- Email: [email protected]
Ethanol causes increases in excitation and inhibition in area CA3 of the dorsal hippocampus
Brain Research, 209 (1981) 113-128 © Elsevier/North-Holland Biomedical Press
I 13
E T H A N O L CAUSES INCREASES IN EXCITATION A N D I N H I B I T I O N IN AREA CA3 OF T H E DORSAL HIPPOCAMPUS
S. A. NEWLIN, JORGE MANCILLAS-TREVINO and F. E. BLOOM
Arthur V. Davis Center for Behavioral Neurobiology, The Salk Institute, P.O. Box 85800, San Diego, Calif. 92138 (U.S.A.)
(Accepted August 28th, 1980) Key words: synaptic effects - - inhibition -- amine modulation - - tolerance - - ethanol metabolite
SUMMARY The effects of ethanol on synaptic transmission in hippocampus were studied by changes in the response of CA3 pyramidal cells to stimulation of two afferent pathways, the dentate (mossy fiber) pathway, and the commissural pathway. Both sources produce an excitatory response as measured either by single unit spiking or by population spike followed by a period of post-stimulus inhibition (PSI). After the injection of 3 g/kg ethanol (i.p.), both the excitatory responses and the duration of PSI are significantly increased. Because these changes occur in both afferent pathways, they are not pathway specific, but may be the result of the local microcircuitry in area CA3. Although the change in excitatory and inhibitory responses can occur simultaneously, detailed statistical analyses show that neither the magnitude nor onset times are correlated. Thus, the two responses are functionally separable. In addition, the increase in the duration of PSI is related to the rate of rise of blood ethanol level and shows short-term tolerance. The maximum change in the PSI occurs after blood ethanol levels plateau suggesting a secondary process is necessary before neurophysiological effects are apparent.
INTRODUCTION Many studies have indicated an electrophysiological effect of ethanol on central neurc.ns 16,22. Both excitatory and inhibitory effects have been described. Most studies have utilized single unit or multi unit recordings of spontaneous rate as an index of excitability. However, we were also interested in possible effects of ethanol on specific synaptic events and on specific neurotransmitters. Our laboratory has, therefore, designed experiments to test the effects of ethanol in several well-defined brain regions.
114 We have recently published observations of the effects of acute and chronic ethanol on cerebellar Purkinje neurons 27. The hippocampus is also an excellent model for the study of neuronal actions of ethanol. Its synaptic connections are well-defined and documented 2,a, transmitters have been well defined in several of the pathways1,5,7, 25, identifiable test cells are spontaneously active 26. Furthermore, preliminary studies have shown that ethanol does have an effect on the neurophysiology of the hippocampus. Changes have been reported in the EEG and in multi-unit activity z3, in spontaneous activity of single units la, and in evoked population responses in vitro1°, 11,14. These changes were usually depressive. In order to locate specific synaptic changes which might underly those general activity changes we have studied the effect of ethanol on evoked single unit and population responses in area CA3 of the dorsal hippocampus. We have found increases both in excitation and inhibition. METHODS Male, Sprague-Dawley rats (Charles Rivers Breeding Company) between 250 and 350 g were used. The rats were anesthetized with halothane (3 ~ during surgery, 1 ~ during the experimental measures) via a tracheal cannula, and held in a stereotactic apparatus. Bone and dura over the dorsal hippocampus were removed. Recording electrodes were placed in area CA3 of the dorsal hippocampus (A 3500/~m, L 3500/~m, V 3300-4000/~m 24); placement was verified during the experiment by field potentials elicited by dentate pathway 2 or commissural pathway 6 stimulation. At the end of each experiment Pontamine sky blue dye was ejected with pulsed DC negative current (2.5 #A, 15 min), and placement was verified by histological examination. Recording electrodes were glass micropipettes filled with Pontamine sky blue in 3 M acetate solution (pH = 7.4) and broken to tip diameters of 1-2#m; resistances were 5-10 MfL Bipolar stimulating electrodes were constructed from two 100 #m teflon-coated stainless steel wires glued in parallel with Stoner-Mudge bond (Mobil Oil Corp.). Stimulating electrodes were placed in the contralateral hippocampus (A3500 /~m, L3500/zm, V3000/tm 24) for commissural pathway stimulation, or in the ipsilateral dentate gyrus (A3500 #m, L2200 #m, V3600 #m ~4)for mossy fiber pathway stimulation. At the end of each experiment a lesion was made by application of constant current (15/~A, 5 min) through the electrode tip, and electrode placement verified by histological examination. Simultaneous recording of evoked population responses and extracellular single unit activity (see Fig. 1) was done by branching the output of a high impedance preamplifier through different filtering systems. Population responses were displayed on a storage oscilloscope after 10 Hz low frequency filtering. The signal for single unit responses of 600--1200/zV amplitude was filtered by a series of filters (10, 300 and 600 Hz low frequency and 30, 10, and 10 kHz high frequency) to eliminate the population response from the signal, then fed through a window discriminator while monitoring
