115-119. 19 Floderus, S., Ultersuchungen uber den bau der menschlichen hypophyse mit besonderer beruck sichtigung der qualitativen mikromorphologischen verhaltnisse, Acta Pathol. Microbiol. Scand., 53, Suppl. (1944) 1-276. 20 Hall, R.D. and Lindhoim, E.P., Organization of motor and sensory neocortex in the albino rat, Brain Res., 66 (1974) 23-28. 21 Hallanger, A.E., Wainer, B.H. and Rye, D.B., Co-localization of y-aminobutyric acid and acetylcholinesterase in rodent cortical neurons, Neuroscience, 19 (1986) 763-769. 22 Holman, R.B., Ethanol and cerebral neuroregulators, Biochem. Soc. Trans., 11 (1983) 62-63.

23 Houser, C.R., Crawford, G.D., Salvaterra, P.M. and Vaughn, J.E., Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses, J. Comp. Neurol., 234 (1985) 17-34. 24 Hunt, W.A. and Majchrowicz, E., Studies of neurotransmitter interactions after acute and chronic ethanol administration, Pharmacol. Biochem. Behav., 18 (1983) 371-374. 25 Johnston, M.V., McKJnney, M. and Coyle, J.T., Neocortical cholinergic innervation: a description of extrinsic and intrinsic components in the rat, Exp. Brain Res., 43 (1981) 159-172. 26 Johnston, M.V., Young, A.C. and Coyle, J.T., Laminar distribution of cholinergic markers in neocortex: effects of lesions, J. Neurosci. Res., 6 (1981) 597-607. 27 Jones, D.G. and Colangelo, W., Ultrastructural investigation into the influence of ethanol on synaptic maturation in rat neocortex. I. Qualitative assessment, Dev. Neurosci., 7 (1985) 94-106. 28 Juliano, S.L., Ma, W. and Eslin, D., Cholinergic depletion prevents expansion of topographic maps in somatosensory cortex, Proc. Natl. Acad. Sci. USA, 88 (1991) 780-784. 29 Kleinrok, A., Tomaszewski, A., Zaczkiewicz, A., Kedziora, E., Gorny, D. and Billewicz-Stankiewicz, J., Acetylcholine metabolism in rat cerebral cortex after joint and separate administration of y-aminobutyric acid and bicuculline, Acta Physiol. Pol., 31 (1980) 225-231. 30 Kochhar, A. and Erickson, C.K., Central cholinergic correlates of low dose ethanol-induced locomotor stimulation, Alcoholism: Clin. Exp. Res., 10 (1986) 595-601. 31 Koelle, G.B., The histochemical localization of cholinesterase in the central nervous system of the rat, J. Comp. Neurol., 100 (1954) 211-235. 32 Kristt, D.A., McGowan, R.A., Jr., Martin-MacKinnon, N. and Solomon, J., Basal forebrain innervation of rodent neocortex: studies using acetylcholinesterase histochemistry, Golgi and lesion strategies, Brain Res., 337 (1985) 19-39. 33 Lamour, Y., Dutar, P. and Jobert, A., Topographic organization of basal forebrain neurons projecting to the rat cerebral cortex, Neurosci. Lett., 34 (1982) 117-122. 34 Lamour, Y., Dutar, P. and Jobert, A., Cortical projections of the nucleus of the diagonal band of Broca and of the substantia innominata in the rat: an anatomical study using the anterograde transport of a conjugate of wheat germ aglutinin and horseradish peroxidase, Neuroscience, 12 (1984) 395-408. 35 Lehmann, J., Nagy, J.I., Atmadia, S. and Fibiger, H.C., The nucleus basalis magnocellularis: the origin of a cholinergic projection to the neocortex of the rat, Neuroscience, 5 (1980) 1161-1174. 36 Levey, A.I., Wainer, B.H., Mufson, E.J. and Mesulam, M.-M., Co-localization of acetylcholinesterase and choline acetyltransferase in the rat cerebrum, Neuroscience, 9 (1983) 9-22. 37 Levey, A.I., Wainer, B.H., Rye, D.B., Mufson, E.J. and Mesulam, M.-M., Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterasepositive neurons, Neuroscience, 13 (1984) 341-353. 38 Lishman, W.A., Alcoholic dementia - a hypothesis, Lancet, 1 (1986) 1184-1185. 