Augmentation of short-term plasticity in CA1 of rat hippocampus after chronic ethanol treatment

Brain Research, 221 (1981) 271-287

271

Elsevier/North-Holland Biomedical Press

A U G M E N T A T I O N OF S H O R T - T E R M P L A S T I C I T Y I N CA1 OF R A T HIPPOCAMPUS AFTER CHRONIC ETHANOL TREATMENT

W. C. ABRAHAM*, B. E. HUNTER**, S. F. ZORNETZER and D. W. WALKER

Veterans Administration Medical Center and Department of Neuroscience, University of Florida, College of Medicine, Gainesville, Fla. 32610 (U.S.A.)

(Accepted February 5th, 1981) Key words: hippocampus - - chronic ethanol - - plasticity - - recurrent inhibition

SUMMARY The neurotoxic effects of chronic ethanol exposure were investigated in rat hippocampus by electrophysiological analysis of the Schaffer collateral-commissural input to stratum radiatum of CA1. Experimental animals were fed an ethanolcontaining liquid diet for 20 weeks but were withdrawn from the special diet at least eight weeks prior to acute electrophysiological recordings. Ethanol treatment had no effect on input-output relationships for either the population EPSP or the population spike (PS). During paired-pulse stimulation, the ethanol group exhibited a greater facilitation of the test pulse PS relative to the control group, although potentiation of the EPSP was unchanged. In addition, the ethanol group showed a trend toward greater facilitation of the PS during 5 and 10 Hz tetani. No differences between groups were observed in the magnitude or duration of the longterm potentiation produced by 5, 10 or 100 Hz stimulus trains. Ethanol treatment did significantly reduce the transient spike depression after low frequency stimulation. This pattern of results is similar to that found for treatments which reduce hippocampal recurrent inhibition. Thus, chronic ethanol treatment may produce a lasting disruption of intrinsic inhibitory neurotransmission in the rat hippocampus.

INTRODUCTION Chronic alcoholism is often associated with pathological deterioration in many organ systems including the central nervous system (CNS). The most severe CNS * Portions of this study were submitted by W.C.A. in partial fulfillment of the Ph.D. degree at the University of Florida. ** To whom all correspondence should be addressed at: Department of Neuroscience, College of Medicine, JHMHC, Box J-244, University of Florida, Gainesville, Florida 32610 (U.S.A.).

272 disorder, Wernicke-Korsakoffsyndrome,is associated with wide-spread neuropathology and a variety of neurological symptoms, the hallmark of which is a debilitating and permanent anterograde amnesia in the absence of a general cognitive declinela, 48, 52. This pathological deterioration has been attributed to several co-existing conditions, especially malnutrition and thiamine deficiency~L However, it seems likely that ethanol exerts direct neurotoxic effects in the CNS, since brain damage and neuropsychological deterioration have been observed in chronic alcoholic patients with no history of malnutrition, head trauma or exposure to other toxic agents 19,24,44,51. Moreover, animal studies have shown that chronic ethanol exposure in the presence of a nutritionally adequate diet results in: (1) retarded acquisition of a variety of behavioral tasks in rodents including shuttlebox avoidance22,45,55, DRL 15,3e,50, go-no-go discrimination57, and complex maze acquisitiong,20; (2) a 15-20 ~ loss of granule cells in the dentate gyrus and pyramidal cells in CA1 and CA2-4 in rat hippocampus54; and (3) spine loss and dendritic atrophy in granule cells and for the basilar dendrites of CA1 pyramidal cells of the rodent hippocampus 4°. On the basis of these morphological results, physiological studies of hippocampal synaptic connections of rats chronically exposed to ethanol might reveal impaired function commensurate with the neuropathology. However, other outcomes are possible, particularly since deafferentation within the hippocampus leads to considerable reorganization of existing connections23,34,35. The present study examined electrophysiologically the persistent effects of chronic ethanol consumption on synaptic function in stratum radiatum of CA1 in the rat hippocampus. Electrical stimulation within stratum radiatum simultaneously activates Schaffer collateral (SCH) and commissural (COM) fibers (from ipsilateral and contralateral CA3, respectively), which course together and terminate on the apical dendrites of pyramidal cells within stratum radiatum of CA1. Repetitive stimulation of SCH/COM afferents to CA1 evokes large extracellular field potentials which are highly labile, exhibiting both short- and long-lasting changes in response to relatively brief tetanic stimulP, 14,16,4a. We have examined such plasticity in order to assess synaptic function within the hippocampus following chronic ethanol treatment. METHODS Male Long-Evans hooded rats (200-250 g) were matched by weight and assigned to the following treatment groups: (1) an experimental group (Group A) which received an ethanol-containing liquid diet; (2) a control group (Group S) which was pair-fed a sucrose-containing liquid diet; and (3) a second control group (Group LC) which received pelleted laboratory chow and water ad libitum. The preparation, contents, and nutritional adequacy of the liquid diet procedure have been documented previously~5. Briefly, the ethanol liquid diet has 35-39~ ethanol-derived calories (8.1-9.4 ~ v/v, ethanol) and was prepared by mixing an ethanol stock solution (63.3 ~, v/v) with Sustacal (Mead-Johnson Co.). The control diet was identical except sucrose was isocalorically substituted for ethanol. Both diets were fortified with Vitamin Diet Fortification Mixture, 0.3 g/100 ml, and Salt mixture XIV, 0.5 g/100 ml (Nutritional

273 Biochemicals Co.). The liquid diets were prepared and consumption measured daily. The liquid diets were administered for a period of 20 weeks. The percentage of ethanol- or sucrose-derived calories was increased by 1 ~o every 4 weeks (35-39 ~o). At the end of this 20 week treatment period, all rats received laboratory chow and water ad libitum.