115 on an oscilloscope. The square wave pulse output from the discriminator integrated over 100 msec intervals was monitored by chart recorder as firing rate, and was fed to a Minc II (Digitimer)computer for on-line production of post-stimulus time histograms (PSTHs) (software by K. Liebold, DECUS). Stimulating currents were expressed as multiples of 'threshold' for all parameters measured, where 'threshold' current was the minimum current necessary to elicit a period of PSI. The range of threshold currents was 50-150/zA. The logn (test measure/control measure; In T/C) was computed for magnitude population responses, spike counts following stimulation, and the period of PSI following afferent pathway stimulation under control conditions; measures were then repeated in the same animal at 15, 30, 45, 60 and 120 min following the injection of ethanol or vehicle. Responses to stimulation were measured as follows: 'post-stimulus inhibition' was measured using post-stimulus time histograms as described in Fig. 2. Population EPSPs were measured in the stratum radiatum as described by Anderson et al.2 and Blackstad6 and population spikes were measured in the striatum pyramidale 2,6 and measured from the first positivity to the following negativity (Fig. 1). Ethanol was diluted to 30~ (w/v) in distilled water and injected i.p. via an indwelling 21-gauge, 0.75 in. needle which had been inserted before experimentation. The injected dose was 3 g/kg in all cases; total volume injected varied from 2.5 to 3.5 ml. The total volume was injected over a period of 10 min to prevent pressure trauma to the gut. We measured 'time post ethanol' from the time point concluding injection. Blood samples were removed from the tail vein at 5 min post-injection and following each experimental measure (i.e. also at 15,30,45,60 and 120 min). Blood samples were spun in a microhematocrit centrifuge, and the plasma decanted for assay, using the Calbiochem-Behring Ethyl Alcohol Reagents Kit. Measures in our laboratory (Shoemaker and Bloom, pers. commun.) and by others 17 have shown that blood ethanol levels do not differ significantly from brain ethanol levels. RESULTS
Blood ethanol levels Blood ethanol levels rose to significant levels within 5 min (Table I). Rates of rise differed greatly between preparations, most reaching a plateau value by 15 min and some reaching a plateau value at 30 or 45 min; on the average maximum blood ethanol levels were reached by 15 min post-injection. Peak blood ethanol concentrations ranged from 110 to 440 mg~o; average peak concentration was 242 :k 30 m g ~ . Blood ethanol levels did not decline throughout the course of the experiments, usually 2-3 h. Increases in the period of post-stimulus inhibition Stimulation of the dentate (mossy fiber) pathway to area CA3 of the dorsal hippocampus produced an excitatory response followed by a period of inhibition, which can be monitored by extracellular single unit recording3 (see Fig. 1). The period of inhibition has been described as being due to a recurrent collateral and interneuron feedback4, but could also receive a contribution from a feedforward pathway1. In this
116 TABLE I Blood ethanol levels Blood was removed from the tail vein. Ethanol assay is described in Methods section, n = number of preparations in which assay was made. Errors are standard error of the mean. Time postinjection (min)
n
Blood ethanol concentration (mg%)
5 15 30 45 60 90 120
(20) (18) (15) (8) (17) (12) (11)
189 242 243 222 274 262 263
A
i 24 ± 30 4- 27 -4- 22 ~ 22 -4- 27 i 29
. III.. IILIII.I~I Ill llllr, llllJll II 11
200yV L I see
'II1",,[ Illrl,, I11111111 [1[ CONTROL
30 MIN POST EtOH 200~V
B t
c,_ !
STIMULUS
t IOOjlV L_. 200 msec
t
STIMULUS
Fig. 1. Recordings from stratum pyramidale of area CA3 of the dorsal hippocampus. A: spontaneous single unit activity. Stimulation of dentate gyrus produced an excitatory response (see B and D) followed by inhibition of spontaneous spiking. One sweep. Stimuli are 1 per 5 sec. See Methods for recording procedures. B: evoked single unit spike following stimulation of dentate gyrus: control and 30 rain post-ethanol injection (3 g/kg). C: recording of single unit activity following dentate stimulation: control and 30 rain post-ethanol injection. Each tracing is the sum of 30 sweeps. Recordings show inhibition following the stimulus artifact, with resumption of spontaneous activity. The length of the inhibitory period is increased in the presence of ethanol. D : population response elicited by stimulation of the dentate gyrus: control and 30 rain post-ethanol injection. The amplitude of the population spike (*) is increased in the presence of ethanol.
117
CONTROL ~C:3
z ~o
ETHANOL
TREATED
DCO D_N.
I0
stimulus
Fig. 2. Post-stimulus-time histograms. On-line, computer-generated post-stimulus-time histograms for one preparation are shown for control and 30 min post-ethanol injection (3 g/kg). Each histogram sums spike counts for 30 stimuli (12 Hz). The length of post-stimulus inhibition, taken as the number of msec following the stimulus artifact in which no spikes are counted, is seen to increase following ethanol injection. paper we will refer to the inhibitory period as 'post-stimulus inhibition' (PSI), and it is taken as the length of time, following 30 consecutive stimuli (Fig. 3), in which no spikes were observed. Post stimulus-time histograms (PSTHs) were used to sum the responses to 30 stimuli at each stimulating voltage. The length of PSI (measured in msec) increased with increasing stimulus voltage. For a given stimulus voltage, the period of PSI was consistently increased following the injection of ethanol (3 g/kg, i.p.), thus shifting the stimulus-response curve to the left (Fig. 2; Table II). The maximum increase in inhibition usually occurred at 30 minutes post ethanol injection. For statistical purposes, changes in the length of inhibition were analyzed in three ways. (1) The length of PSI (measured in msec) was measured for each stimulus intensity under control conditions and at 30 min following the administration of ethanol, and then averaged for all cells. By this measure, increases were observed at
* P < 0.025;
** P < 0.01;
9.4 (15) 18.6 (15) 26.4 (13) 46.4 (11)