39 Lishman, W.A., Alcohol and the brain, Br. J. Psychiatr., 156 (1990) 635-644. 40 Luiten, P.G., Spencer, D.G., Jr., Traber, J. and Gaykema, R.P., The pattern of cortical projections from the intermediate parts of the magnocellular nucleus basalis in the rat demonstrated by tracing with Phaseolus vulgaris-leucoagglutinin, Neurosci. Lett., 57 (1985) 137-142. 41 Mancillas, J.R., Siggins, G.R. and Bloom, F.E., Systemic ethanol: selective enhancement of responses to acetylcholine and somatostatin in hippocampus, Science, 231 (1986) 161-163. 42 McCormick, D.A. and Prince, D.A., Mechanisms of action of acetylcholine in the guinea-pig cerebral cortex in vitro, J. Physiol., 375 (1985) 169-194. 43 McMullen, P.A., Saint-Cyr, J.A. and Carlen, P.L., Morphological alterations in the rat CA1 hippocampal pyramidal cell dendrites resulting from chronic ethanol consumption and withdrawal, J. Comp. Neurol., 225 (1984) 111-118.

112 44 Melgaard, B., The neurotoxicity of ethanol, Acta Neurol. Scand., 67 (1983) 131-142. 45 Mesulam, M.M. and Dichter, M., Concurrent acetylcholinesterase staining and y-aminobutyric acid uptake of cortical neurons in culture, J. Histochem. Cytochem., 29 (1981) 306308. 46 Mesulam, M.M., Mufson, E.J., Wainer, B.H. and Levey, A.I., Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neuroscience, 10 (1983) 1185-1201. 47 Miller, M.W., Fetal alcohol effects on the generation and migration of cerebral cortical neurons, Science, 233 (1986) 1308-1311. 48 Miller, M.W., Effect of prenatal exposure to ethanol on the distribution and time of origin of corticospinal neurons in the rat, J. Comp. Neurol., 257 (1987) 372-382. 49 Miller, M.W., Effect of prenatal exposure to ethanol on the development of cerebral cortex: I. Neuronal generation, Alcoholism: Clin. Exp. Res., 12 (1988) 440-449. 50 Miller, M.W. and Muller, S.J., Structure and histogenesis of the principal sensory nucleus of the trigeminal nerve: effects of prenatal exposure to ethanol, J. Comp. Neurol., 282 (1989) 570580. 51 Miller, M.W. and Potempa, G., Numbers of neurons and glia in mature rat somatosensory cortex: effects of prenatal exposure to ethanol, J. Cornp. Neurol., 293 (1990) 92-102. 52 Miller, M. and Vogt, B., Direct connections of rat visual cortex with sensory, motor and association cortices, J. Comp. Neurol., 226 (1984) t84-204. 53 Orona, E., Hunter, B.E. and Walker, D.W., Ethanol exposure following unilateral entorhinal deafferentation alters synaptic reorganization in the rat dentate gyrus: a quantitative analysis of acetylcholinesterase histochemistry, Exp. Neurol., 101 (1988) 114131. 54 Phillis, J.W., Jiang, Z.G. and Chelack, B.J., Effects of ethanol on acetylcholine and adenosine efflux from the in vivo rat cerebral cortex, J. Pharrn. Pharmacol., 32 (1980) 871-872. 55 Rieck, R. and Carey, R.G., Evidence for a laminar organization of basal forebrain afferents to the visual cortex, Brain Res., 297 (1984) 374-380. 56 Rivera-Calimlim, L., Hartley, D. and Osterhout, D., Effects of ethanol and pantothenic acid on brain acetylcholine synthesis, Br. J. Pharmacol., 95 (1988) 77-82. 57 Robertson, R.T., Hanes, M.A. and Yu, J., Investigations of the origins of acetylcholinesterase activity in developing rat visual cortex, Dev. Brain Res., 41 (1988) 1-23. 58 Saper, C.B., Organization of cerebral cortical afferent systems in the rat. II. Magnocellular basal nucleus, J. Comp. Neurol., 222 (1984) 313-342. 59 Shaw, C., Needler, M.E. and Cynader, M., Ontogenesis of muscimol binding sites in cat visual cortex, Brain Res. Bull., 13 (1984) 331-334.