Electrophysiological methods Electrophysiological recordings were obtained within a 10 week period, commencing 8 weeks after discontinuation of liquid diet treatment. This extended abstinence period was used to eliminate or at least minimize the residual effects of ethanol intoxication or acute alcohol withdrawal. Moreover, both the duration of ethanol treatment (20 weeks) and ethanol abstinence (8 weeks) were chosen to facilitate comparisons with previous morphological studies (cf. ref. 54). Rats from each group were coded in order to prevent experimental bias in final electrode placement and data collection. Recordings were made in urethane-anesthetized preparations (1.5 g/kg i.p.), with supplemental doses of urethane administered as required. The rat was placed in a stereotaxic instrument and the skull exposed and leveled. A heating pad was used to maintain rectal temperature at 37 4- 0.5 °C. Stimulating and recording electrodes were remotely controlled via hydraulic microdrives and placed in the brain through small burr holes in the skull. The coordinates for the stimulating and recording electrodes were (relative to bregma): 3.2 mm posterior and 2.8 mm lateral, and 4.2 mm posterior and 3.0 mm lateral, respectively. Concentric bipolar electrodes were used for electrical stimulation. Glass micropipettes filled with 4 M NaCI (1-2 #m tip diameter, 1-3 Mf~ at 1 kHz) were used for extracellular field potential recordings. Field potentials were amplified, filtered at 0.3-10 kHz, and analyzed on-line or recorded on magnetic tape for later analysis. Potentials were in some cases averaged with a Dagan 4800 Signal Averager. All evoked responses were displayed on a Gould X-Y plotter for quantitative analysis. Electrical stimulation was delivered by a Nuclear Chicago constant current stimulator and consisted of monophasic square wave pulses (0.1 ms duration). High frequency stimulation employed biphasic pulses (0.1 ms duration each half phase) to reduce electrode polarization.

Experimental protocol The recording electrode was lowered into the hippocampus and localized to the pyramidal cell layer of CA1 through monitoring of extracellular single unit activity. The stimulating electrode was then lowered into the stratum radiatum of CA1 and adjusted to produce maximal activation of SCH/COM-CA1 synapses. In the synaptic region (stratum radiatum) this response consists of a short latency, negative field potential, which is indicative of excitatory synaptic activity and is termed the population EPSP zg. The orientation of cellular elements within CA1 results in the ability to record a near mirror-image positive field potential in stratum oriens and stratum pyramidale. At stronger current intensities an extracellularly recorded negative potential develops superimposed on the synaptic wave (see Fig. 1A). This negative

274 potential or population spike (PS) reflects the synchronous activation of the pyramidal cellsZ,29. Following determination of the optimal electrode configuration, synaptic function in the SCH/COM-CA1 path was examined through 5 separate series in each rat which were evaluated in the following sequence. (1) Laminar analysis. A laminar analysis was conducted in 25-#m steps through CA1 in order to determine the distribution of SCH/COM afferents as revealed by extracellular field potential and current-source density analyses. Evoked responses typically observed for this analysis are shown in Fig. 1C. These data will be presented in a separate publication. (2) Input-output relations (I/0). I/0 curves were generated by systematic variation of the stimulus current (20-1000 #A) in order to evaluate the potency of SCH/COM-CAI synapses. Stimulus pulses were delivered at 0.1 Hz and 4 responses were averaged at each current level. (3) Paired-pulse potentiation (PPP). PPP was evaluated at low (subthreshold for a PS) or high (suprathreshold for a PS) stimulus current intensities. Inter-pulse intervals (IPI) were varied from 20 to 180 ms. Stimulus pairs were delivered at 0.1 Hz and 4 responses were averaged at each inter-pulseinterval. (4) Frequency potentiation (FP). A total of 5 FP series were conducted at varying stimulation frequencies: 1 Hz/5 s, 1 Hz/25 s, 5 Hz/5 s, 10 Hz/2.5 s and 10 Hz/5 s. This design allowed for comparisons of FP at equivalent tetanus duration (5 s) or at equal numbers of stimuli within the train (25 pulses). Every third response within the tetanus was evaluated for each stimulus condition and compared to a baseline response generated prior to each series. (5)Long-term potentiation (LTP). Following each FP series, test pulses were systematically delivered for 15-30 min in order to assess the development of LTP. In addition, LTP was examined after a single tetanus (100 Hz/10 s) at the end of each experiment. Following this data collection period, small electrolytic lesions (10 #A, 10 s) were made at the stimulation site and at the isopotential point (inversion point) as determined during the laminar analysis. Electrode placements were verified in all animals through microscopic examination of the lesions in myelin-stained sections.

Data analysis A major difficulty in making quantitative comparisons between treatment groups is the extreme within- and between-group variance normally observed in the intact hippocampus6, 27. We employed several standardization procedures to reduce this variability. First, following the laminar analysis, the recording electrode was fixed at a point 125/zm dorsal to the inversion point. I/O curves, PPP, FP, and LTP series were all obtained at this fixed reference point in each animal. Second, stimulus current levels for PPP, FP and LTP series were also normalized with respect to individual I/O curves. For the first PPP series, current strength was adjusted to obtain a baseline EPSP peak amplitude 5 0 ~ of that observed at PS threshold. In the second PPP series, stimulus current strength was adjusted to obtain a PS 40 ~ of the asymptotic value observed on the I/O curve. For both FP and LTP, current strength was adjusted to produce a PS which was 20 ~ of the asymptotic PS value. This baseline stimulus current value was re-adjusted at the beginning of each FP series when long-term effects of the