24.7(15)** 44.0 (15)*** 58.7 (13)* 59.9 (I1)***
0.599 0.361 0.332 0.328
i 4± 4-
15 min
In TIC
0.070(13) 0.144 (12)* 0.127 (10)* 0.113 (10)*
*** P < 0.005, paired t-test.
± ± 44-
111.5 252.3 339.8 519.3
± ± 4±
T 2T 3T 4T
42.0 115.2 227.6 328.9
30 min Post-EtOH ( msec )
Stimulus Control (msec) intensity
0.861 1.329 0.514 0.427
q- 0.252(15)*** ± 0.841 (15)* d_ 0.111 (13)*** -4- 0.116 (11)***
30 min 0.384 0.484 0.481 0.299
± 0.206(15)* -+ 0.146 (14)*** 4- 0.104 (13)*** 5_ 0.088 (11)***
60 min
0.525 0.380 0.491 0.271
~± i ±
120 min 0.209 0.153 0.073 0.174
(10)* (9)* (9)*** (9)
Length of post-stimulus inhibition is the period of time (msec) following the stimulus in which no spike activity occurred. Measurements were taken from post-stimulus-time histograms (see Fig. 2); 30 stimuli were given at a rate of 12 Hz. Stimulus intensities were expressed as multiples of threshold (T), where threshold was the minimum current necessary to produce inhibition. Length of post-stimulus inhibition is represented in msec for control measures and for 30 min post-injection of 3 0 ~ ethanol (3 g/kg, i.p.), and also as a normalized value. Normalized values were computed as In (test msec/control msec or (In T/C) for each preparation at 4 different times post-ethanol injection, and then averaged. Numbers in parentheses indicate numbers of preparation in which measurements were taken.
Length o f post-stimulus inhibition dentate to C.43 pathway
TABLE II
Oo
119 each stimulus intensity (T through 4T) and were significantly different from control measures (Table II). (2) Normalized changes were computed by the formula ln[test msec/control msec], (ln T/C); this computation gives equal arithmetical weight to increases and to decreases in the length of PSI. Such normalized computations were made for each stimulus voltage for each testing time; the averages are reported in Table II and show increased PSI after alcohol. There is an increase in the time of inhibition for each stimulus voltage tested. (3) The average normalized increase (In T/C) in PSI for all stimulus intensities, measured 15-45 min post-ethanol injection, for 18 cells, was 0.553 4- 0.084, n = 153. As a control measure a similar volume of vehicle (distilled water) was injected in 5 preparations. The normalized increase in PSI was 0.089 ~: 0.042, n ---- 52. Measures for the ethanol injected preparations differed significantly from that for the water-injected preparations (P < 0.001, Student's t-test). To test for pathway specificity, similar measurements were made for the commissural to CA3 pathway, where stimulation also produces an excitation in CA3 neurons, followed by a period of inhibition. By all statistical measures, the period of PSI was significantly increased for stimulus voltages of threshold and three times threshold (Fig. 3 and Table III) and differed significantly from water-injected controls (P < g
CONTROL G
m-,
.... I ...... h,~ ........... °o'. oo 7~.]q [~6.29 ~ 2~9.U3
/
,,=,
292.57
3~5.71
~8.86 ---
0-
_~-
ID (_)
ETHANOL
TREATED
"-t-
III
~:oo
.
.
.
.
.
.
7~.1q
.
.....
IJ..L
t~s.29 t 2'19.~3 /
stimulus
2b;.s7
3'6s.TJ
,~.~s
TIME - MSEC
Fig. 3. Expanded time scale post-stimulus-time histograms, control and 30 min post-ethanol injection (3 g/kg, i.p.). Each histogram sums the response to 30 stimuli (12 Hz). Spontaneous activity can be observed for 200 msec prior to the stimulus artifact; spikes can be counted following the stimulus artifact (0.2 msec), followed by the period of inhibition. A greater number of stimulus-evoked spikes occur in the ethanol-treated animal.
120 TABLE III Length of post-stimulus inhibition, commissural to CA3 pathway Length of post-stimulus inhibition was measured as described in caption to Table II and is expressed as raw data (msec) and as averaged, normalized data (In T/C) at 30 rain post-injection of 30 ~ ethanol (3 g/kg ip.). Numbers in parentheses indicate numbers of preparations. Intensity
Control (msec)
30 min post-EtOH ( msec)
In TIC 30 min
T 3T 5T
84.6 :k 26.4 (4) 283.8 i 77.0 (4) 508.6 ::k 138.8 (4)
182.3 ± 22.9 (4)** 462.2 -- 102.8 (4)* 506.6 ± 113.9 (4)
1.293 ± 0.461 (4)** 0.540 ± 0.157 (4)*** 0.096 ± 0.189 (4)
* P < 0.10;