60 Siggins, G.R., Bloom, F.E., French, E.D., Madamba, S.G., Mancillas, J., Pittman, Q.J. and Rogers, J., Electrophysiology of ethanol on central neurons, Ann. N.Y. Acad. Sci., 492 (1987) 350-366. 61 Smolen, A.J., Wright, L.L. and Cunningham, T.J., Neuron numbers in the superior cervical sympathetic ganglion of the rat: a critical comparison of methods for cell counting, J. Neurocytol., 12 (1983) 739-750. 62 Sut.ton, I. and Simmonds, M.A., Effects of acute and chronic ethanol on the gamma-butyric acid in the actions of ethanol, Eur. J. Pharmacol., 89 (1983) 53-62. 63 Sytinsky, I.A., Guzikov, B.M. and Gomanko, M.V., The gammabutyric acid (GABA) system in brain during acute and chronic intoxication, J. Neurochem., 25 (1975) 43-48. 64 Strejckova, A., Mares, P. and Raevsky, V., Changes of activity of cortical neurons induced by acetylcholine in adult and young rats, Exp. Neurol., 98 (1987) 555-562. 65 Tavares, M.A., Paula-Barbosa, M.M. and Gray, E.G., Dendritic spine plasticity and chronic alcoholism in rats, Neurosci. Lett., 42 (1983) 235-238. 66 Volicer, L., GABA levels and receptor binding afetr acute and chronic ethanol administration, Brain Res. BulL, 5 (1980) 809-813. 67 Walker, D.W., Hunter, B.E. and Abraham, W.C., Neuroanatomical and functional deficits subsequent to chronic ethanol administration in animals, Alcoholism: Clin. Exp. Res., 5 (1981) 267-282. 68 Watson, M., Vickroy, T.W., Fibiger, H.C., Roeske, W.R., and Yamamura, H.I., Effects of bilateral ibotenate-induced lesions of the nucleus basalis magnocellularis upon selective cholinergic biochemical markers in the rat anterior cerebral cortex, Brain Res., 346 (1985) 387-391. 69 Webster, H., Hanisch, U.K., Dykes, R.W. and Biesold, D., Basal forebrain lesions with or without reserpine injection inhibit cortical reorganization in rat hindpaw primary somatosensory cortex following sciatic nerve section, Somatosen. Mot. Res., 8 (1991) 327-346. 70 Welker, C. and Sinha, M.M., Somatotopic organization of SmlI cerebral neocortex in albino rat, Brain Res., 37 (1972) 132-136. 71 Welker, C. and Woolsey, T.A., Structure of layer IV in the somatosensory neocortex of the rat: description and comparison with the mouse, J. Comp. Neurol., 158 (1974) 437-454. 72 West, J.R., Lind, M.S., Demuth, R.M., Parker, E.S., Alkana, R.L., Cassell, M. and Black, Jr., A.C., Lesion-induced sprouting in the rat dentate gyrus is inhibited by repeated ethanol administration, Science, 218 (1982) 808-810. 73 White, E.L., Cortical Circuits: Synaptic Organization of the Cerebral Cortex - Structure, Function and Theory, Birkhauser, Boston, 1989. 74 Woolf, N.J., Cholinergic systems in mammalian brain and spinal cord, Prog. Neurobiol., 37 (1991) 475-524.