275 previous tetanus were observed. Finally, all electrodes were carefully selected to fall within a narrow range of tip size, impedance and shaft taper requirements. Field potentials were quantified with a Numonics Digitizer. Four measures were commonly assessed: EPSP onset latency, PS peak latency, EPSP amplitude, and PS amplitude. Because the precise onset of the EPSP was difficult to determine due to its shallow initial slope, the EPSP onset was arbitrarily chosen to be that point where the evoked potential rose 200 #V above baseline. In an attempt to avoid contamination by the PS, EPSP amplitude was evaluated 0.75 ms following the EPSP onset. The latency to the peak of the PS was measured with respect to the EPSP onset. Finally, PS amplitude was assessed by averaging the distance from the peak negativity to the preceding and following positive peaks (cf. ref. 1). The basic waveform upon which these measures were made is illustrated in Fig. 1A. Following this analysis, the code was broken and the data grouped by treatment. The potentiation data were expressed as either difference from baseline (latency measures) or percentage of control (amplitude measures). In an effort to reduce within-group variance, data from the two control groups (which did not differ) were pooled. The data were analyzed by 2-way analysis of variance (ANOVA) with treatment as one factor and either stimulus current (I/O), inter-pulse interval (PPP), stimulus number (FP) or time following tetani (LTP) as the second factor. Subsequent analyses were performed using Duncan's multiple range and Student's t-tests. RESULTS The following results were based on a total sample of 29 rats which were distributed as follows: Group A = 12, Group S = 10, Group LC = 7. Because Group S and Group LC rats did not differ on any measure, these groups were combined into a single control group (Group C = 17). Group A animals consumed a mean daily ethanol dosage (14.14 g/kg) comparable to previous experiments in which either associative deficits or neuronal loss in hippocampus have been observed. Body weight did not differ among the three groups at any point during the experiment. Mean body weights at the end of the experiment were: Group A = 553.1 4- 15.4 g, Group C = 519.1 4- 10.0 g.

Histological analysis Chronic ethanol treatment has been shown to produce cell loss in the rat hippocampus 54. If one consequence of this cell loss is a selective shrinkage of the volume of the hippocampus, then the relative positions of electrodes placed within the hippocampus using fixed stereotaxic coordinates might be differentially altered in Group A. In order to address this issue, electrode tracts were localized and plotted in three dimensions relative to the brain surface, midline and the septal pole of the hippocampus. This analysis revealed no differences among the three groups. Stimulating electrode sites were in stratum radiatum near the CA1-CA2 border (Fig. 1A). Recording electrode tracts were localized to CA1 (Fig. 1A). Electrolytic lesions placed at the inversion point of the laminar analysis were consistently found at the

A B

SM

sR

SP

SO

ALV

/

f

300 .4o0~ _ j ~

2oo

IOO

DEPTH(UM) o

C

277 stratum pyramidale-radiatum border. The location of the inversion point, with respect to the cell layer, also did not differ across groups. Finally, detailed measures (corrected for tissue shrinkage) were made of the major laminae of CA1 including stratum oriens, pyramidale and radiatum. These measures were made along the trajectory of the electrode track and also did not differ between groups.

1/0 Relationships Chronic ethanol treatment did not produce statistically significant alterations in the basic synaptic responses to single pulse stimulation. The threshold current (#A) required to elicit an EPSP was: Group A: ~ = 61.7, S.E.M. = 3.8; Group C: ~ = 61.2, S.E.M. = 6.1. PS threshold current values were also virtually identical across groups (Group A: ~ = 239.5, S.E.M. = 23.9; Group C: ~ = 251.8, S.E.M. -- 27.9). Moreover, the EPSP amplitude at PS threshold (A: 2.96 4- 0.39 mV; C: 2.91 q- 0.34 mV) and the PS amplitude at asymptote (A: 5.78 4- 1.04 mV; C: 6.23 4- 0.62 mV) did not differ between groups. These results are important, since these values were used to standardize the stimulus current during subsequent potentiation series in each rat. Finally, two way analysis of variance revealed no statistically significant group differences over the range of stimulus current examined (20-1000 #A) for latency to EPSP onset, latency to the peak of the PS, EPSP amplitude or PS amplitude.

Paired-pulse potentiation Chronic ethanol treatment failed to alter the pattern of response to paired-pulse stimulation. In both groups facilitation of the test pulse peak EPSP amplitude (at current levels subthreshold for PS) was maximal at inter-pulse intervals of 30 ms, where potentiation 150-175 % of control was observed (Fig. 2B). In contrast, test pulse PS amplitude was dramatically inhibited at these short inter-pulse intervals, but exhibited facilitation at pulse intervals of 80 ms or greater (Fig. 2B). This differential action of paired stimuli on EPSP and PS amplitude is identical to that observed in previous studies of CA1 and the dentate gyrus14,27,30,46. While this pattern of response to paired stimuli was preserved, chronic ethanol treatment did produce significant changes in the magnitude of PPP. Two-way A N O V A revealed a significant treatment effect for PS amplitude (F(1,39) ~ 6.58, P < 0.02) and a significant group × IPI Fig. 1. A: representative hippocampal lameUa illustrating the position of the stimulating and recording electrodes relative to the SCH and COM afferents and the major laminae of CA1. Insert illustrates basic evoked potential. Note that the PS (peak at second arrow) is superimposed upon the positive extracellular EPSP (onset indicated by first arrow). Waveform is preceded by a calibration pulse (1.0 mV, 2.0 ms) and stimulus artifact. B: representative CA1 pyramidal cell (modifiedfrom Scheibel4e) shown in comparison to major laminae of CA1. The widths of the major laminae were derived from actual measurements in the histological material from Group E and S rats. C: typical laminar analysis through CA1 of rat hippocampus employing SCH/COM stimulation. Tracings are positioned at appropriate depths, relative to ventral surface of alveus. Excitation in stratum radiatum produces an extracellularly recorded negative population EPSP and a corresponding positive potential in stratum pyramidale and stratum oriens. (Calibration: 1.0 mV, 2.0 ms; negativity down). Abbreviations: ALV, alveus; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum; SM, stratum lacunosum-moleculare; HF, hippocampal fissure, COM, commissural, SCH, Schaffer collateral.