** P < 0.05;
*** P < 0.025, paired t-test.
0.025, Student's t-test). We were able to test higher stimulus intensities (5 times threshold) with the stimulating electrode in the contralateral hippocampus, without significant current spread to the site of the recording electrode. N o significant increase in the length of PSI was noted at this higher stimulus intensity. Increases in excitability Excitation of pyramidal cell n e u r o n s was measured in two different ways: (1) single unit responses a n d (2) p o p u l a t i o n responses. In 4 of the 18 cells studied we could observe single unit spikes in response to dentate stimulation. Spikes could be counted with the aid of expanded-time P S T H s such as seen in Fig. 3. In all cells where such excitatory responses were obtained, we observed, after 30 stimuli, a n increase in the total n u m b e r of spikes for any given stimulus voltage following the injection of ethanol (Table IV). TABLE 1V Increases in excitability following ethanol administration dentate to CA3 path way All measures are represented as the average normalized increase (In T/C). Spike counts were read by the computer from expanded time post stimulus time histograms (such as shown in Fig. 4). Spike counts were summed for 30 stimuli at each stimulus intensity for 6 different preparations. Average counts were: T, 70.5 ± 11.5; 2T, 128.3 i 20.8; 3T, 134.0 ± 15. Measures were then taken at 30 and 45 min post-ethanol injection;normalized increases (In T/C) were computed and then averaged. The population spike (see Fig. 1) was measured at the pyramidal cell layer of area CA3 of the dorsal hippocampus. Responses were recorded as the average of three measures at each of 3 different stimulus intensities. Average control amplitudes were: T 0.22 ± 0.79 mV; 2T, 1.29 ± 0.08 mV; 3T, 1.67 ± 0.18 mV. Measures were then taken at 30 and 45 rain post-ethanol in 12 different preparations, normalized increases (In T/C) were computed and then averaged. The population EPSP was measured in the stratum radiatum of area CA3 in 5 different preparations. Average control amplitudes were: T, 2.31 ± 0.93 mV; 2T, 2.99 ± 1.07 mV; 3T, 3.83 ± 1.49 inV. Stimulus intensities were in multiples of threshold (T), which was the minimum intensity necessary to elicit a period of PSI in each preparation. Response measured
In T/C
P (paired t-test)
Spike counts Population spike Population EPSP
0.25 ± 0.11 (28)** 0.09 ± 0.07 (69)* 0.15 ± 0.10 (28)
<0.025 < 0.05 <0.20
121 Population responses were observed in all preparations following dentate pathway stimulation. Population responses were measured with stimulus frequencies of 1/5 sec and 1/20 sec. No facilitation of responses was noted at either stimulus frequency. The 'population EPSP' was measured with the recording electrode in the stratum radiatum of area CA3, and the 'population spike' with the recording electrode in the stratum pyramidaleL In 8 of 11 preparations the population spike increased following ethanol administration, in 2 preparations the population spike decreased, and there was no change in one preparation. The average change in the size of the population spike was a small, but significant, increase (Table IV). The size of the population EPSP showed no consistent trend across or within preparations. On the average it showed a small and insignificant, increase. Preparations receiving water injections showed no significant increase in any measure of excitability. Similar increases in excitability were noted with comlnissural pathway stimulation in the presence of ethanol, but were not formally measured. Changes in excitatory responses do not correlate with changes in the inhibitory response Since the increases in both excitation and inhibition were noted, one might hypothesize that the effect of ethanol was to increase pyramidal cell responsiveness to afterent excitatory drive, and thus increase the amount of recurrent inhibitory interneuron activation, or, alternatively, that the effect of ethanol was to increase pyramidal cell responsiveness to all synaptic input simultaneously. In either case, one might then expect a correlation between the change in amount of excitation and the amount of inhibition. However, such a correlation was not observed. There was no statistical correlation for the amount of change between either measure of increased excitability (population spike or single unit counts) and the increase in length of PSI. The correlation factor for In T/C for the length of PSI vs In T/C for population spike amplitude, measured at the same voltages, was 0.119 (n -- 16); and the correlation factor for the length of PSI vs spike counts (In test/control) was 0.129 (n =: 27). Furthermore, we noted that the peak increase in measures of excitability did not necessarily occur at the same time post ethanol injection as the increase in inhibition and in some preparations the maximum change in population spike occurred at a later time. Time course of effects The duration of PSI was noticeably increased at the first measurement after ethanol injection (15 min) and continued to increase, usually reaching a maximum length at 30 min post-injection. Fig. 4 shows a time plot of the average of changes in PSI (expressed as In T/C). Individual preparations typically followed the same pattern. The peak change in population spike amplitude (whether increase or decrease) occurred between 30 and 60 min, and the same was true for increases in spike counts. Increases in excitability and the increase in length of PSI both occurred at a time point later than the time where the plateau in blood ethanol levels was reached (Table I and Fig. 4). We considered the possibility that stimulus history might be important for the expression of ethanol effect or in the development of tolerance, as has been shown for other preparationsa2,a6,a7. In several preparations we did not stimulate the dentate
122 BLOOD ETHANOL LEVEL ,,~ 300 F o
/
~ 200 II50 . ' / ' " o, i" , E
J
1.2
F- 0.8 z
~.O 0.6
PERIOD OF RECURRENT INHIBITION
-":. :: /"
1.0
0
J
• '. ..
~-
~
T'-
STIMULUS
'... ~
INTENSITY
".
/T
(THRESHOLD)
-' ,'~. . . . . \-a, ..... = . = - - - - 7 " ~ - - 3 T 0.4
ZT~
............X-- 2T
3TX" / /
0.2 0 0
"~'-o
.......
o--4T
/ aT o/ i 15
i 30
i 60
TIME POST ETHANOL INJECTION
i 120 (min)
Fig. 4. "lime plot of increase in period of PSI compared to blood ethanol levels. Period of PSI induced by stimulation of the dentate to CA3 pathway was measured from post-stimulus-time histograms at the indicated times for 4 different stimulus intensities, expressed as multiples of threshold (T) intensity. The increases were normalized (In T/C). A value of In T/C = 0 would indicate no change. The peak average increase in the period of PSI was seen at 30 rain and began to reverse by 60 min. Blood ethanol levels, however,rose to near maximum values by 15 min and remained high throughout the experiment.
pathway at 15 rain, but took measurements at 30 and 45 min or at 30 and 60 rain. The maximum change in the period of PSI was still noted at 30 min post-ethanol injection. Therefore, we conclude that the amount of prior stimulation had no effect on the measures.
Recovery The increase in the duration of PSI and the increases in excitability declined to at least 50 ~ of peak values by 60 rain post-injection. In most preparations recovery was complete by 120 min post injection. Recovery time was, however, highly dependent upon the levels of blood ethanol at 15 min after ethanol injection (Fig. 5). When the data from individual preparations were analyzed it was found that the more rapid recovery occurred in preparations in which blood ethanol levels reached 200 mg ~ or more during the first 15 min post-injection. Recovery in the length of PSI occurred even though blood ethanol levels remained high (Table II; Fig. 4). Preparations with low blood ethanol levels (i.e. < 200 mg ~ ) at 15 min post-injection did not recover significantly during the time we were able to follow the cell, contributing to a deceptively high average PSI duration at 120 min (see Table II).
Dose-response relationships The observed change in PSI duration did not follow the traditional S-shaped dose-response curve. Examination of Fig. 6 shows that there was no relationship be-
123 >120
~
o,
~: 1 2 0 w
•
•
N
N,,,o
,,o
z
@
o81
§ 9D w
o
6D
0
~
•
•
•
w
~
n ~
3c
_W
~'0
,$0
,~0 2~0
2&o 3~0
BLOOD E T H A N O L
AT
,~o
~0
,~o
(mg%)
15 MINUTES
Fig. 5. Time to 50% reversal of the increase in the length of post-stimulus inhibition as a function of 15 rain blood ethanol levels. Measurements of PSI induced by stimulation of the dentate to CA3 pathway were made prior to and at 30, 45, 60, 90 and 120 rain post-ethanol injection (3 g/kg i.p.). Data points represent individual measures in 15 different preparations. A value of one-half the maximum value measured was taken as a 50 % recovery of the ethanol-induced response. Measures were made for 4 different stimulus voltages (T to 4T) in 16 different preparations. Preparations in which blood ethanol levels reached values of greater than 200 mg % by 15 min were seen to recover from the increase in poststimulus inhibition much sooner than those preparations which achieved lower blood ethanol levels within 15 min.
tween the magnitudes of increase in inhibition measured at 15, 30, and 45 min and the different blood ethanol levels measured at the same time (open circles). However, if the blood ethanol level at 15 min was plotted against the maximum observed increase in PSI for any given preparation, then a convex function was noted; the magnitude of DOSE-RESPONSE MEASUREMENTS FOR LENGTH OF RECURRENT INHIBITION 28 J5 _~
24 20
"6
•
BLOOD ETHANOL CONCENTRATION VS O-LENGTH OF RECURRENT INHIBITION,
0
BLOOD ETHANOL CONCENTRATION OD
12
•
o8
o
•
VS @--PEAK LENGTH OF RECURRENT INHIBITION
o
eo
•
o
0.4 o 0
af 15 mln
•
i
# -&
o
o
.
.
.
.
.
.
.
.
• .
.
o .
.
.
.
.
-04
-:
.~o
,~o
,~o
'
266
'
z~o
360
. . 3 5.0 . . .400 . .
450
rncj % BLOOD EIOH
Fig. 6. Dose-response measurements for increases in the period of inhibition. The normalized increase (In T/C) in the period of post-stimulus inhibition induced by stimulation of the dentate to CA3 pathway is plotted as a function of two different measures of blood ethanol concentration. Data represent individual measures in 15 different preparations as indicated: Open circles ( © ) plot the length of PSI vs blood ethanol concentration at the time the measurement was made. There is no dose-response correlation. Closed circles (O) plot the peak length of inhibition (which usually occurred at 30 min - - see text) vs the blood ethanol concentration at 15 min. This plot shows a convex function. The length of inhibition is seen to rise as the 15 min blood ethanol levels rise between values of 50 mg % to 200 mg %. At higher values of 15-rain blood ethanol concentration the increase in inhibition is seen to decline as a function of blood ethanol concentration.