278

AI

Cond Pulse IPI

Test Pulse

20

40

60

80

I00

125

150

175

0EE Z 0 L)

\

O T O O

B°

4oo

• EPSP " PS

T Alc°h°I

I

o EPsP

o

{3 PS

T Control

l j

500

J i

S O_ 200

200 x

"

v I00

150

N -7

(3. 0

20

40

60

80

I00

120

140

160

180

I00

CONDITION-TEST INTERVAL(reset )

Fig. 2. Paired-pulse potentiation in CA1 after chronic ethanol treatment. A: recordings from individual alcohol and control rats at varying inter-pulse intervals (IPI). For each rat responses to the conditioning stimulus are superimposed. Calibration: 1.0 m V ; 5.0 ms. B: group comparisons (mean ± S.E.M.) o f PS and EPSP amplitude as a function of IPI. Note the different scales for EPSP and PS. Asterisks denote statistically significant (P < 0.05) group differences as indicated by Student's t-tests.

interaction for the latency to PS (F(1,39) = 2.47, P < 0.03). Subsequent t-tests revealed these effects to be maximal at 100-175 ms inter-pulse intervals. The chronic ethanol-induced enhancement o f PPP is illustrated in Fig. 2A in representative rats from alcohol and control groups. Although there was a trend toward enhancement of the test pulse EPSP amplitude at short inter-pulse intervals in ethanol-treated rats, the

279 A N O V A was not statistically significant (F(1,44) 1.30, P > 0.2). These effects could not be related to differences in the conditioning pulse current levels across groups. The standardization procedure designed to produce equivalent baseline responses across animals resulted in baseline EPSP (A: 1.23 q- 0.17 mV, C: 1.39 ± 0.13 mV) and PS (A: 1.59 ± 0.41 mV, C: 1.56 q- 0.16 mV) amplitudes which did not differ across groups. =

I

HZ

400

200

i

0

5

Z -J

W (/)

I'6

;9

22

I

25

800

C{3 (,r) Z

600 W w ca LI. 0 I..Z w o

40()

w 11_ W o

200

.J

n

I

I

I

i

I

I

i

4

7

I0

13

16

19

22

25

~.

"}

,b

,%

,~

~9

12

2'5

600

400

200

STIMULUS NUMBER WITHIN TETANUS

Fig. 3. Frequency potentiation in CA1 :(mean i S.E.M.) compared across alcohol and control groups as a function of stimulus frequency (1, 5, 10 Hz). A statistically significant treatment effect was observed only at 10 Hz. Intermediate effects were observed at 5 Hz, whereas no group differences were noted at 1 Hz.

280

Frequency potentiation As was the case with PPP, chronic ethanol treatment failed to alter the frequency de-

pendent pattern of responses during FP. At I Hz, potentiation of the PS rapidly grew to asymptotic values (within 4 stimuli) and remained at a stable level (400 ~ of control) thereafter. Both 5 and 10 Hz stimulation produced more robust levels of potentiation (5 Hz > 10 Hz), including the development of multiple population spikes. However, FP at these frequencies was more unstable, exhibiting a definite waxing and waning which was clearly more related to the number of stimuli presented than to the frequency of stimulation (Fig. 3). Since both 5 and 10 Hz tetani lead to the development of multiple population spikes, separate analyses were conducted on either the amplitude of the first spike alone or on the grand sum of all the spikes. Identical results were obtained with each measure. Only the data for the combined PS measures will be presented. Fig. 3 compares 1 Hz, 5 Hz and 10 Hz FP across groups at identical numbers of stimuli (25 pulses). Chronic ethanol treatment did not influence FP at 1 Hz stimulation. However a 2-way ANOVA revealed that FP was enhanced in ethanoltreated rats at 10 Hz (F(1,44) = 4.33, P < 0.05). Stimulation at 5 Hz produced intermediate results although the effect at this frequency did not reach statistical significance. Thus, chronic ethanol treatment augments FP at higher frequencies over a relatively narrow range of stimulation. No measures other than the amplitude of the PS were significantly influenced by ethanol treatment. Neither the latency to EPSP onset or to PS peak were affected. Since EPSP amplitude was measured at a fixed latency (0.75 ms) from onset, this measure was often contaminated by the PS. Especially during FP, the measures of EPSP amplitude and PS amplitude were often inversely correlated. FP produced a dramatic reduction in PS latency, even to the point of obliterating the EPSP completely. We therefore abandoned this measure in both the FP and LTP series.

Long-term potentiation We investigated the long-term effects of repetitive stimulation by presenting 6 separate tetani which were systematically varied by frequency and duration. The longterm effects of each of the stimulus trains on PS amplitude are presented in Figs. 4 and 5. The pattern of long-term effects were dependent on both the frequency and duration of the tetanus. Stimulation at 1 Hz failed to produce LTP. Rather, when of sufficient duration (25 s), 1 Hz stimulation produced a brief (1-2 rain) depression of PS amplitude immediately following the tetanus (Fig. 4). Stimulation at 5, 10 and 100 Hz (Fig. 5) produced LTP. These results agree well with the parametric in vitro study of Dunwiddie and Lynch 16. Stimulation at 5, 10 (5 s) and 100 Hz produced characteristic LTP of the PS which reached a peak at 5 min post-tetanus. In contrast, 10 Hz(2.5 s) produced a robust post-tetanic potentiation but showed little evidence of LTP 15 min following tetanus (Fig. 5). These results suggest that a rather complicated interaction may exist between the frequency and duration of the tetanus in producing LTP in CA 1 of the rat hippocampus. Under the conditions of this experiment, considerable decay of LTP was observed by 30 min following even the 100 Hz tetanus (Fig. 5).

281 200 I Hz

5 sec.

150

o ta

I00

i

& 5o

• Alcohol 0 Control

N 2

o

,~

I00

,

2=

i

0

,

'

40

• ,/

60

=

I

I

I

2

5

I0

15

I I0

I 15

I Hz 25 sec.

~

•

I

Alcohol

13 C o n t r o l 0

i 40

0

, 60

.,/

I 2

I 5

Sec.

Min.

TIME P O S T - T E T A N U S

Fig. 4. Post-tetanic depression of PS amplitude after low frequency stimulation. Response depression was observed only after 25 s of 1 H z stimulation. Ethanol treatment significantly reduced the magnitude of this post-tetanic depression. Asterisks denote statistically significant group differences (P < 0.05). Hz 2.5 sec.

300

200

'00

m

i

°

600

O.

I0 Hz 5 s e c .

I0 ~o

,'o

~'~

• Alcohol 300

0 Cor~trol

200

~00

"

2

,

sec.

,o

0

,,

min.

;-

500

500

~

4oo

400

~.

300

300

200

200

i00

I00

I

20

.

40--

sec.

610

//

~ t

2 5

min.

sec.

600

5 Hz 5 sec.

,

,

15

,

,

h

30

,oo.

0

rain.

,o . . . .

i

0

,

i

0 sec.

,

,1

6o

i

,

2 5

i

i

15

i

,

i

30

mi~

TIME POST- TETANUS

Fig. 5. Long-term potentiation in alcohol and control groups compared (mean -4- S.E.M.) as a function of the frequency and duration of the tetanus. When the duration of the tetanus was varied at a constant frequency (10 Hz), strikingly different patterns of potentiation were observed in both groups. Ethanol treatment failed to significantly affect long-term potentiation at the frequencies tested.

282 Chronic ethanol treatment also produced effects which were dependent on the frequency and duration of stimulation. The transient depression of the PS induced by 1 Hz stimulation (25 s) was significantly reduced in ethanol-treated animals (F(1,44) = 4.33, P < 0.05). Subsequent t-tests revealed that this effect was present only at time points immediately following the tetanus (20-50 s). Ethanol treatment failed to alter the pattern of LTP produced by 5, 10 or 100 Hz stimulation. ANOVA were not statistically significant for any of the results shown in Fig. 5. However, at 100 Hz stimulation chronic ethanol treatment, while failing to alter the development of LTP of the PS, nevertheless appeared to produce a trend toward a reduction in the magnitude of LTP starting approximately 5 min post-tetanus (Fig. 5). DISCUSSION We have used a nutritionally controlled liquid diet preparation to produce an animal model of chronic ethanol exposure. At comparable durations of exposure, chronic ethanol results in a 15-20% loss of granule and pyramidal cells of the rat hippocampal formation 54 as well as dendritic atrophy and reduction of dendritic spines of CA1 pyramidal cells and dentate granule cells of the mouse hippocampus 4°. Taken together, these results suggest that chronic ethanol exposure induces a progressive morphological deterioration of neurons in the hippocampus ultimately ending in cell death. Despite these findings, 20 weeks of ethanol exposure failed to produce significant alterations in basic waveforms, EPSP or PS thresholds, I/O functions, or the production of LTP of CA3-derived fibers terminating in stratum radiatum of CAl. The predominant effect of chronic ethanol treatment was an enhancement of the potentiation of PS responses to paired stimuli or repetitive stimulation at 5 and 10 Hz in the absence of changes in the synaptic response (EPSP). Even though a trend toward enhancement of EPSP potentiation was observed during PPP (Fig. 2B), this enhancement was only apparent at short inter-pulse intervals. Both ethanol and control groups reached a similar asymptote at longer inter-pulse intervals where ethanol treatment produced its greatest enhancement of PS amplitude. The existence of alterations of potentiation of the PS without concomitant changes in the EPSP suggests that ethanol did not act primarily on the SCH/COM-CA 1 synapses themselves. Rather, ethanol treatment appeared to enhance the ability of CAI pyramidal cells to fire synchronously in response to repetitive stimulation. While a number of hypotheses could account for these results, recent evidence indicates that recurrent inhibition plays a significant role in PP facilitation of the PS. Experimental treatments which directly reduce recurrent inhibition in the hippocampus (anoxia, picrotoxin, enkephalins), enhance the facilitation of PS amplitude to paired stimuli and in some cases directly facilitate PS responses to single shock stimuli2,17, ~s without significantly altering the synaptic response. Thus, under ordinary conditions recurrent inhibition antagonizes PP facilitation within the hippocampus. This evidence coupled with the present results support the view that chronic ethanol treatment reduces the effectiveness of recurrent feedback inhibition of pyramidal cells of CA 1. Facilitation of the PS amplitude to paired stimuli was enhanced in ethanol-