124 increase in inhibition rose at lower doses and fell at higher doses:i.e, the amount of increase in the length of PSI increased as a function of blood ethanol levels up to a blood ethanol level of 175 mg ~ ; at higher blood levels the function decreased with increasing ethanol concentrations. No relationship was found between measures of excitability (population spike or single unit spike counts) and blood ethanol levels. DISCUSSION Cellular actions o f ethanol on CA3 synaptic responses Injections of ethanol (3 g/kg, i.p.) increased the duration of inhibition in area CA3 after dentate stimulation in a dose-dependent manner. In contrast, the same treatments led to less consistent increases in excitability, whether measured by the population spike or by single unit spike frequencies, and changes in excitation were not dose-dependent: evoked single unit spikes were increased after ethanol in every preparation, and small increases in the population spike were observed occasionally. Our observed changes in population spike are opposite to those reported for CA1 by others10,11,14,15; these differences may be attributed to our in vivo preparation or the existence there of biochemical or functional processes not active in the slice preparation. The difference in area (CA3 as opposed to CA1) might also account for the different observations. These results indicate that parenteral treatment with ethanol influences synaptic transmission in hippocampus, but does not affect all pathways equally. Similar increases in both excitatory and inhibitory responses were noted after ethanol with stimulation of the commissural-CA3 pathway. Therefore, the effects of ethanol were not either pathway specific or transmitter specific. The transmitter of the commissural pathway is reported to be glutamate or aspartate25; the transmitter of the mossy fiber pathway remains unknown. The transmitter mediating recurrent inhibition for both pathways is reported to be GABA1, 5,7, as is the transmitter which is thought to mediate feedforward inhibition1, 5. A few measures (not reported here) were also made from the commissural to CA 1 pathway, the CA3 to CA 1 (Schaeffer collateral) pathway, and the commissural to dentate pathway. In each of these cases, increases of similar magnitudes in the length of PSI as well as increases in population spike amplitude were observed following ethanol, suggesting that these responses may be generalized throughout the hippocampus. The increases in excitability showed no correlation with the increase in PSI - - the magnitudes of change varied independently, and their time courses were also quite different. We conclude, therefore, that the increases in excitation and the increases in inhibition are functionally separable, and cannot, for example, be explained as a generalized increase in pyramidal cell responsiveness to all synaptic input. Both the increases in excitability and the increases in the period of PSI returned towards control levels within 2 h; however, blood ethanol levels remained high throughout the course of the experiments. Furthermore, we observed that the higher the blood ethanol levels at 15 rain post-injection, the sooner the length of PSI returned
125 towards control measures. These observations suggest that the whole animal preparations may have acutely adapted to ethanol; a similar adaptation with loss of response to an ethanol effect was observed on synaptic post-tetanic potentiation in Aplysia3z. Recent studies in our laboratory by Rogers et al. 27 have shown that in rats chronically exposed to ethanol, cerebellar Purkinje neurons clearly exhibit compensatory changes in firing patterns which may reflect adaptive changes in response to ethanol. Animals exposed to intoxicating doses of ethanol over a period of several weeks 2s and then withdrawn, showed a considerably shortened period of PSI in CA3 in response to dentate stimulation. Thus the return of the duration of PSI towards control values which is noted within two hours may be an early manifestation of this compensatory adjustment.
Time relationships Although blood ethanol levels reached a plateau at 15 min, the maximum increases in PSI did not occur until 30 min post-ethanol injection. The lag in time between maximum blood ethanol concentrations and peak response suggests the possibility that some biochemical by-product or physiological processg,12,~6,37 is necessary to elicit the maximum effect. Whole brain ethanol levels closely follow blood ethanol levels31. However, uptake of ethanol into some critical synaptic or lipid components13,18,21,29,35-37 may not parallel blood ethanol levels and could also account for the lag time in effect onset. There also remains the possibility that either an ethanol metabolite or an ethanol-induced novel metabolite of some endogenous factor must be produced 9,12, and that its concentration is the critical determinant in the magnitude of effect measured.
Dose-response relationships The levels of blood ethanol achieved in individual preparations varied among subjects even though a similar dose was injected. Study of dose-response relationships was therefore possible. When plotting the peak increase in PSI against the level of blood ethanol at 15 min post-injection, we observed a convex function ; the amount of change increased with blood ethanol concentrations up to 200 mg ~, and then declined with higher blood ethanol levels. There was no relationship between the increase in inhibition and the blood ethanol levels at the time the measurement of inhibition was made. Investigators using rat performance on a tilting plane as a measure of intoxication have also found that their behavioral measure (presumably mediated by GABA) is similarly related to the levels of blood ethanol reached shortly after injection19. Additional experiments will be required to explore the changes in response when lower doses yield lower blood levels at longer post-injection periods. Grupp and Perlanski TM have reported that lower doses of ethanol produce excitatory effects (i.e. speeding of single unit spontaneous firing) in the hippocampus, while higher doses produce inhibitory effects (i.e. slowing of firing rate). Our observations do not follow this trend. The excitatory effects which we observed showed no dose relationship; the inhibitory effects are greatest at a middle range of 15-min blood ethanol concentration, and smaller at both low and high 15 min blood ethanol levels.