283 treated rats in the absence of significant effects on potentiation of the synaptic response. Further, chronic ethanol treatment enhanced FP of the PS only at those frequencies (5, 10 Hz) in which recurrent inhibition would exert an influence. PS responses to 1 Hz stimulus trains were identical across groups. Finally, ethanol treatment significantly reduced the post-tetanic depression produced by 1 Hz stimulation (Fig. 4). Although ethanol treatment failed to influence the more brief depression produced by higher frequency stimulation (Fig. 5), it is noteworthy that such depression may have causes unrelated to recurrent inhibition, for example, transmitter depletion59. Recurrent inhibition in the hippocampus is mediated by GABAergic interneurons which receive axon collaterals and project back onto the parent pyramidal and granule cells. These interneurons are believed to be basket cells4,5,31. Since relatively few basket cells exist in the hippocampus (relative to principal cell types), the powerful and widespread recurrent inhibition in the hippocampus must reflect a great divergence of synaptic contacts. Even a modest alteration in basket cells would be expected to produce effects which are more potent than the loss of principal cell types54. Thus, ethanol treatment could reduce recurrent inhibition by reducing the population of basket cells or altering GABAergic neurotransmission. Chronic ethanol treatment has been reported to decrease GABA concentrations by some39,5a but not all investigators47 and reduce the density of low-affinity GABA binding sites4L Moreover, chronic ethanol treatment decreases the affinity and density of receptor binding for benzodiazepines21, which appear to augment GABAergic inhibition in widespread brain areas. However, in many of these studies the duration of ethanol treatment was substantially shorter (2-3 weeks) than in the present study and GABAergic function was assessed within 24 h of ethanol withdrawal. There is little doubt that these effects are dependent upon the duration of ethanol exposure. For example, while 15-19 days of ethanol exposure failed to alter benzodiazepine receptor binding 21,26, 7 months of exposure decreased the density of benzodiazepine receptors for at least one month following abstinence21. Thus, it remains unclear whether longer durations of ethanol exposure would decrease GABAergic neurotransmission after 8 weeks of ethanol abstinence, the time period assayed in this study. While reduced recurrent inhibition may be best explained by a direct action of chronic ethanol treatment on basket cells, at least one alternative should be considered. Anesthetic agents including ethanol have been reported to augment recurrent inhibition of single unit activity in the hippocampus36,50,~8. Since ethanol exhibits crosstolerance to other depressants 25, the apparent reduction of recurrent inhibition could result from differential group responses to general anesthesia. This hypothesis must be seriously considered since chronic ethanol treatment has recently been shown to produce tolerance of the response to ethanol of SCH/COM-CA 1 synapses in a hippocampal slice preparation 12. However, we consider this alternative unlikely for the following reasons. Neither the initial nor supplemental doses of urethane required to produce and maintain anesthetic levels differed between groups. Behavioral tolerance 25 to ethanol normally dissipates over a brief time course (2-3 days) and ethanol tolerance in hippocampal slices was only observed immediately upon ethanol with-

284 drawal ~2. Since our data were collected at least 8 weeks after ethanol withdrawal, it is likely that the residual effects of ethanol tolerance would have dissipated. This possibility, while still unlikely, cannot be completely ruled out (cf. ref. 7). The effects of chronic ethanol treatment on PPP, FP and post-tetanic depression are significant insofar as they persisted for at least 8 weeks after ethanol abstinence. Nevertheless, the failure to observe significant alterations in basic synaptic responses (thresholds, I/O curves) is surprising, since both the pre- and postsynaptic elements of the SCH/COM-CA1 path are reduced by chronic ethanol treatment 54. However, it is unlikely that a simple relationship exists between the number of CA1 pyramidal cells and the size of the extracellular EPSP and PS. Further, it is plausible that the varied effects of ethanol treatment could interact. For example, since picrotoxin, a GABA receptor blocker, has been shown to facilitate PS responses to single shock stimuli 17, it is possible that a loss of recurrent inhibition may have masked a reduction in PS responses (I/O curves) produced by deafferentation of CA1. Alternatively, the failure to observe changes in basal synaptic response strength may reflect morphological reorganization and recovery, since reactive synaptogenesis has been documented in CA1 following destruction of afferents originating in CA334, aS. At present, a connection between long-term potentiation in the hippocampus and normal memory formation is clearly speculative. However, the issue may be addressed through correlative analysis of synaptic potentiation in memory deficient animals. Studies in aged, memory deficient rats have indicated that LTP is reduced in both CA1 z7 and the dentate gyrus6 of the hippocampus. The similarity of the mnemonic deficits associated with alcoholism and aging have led to the hypothesis that alcohol accelerates the aging processS,4L Studies of chronic ethanol-treated and aged animals indicate a remarkable similarity in the nature of morphological deterioration in the hippocampusl0,11,4L However, aged animals exhibit a reduction in FP and LTP in SCH/COM-CA 1 connections 27 whereas ethanol-treated rats exhibit enhanced PPP and FP. Insofar as the hippocampus is involved in memory formation 3a,37, these results suggest that very different mechanisms may underlie the mnemonic deficits associated with alcoholism and aging. This conclusion must be considered preliminary, particularly since a trend toward a greater decay of LTP was observed in ethanol-treated rats (Fig. 5), and a recent study with hippocampal slices has reported an impairment of LTP formation after chronic ethanol treatment assayed immediately following ethanol withdrawal is. ACKNOWLEDGEMENTS We gratefully acknowledge the technical assistance of Pat Burnett, Larry Ezell, Dot Robinson, Don Morris and Mike King. Data were analyzed using the Statistical Analysis System on an Amdahl 470 V/6-11 with OS/MVS Release 3.8 and JES2/NJE Release 3. Computing was done using the facilities of the Northeast Regional Data Center of the State University System of Florida. W.C.A. was supported by an NSF Predoctoral Fellowship. This research was supported by the Veterans Administration and NIAAA Grant AA-00200.

285 REFERENCES 1 Alger, B. E. and Teyler, T. J., Long-term and short-term plasticity in the CA1, CA3, and dentate regions of the rat hippocampal slice, Brain Research, 110 (1976) 463-480. 2 Andersen, P., Interhippocampal impulses II: apical dendritic activation of CA1 neurons, Acta physiol., scand., 48 (1960) 178-208. 3 Andersen, P., Bliss, T. V. P. and Skrede, K. K., Unit analysis of hippocarnpal population spikes, Exp. Brain Res., 13 (1971) 208-221. 4 Andersen, P., Eccles, J. C. and Loyning, Y., Location of postsynaptic inhibitory synapses on hippocampal pyramids, J. NeurophysioL, 27 (1964) 592-607. 5 Andersen, P., Eccles, J. C. and L~yning, Y., Pathway of postsynaptic inhibition in the hippocampus, J. Neurophysiol., 27 (1964) 608-619. 6 Barnes, C. A., Memory deficits associated with senescence: a neurophysiological and behavioral study in the rat, J. comp. physiol. Physiol., 93 (1979) 74-104. 7 Begleiter, H., DeNoble, V., Porjesz, B., Protracted brain dysfunction after alcohol withdrawal in monkey, Advanc. exp. Med. Biol., 126 (1980) 237-250. 8 Blusewicz, M. J., Dustman, R. E., Schenkenberg, T. and Beck, E. C., Neuropsychological correlates of chronic alcoholism and aging, J. nerv. ment. Dis., 165 (1977) 348-355. 9 Bond, N. W. and Di Giusto, E. L., Impairment of Hebb--Williams maze performance following prolonged alcohol consumption, Pharmacol. Biochem. Behav., 5 (1976) 85-86. 10 Bondareff, W., Synaptic atrophy in the senescent hippocampus, Mech. Ageing Develop., 9 (1979) 163-172. 11 Brizzee, K. R. and Ordy, J. M., Age pigments, cell loss and hippocampal function, Mech. Ageing Develop., 9 (1979) 143-162. 12 Carlen, P. L. and Corrigall, W. A., Ethanol tolerance measured electrophysiologically in hippocampal slices and not in neuromuscular junctions from chronically ethanol-fed rats, Neurosci. Lett., 17 (1980) 95-100. 13 Courville, C. B., Effects of Alcohol on the Nervous System of Man, San Lucas Press, Los Angeles, 1966, pp. 102. 14 Creager, R., Dunwiddie, T. and Lynch, G., Paired-pulse and frequency facilitation in the CA1 region of the in vitro rat hippocampus, J. Physiol. (Lond.), 299 (1980) 409-424. 15 Denoble, V. J. and Begleiter, H., Impairment of acquisition of a DRL schedule following prolonged ethanol consumption, Pharmacol. Biochem. Behav., 10 (1979) 393-396. 16 Dunwiddie, T. and Lynch, G., Long-term potentiation and depression of synaptic responses in the rat hippocampus: localization and frequency dependency, J. Physiol. (Lond.), 276 (1978) 353-368. 17 Dunwiddie, T., Mueller, A., Palmer, M., Stewart, J. and Hoffer, B., Electrophysiological interactions of enkephalins with neuronal circuitry in the rat hippocampus. I. Effects on pyramidal cell activity, Brain Research, 184 (1980) 311-330. 18 Durand, D., Cohen, P. L. and McMullen, P., Impairment of long-term potentiation following chronic ethanol consumption in rats, Neurosci. Abstr., 6 (1980) 89. 19 Epstein, P. S., Pisani, V. D. and Fawcett, J. A., Alcoholism and cerebral atrophy, Alcoholism: Clin. Exp. Res., 1 (1977) 61-65. 20 Fehr, K. A., Kalant, H. and LeBlanc, A. E., Residual learning deficit after heavy exposure to cannabis or alcohol in rats, Science, 192 (1976) 1249-1251. 21 Freund, G., Benzodiazepine receptor loss in brains of mice after chronic alcohol consumption, Life Sci., 27 (1980) 987-992. 22 Freund, G. and Walker, D. W., Impairment of avoidance learning by prolonged ethanol consumption in mice, J. Pharmacol. exp. Ther., 179 (1971) 284-292. 23 Goldowitz, A., Scheff, S. W. and Cotman, C. W., The specificity of reactive synaptogenesis: a comparative study in the adult rat hippocampal formation, Brain Research, 170 (1979) 427--442. 24 Haug, J. O., Pneumoencephalographic evidence of brain damage in chronic alcoholics, Acta psychiat, scand., 203, Suppl. (1968) 135-143. 25 Kalant, H., Leblanc, E. and Gibbins, R., Tolerance to, and dependence on, ethanol. In Y. Israel, J. Mardones (Eds.,), Biological Basis of Alcoholism, Wiley Interscience, New York, 1971, pp. 235-261. 26 Karobath, M., Rogers, J. and Bloom, F. E., Benzodiazepine receptors remain unchanged after chronic ethanol administration, Neuropharmacology, 19 (1980) 125-128. 27 Landfield, P. W., McGaugh, J. L. and Lynch, G., Impaired synaptic potentiation processes in the