126 Possible interactions o f halothane and ethanol Previous reports have indicated pharmacological and biochemical interactions between halothane and ethanol and that halothane may lower blood ethanol concentrations over time~0, 84. Such effects are unlikely to alter our experiments, however, since we studied only acute ethanol administration and blood ethanol levels thereafter remained high. Furthermore, we observed that the level of halothane anesthesia (between 0.75 ~ and 3.0 Y/o)had no bearing on the magnitude of the evoked responses we report. Further testing in unanesthetized preparations is, of course, necessary to verify the conclusion that halothane does not modify the effects of ethanol.
CONCLUSIONS Ethanol can increase the length of inhibition following afferent pathway stimulation. The effect shows distinct dose relationships and acute tolerance. This increase in PSI could be due to increases in the function of either of two GABA-mediated pathways which have been described for the dorsal hippocampus 1,4,5. However, other transmitters are known to have an effect on PSI. For example, Zieglgansberger et al. 3s have shown that enkephalin can shorten and block the period of PSI. In preliminary studies (not published) we f o u n d that serotonin may produce a similar effect. Norepinephrine is also known to be active in the hippocampus3°; and changes in norepinephrine sensitivity have been implicated in ethanol effects in the cerebellum 27. Bloom has suggested that any of these transmitter systems can heterosynaptically modulate primary synaptic events a. Therefore, any of the 'modulating' transmitter systems could also be implicated in the effect of ethanol on PSI. The effects could also be due to general changes in membrane resistance caused by ethanol zl. Pharmacological studies with antagonists and neurotransmitter analogues will be necessary in order to determine if a specific neurotransmitter system is responsible for the effects reported here. Intracellular recording will help determine possible membrane actions of ethanol and any contribution of an after hyperpolarization to the response. The hippocampal model could be a useful tool in testing for specific transmitter interactions. ACKNOWLEDGEMENTS We wish to thank Sam Madamba for excellent technical assistance, Drs. Steven Henriksen, George Siggins, Ed French and Ted Berger for technical advice and comments on the manuscript, Klaus Liebold for computer programming and Nancy Callahan for typing the manuscript. S.A.N. was supported by a grant from the National Institute on Alcohol Abuse and Alcoholism and the Ruth Harvey Foundation.
REFERENCES 1 Alger, B. E. and Nicoll, R. A., Biphasic IPSPs in rat hippocampal slice, Neurosci..4bstr., 5 (1979) 2472. 2 Andersen, P., Blackstad, T. W. and Lomo, T., Location and identification of excitatory synapses on hippocampal pyramidal ceils, Ex. Brain Res., 1 (1966) 236-248.
127 3 Andersen, P., Bliss, T. V. P. and Skred¢, K. K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208-221. 4 Andersen, P., Eccles, J. C. and Loyning, Y., Pathway of postsynaptic inhibitionin the hippocampus, J. Neurophysiol., 27 (1964) 608-619. 5 Ayala, G. F. and Thalmann, R. H., A biphasic IPSP in pyramidal neurons of hippocampal slices in the presence of phenobarbital, Neurosci. Abstr., 5 (1979) 2476. 6 Blackstad, T. W., Commissural connections of the hippocampal region of the rat, with special reference to their mode of termination, J. comp. Neural., 105 (1956) 417-536. 7 Bloom, F. E. and iversen, L. L., Localizing 3H-GABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography, Nature (Lond.), 229 (1971) 628-630. 8 Bloom, F. E., Is there a neurotransmitter code in the brain? Advanc. Pharmacol. Ther., 2 (1979) 205-213. 9 Cohen, G., Alkaloid products in the metabolism of alcohol and biogenic amines, Biochem. Pharmacol., 25 (1976) 1123. 10 Corrigal, W. A., Carlen, P. L. and Stainman, A. L., Acute effects of ethanol on hippocampal CA1 field potentials recorded in vitro, Neurosci. Abstr., 3 (1977) 288. 11 Corrigai, W. A. and Carlen, P. L., Ethanol: electrophysiological evidence of tolerance remains after isolation of the hippocampus in vitro, Neurosci. Abstr., 4 (1978) 1329. 12 Deitrich, R. A. and Erwin, V. G., Involvement of biogenic amine metabolism in ethanol addiction, Fed. Proc., 34 (1975) 1962-1967. 13 Denovo, E. C., Gruber, B. and Noble, E. P., Alterations of fast-reacting sulfhydryl groups of rat brain microsomes by ethanol, Biochem. biophys. Res. Commun., 68 (1976) 975-981. 14 Doller, H. J., Eckhardt, M. J. and Weight, F. F., Effects of ethanol on the hippocampal slice preparation, Fed. Proc., (1980) No. 80. 15 Durand, D. and Carlen, P., Slow potentiation of CA1 hippocampal slice field potentials and acute effects of low-dose ethanol, Neurosci. Abstr., 5 (1979) 1875. 16 Faber, D. S. and Klee, M. R., Actions of ethanol on neuronal membrane properties and synaptic transmission. In Alcohol and Opiates: Neurochemical and Behavioral Mechanisms, Academic Press, New York, 1977, pp. 41-63. 