286 hippocampus of aged, memory-deficient rats, Brain Research, 150 (1978) 85-102. 28 Lee, H. K., Dunwiddie, T. and Hoffer, B., Electrophysiological interactions of enkephalins with neuronal circuitry in the rat hippocampus. II. Effect on interneuron excitability, Brain Research, 184 (1980) 331-342. 29 L~mo, T., Patterns of activation in a monosynaptic cortical pathway: the perforant path input to the dentate area of the hippocampal formation, Exp. Brain Res., 12 (1971) 18-45. 30 L~mo T., Potentiation of monosynaptic EPSPs in the perforant path-dentate granule cell synapse, Exp. Brain Res., 12 (1971) 46-63. 31 Lorente de No., R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. Psychol. Neurol., 46 (1934) 113-177. 32 MacDonell, J. S. and Marcuella, H., Short-term memory deficits following prolonged ethanol consumption by rats. In F. A. Seixas (Ed.), Currents" in Alcoholism VoL, 3, (Grune and Stratton, New York, 1978, pp. 245-254. 33 Milner, B., Corkin, S. and Teuber, H. L., Further analysis of the hippocampal amnestic syndrome: 14 year follow-up study of H. M., Neuropsychologia, 6 (1968) 267-282. 34 Nadler, J. V., Perry, B. W. and Cotman, C. W., Selective reinnervation of hippocampal area CA1 and the fascia dentata after destruction of CA3-CA4 afferents with kainic acid, Brain Research, 182 (1980) 1-10. 35 Nadler, J. V., Perry, B. W., Gentry, C. and Cotman, C. W., Loss and reacquisition ofhippocampal synapses after selective destruction of CA3-CA4 afferents with kainic acid, Brain Research, 191 (1980) 387-404. 36 Newlin, S. A., French, E. D., Berger, T. and Bloom, F. E., Ethanol prolongs the time course of recurrent inhibition in the hippocampus in response to commissural fiber activation, Neurosci. Abstr. 5 (1979). 37 O'Keefe, J. and Nadel, L., The Hippocampus as a Cognitive Map, Clarendon Press, Oxford, 1978. 38 Oliver, A. P., Hoffer, B. J. and Wyatt, R. J., Kindling induces long-lasting alterations in response of hippocampal neurons to elevated potassium levels in vitro, Science, 208 (1980) 1264-1265. 39 Patel, G. J. and Lal, H., Reduction in brain )'-aminobutyric acid and in barbital narcosis during ethanol withdrawal, J. Pharmacol. exp. Ther., 186 (1973) 625-629. 40 Riley, J. N. and Walker, D. W., Morphological alterations in hippocampus after long-term alcohol consumption in mice, Science, 201 (1978) 646-648. 41 Ryan, C. and Butters, N., Learning and memory impairments in young and old alcoholics. Evidence for the premature-aging hypothesis, Alcoholism: Clin. Exp. Res., 4 (1980) 288-293. 42 Scheibel, A. B., The hippocampus: organizational patterns in health and senescence, Mech. Ageing Develop., 9 (1979) 89-102. 43 Schwartzkroin, P. A. and Wester, K., Long-lasting facilitation of a synaptic potential following tetanization in the in vitro hippoeampal slice, Brain Research, 89 (1975) 107-119. 44 Smith, J. W., Burt, D. W. and Chapman, R. F., Intelligence and brain damage in alcoholics: a study of patients of middle and upper social class, Quart. J. Stud. Alcohol., 34 (1973) 41~422. 45 Sotzing, J. H. and Brown, T. S., Chronic intermittant ethyl alcohol inhalation and avoidance learning, Pharmacol. Bioehem. Behav., 5 (1976) 417-421. 46 Steward, O., White, W. F. and Cotman, C. W., Potentiation of the excitatory synaptic action of commissural, associational and entorhinal afferents in dentate granule cells, Brain Research, 134 (1977) 551-560. 47 Sutton, I. and Simmonds, M. A., Effects of acute and chronic ethanol on the~'-aminobutyric acid system in rat brain, Biochem. Pharmacol., 22 (1973) 1685-1692. 48 Talland, G. A., Deranged Memory, Academic Press, New York, 1965, p. 356. 49 Ticku, M. K. and Burch, T., Alterations in ~,-aminobutyric acid receptor sensitivity following acute and chronic ethanol treatments, J. Neurochem., 34 (1980) 417-423. 50 Tsuchiya, T. and Fukushima, H., Effects of benzodiazepines and pentobarbitone on the GABAergic recurrent inhibition of hippocampal neurons, Europ. J. Pharmaeol., 48 (1978) 421-424. 51 Tumarkin, B., Wilson, J. D., and Snyder, G., Cerebral atrophy due to alcoholism in young adults, US Armed Forces med. J., 6, (1955) 64-74. 52 Victor, M., Adams, R. D. and Collins, G. H., The Wernicke-Korsakoff Syndrome, F.A. Davis, Philadelphia, 1971. 53 Volicer, L., Hurter B. P., Williams, R., Puri, S. K. and Volicer, B. J., Relationship between brain levels of cyclic nucleotides and y-aminobutyric acid during ethanol withdrawal in rats, Drug Alcohol. Depend., 2 (1977) 317-327. 54 Walker, D. W., Barnes, D. A., Zornetzer, S. F., Hunter, B. E. and Kubanis, P., Neuronal loss in

287 hippocampus produced by prolonged ethanol consumption in rats, Science, 109 (1980) 711-713. 55 Walker, D. W. and Freund, G., Impairment of shuttle box avoidance learning following prolonged alcohol consumption in rats, Physiol. Behav., 7 (1971) 773-778. 56 Walker, D. W. and Freund, G., Impairment of timing behavior after prolonged alcohol consumption in rats, Science, 182 (1973) 597-599. 57 Walker, D. W. and Hunter, B. E., Short term memory impairment following chronic alcohol consumption in rats, Neuropsychology, 16 (1978) 545-554. 58 Wolf, P. and Haas, H. L., Effects of diazepines and barbiturates on hippocampal recurrent inhibition, Arch. Pharmacol., 299 (1977) 211-218. 59 Yamamoto, C., Matsumoto, K. and Takagi, M., Potentiation of excitatory postsynaptic potentials during and after repetitive stimulation in thin hippocampal sections, Exp. Brain Res., 38 (1980) 469-477.