17 Fischer, E. and Wallgrew, H., Cerebral blood flow and cerebral alcohol uptake of anesthetized dogs, Physiologist, 1 (1957) 27. 18 Grupp, L. A. and Perlanski, E., Ethanol-inducedchanges in the spontaneous activity of single units in the hippocampus of the awake rat: a dose-response study, Neuropharmacology, 18 (1979) 63-70. 19 Hakkinen, H.-M. and Kulonen, E., Ethanol intoxication and ~'-aminobutyric acid, J. Neurochem., 27 (1976) 631-633. 20 Han, Y. H., Why do chronic alcoholics require more anesthesia? Anesthesiology, 30 (1969) 341-2. 21 Israel, Y. and Kuriyama, K., Effects of in vivo ethanol administration on adenosine triphosphatase activity of subcellular fraction of mouse brain and liver, Life Sci., 10 (1971) 591-599. 22 Kalant, H., Ethanol and the nervous system, Exp. neurophysiol., Asp., 9 (1974) 111-124. 23 Klemm, W. R. and Stevens, R. E., IlI, Alcohol effects on EEG and multiunit activity in various brain regions of rats, Brain Research, 70 (1974) 361-368. 24 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain andLower Parts of the brain Stem, Williams and Wilkins, 1963. 25 Nadler, J. V., Vaea, K. W., White, W. F., Lynch, G. S. and Cotman, C. W., Aspartate and glutamate as possible transmitter of excitatory hippocampal afferents, Nature (Lond.), 260 (1976) 538-540. 26 Ranck, J. B., Jr., Behavioral correlates and firing repertoires of neurons in the dorsal hippocampal formation and septum of unrestrained rats. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. 2, Plenum Press, New York, 1975, pp. 207-244. 27 Rogers, J., Siggins, G. R., Schulman, J. A. and Bloom, F. E., Physiological correlates of ethanol intoxication, tolerance, and dependence in rat cerebellar Purkinje cells, Brain Research, in press. 28 Rogers, J., Weiner, S. G. and Bloom, F. E., Long term ethanol administration methods for rats: advantages of inhalation over intubation or liquid diets, Behav. neur. Biol., 27 (1979) 466-486. 29 Sceman, P., The membrane actions of anesthetics and tranquilizers, Pharmacol. Rev., 24 (1972) 583-655. 30 Segal,M. and Bloom, F. E., The action of norepinephrine in the rat hippocampus. IV. The effects
of locus coeruleus stimulation on evoked hippocampal unit activity, Brain Research, 107 (1976) 513-525. 31 Siggins, G. R. and Bloom, F. E., Alcohol-related electrophysiology. In Proc. 5th Biennial Int. Syrup. Alcoholism, Drug Alcohol Dep., in press.
128 32 Traynor, A. E., Schlapfer, W. T. and Barondes, S. H., Stimulation is necessary for the development of tolerance to a neuronal effect of ethanol, in preparation. 33 Traynor, M. E., Woodson, P. B. J., Schlapfer, W. T. and Barondes, S. H., Sustained tolerance to a specific effect of ethanol on post-tetanic potentiation in Aplysia, Science, 193 (1976) 510-511. 34 Ugarte, G., Pino, M. E., Peroda, T. and Iturriagia, H., Increased blood ethanol elimination in rats treated with halothane, Pharmacology, 9 (1973) 275-280. 35 Volicer, L. and Hurter, B. P., Effects of acute and chronic ethanol administration and withdrawal on adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate levels in the rat brain, J. Pharmacol. exp. Ther., 200 (1977) 298-305. 36 Wallgreen, H., Nikander, P., von Boguslawsky, P. and Linbola, J., Effects of ethanol, butanol and clomethiazole on net movements of sodium and potassium in electrically stimulated cerebral tissue, Actaphysiol. scand., 91 (1974) 83-93. 37 Wallgreen, I-I., Nikander, P. and Virtanero, P., Ethanol-induced changes in cation-stimulation adenosine triphosphatase activity and lipid-proteolipid labeling in brain microsomes, Advanc. exp. Med. BioL, 59 (1975) 23-36. 38 Zieglg~nsberger, W., French, E. D., Siggins, G. R. and Bloom, F. E., Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory neurons, Science, 205 (1979) 415--417.
I 13
E T H A N O L CAUSES INCREASES IN EXCITATION A N D I N H I B I T I O N IN AREA CA3 OF T H E DORSAL HIPPOCAMPUS
S. A. NEWLIN, JORGE MANCILLAS-TREVINO and F. E. BLOOM
Arthur V. Davis Center for Behavioral Neurobiology, The Salk Institute, P.O. Box 85800, San Diego, Calif. 92138 (U.S.A.)
(Accepted August 28th, 1980) Key words: synaptic effects - - inhibition -- amine modulation - - tolerance - - ethanol metabolite
SUMMARY The effects of ethanol on synaptic transmission in hippocampus were studied by changes in the response of CA3 pyramidal cells to stimulation of two afferent pathways, the dentate (mossy fiber) pathway, and the commissural pathway. Both sources produce an excitatory response as measured either by single unit spiking or by population spike followed by a period of post-stimulus inhibition (PSI). After the injection of 3 g/kg ethanol (i.p.), both the excitatory responses and the duration of PSI are significantly increased. Because these changes occur in both afferent pathways, they are not pathway specific, but may be the result of the local microcircuitry in area CA3. Although the change in excitatory and inhibitory responses can occur simultaneously, detailed statistical analyses show that neither the magnitude nor onset times are correlated. Thus, the two responses are functionally separable. In addition, the increase in the duration of PSI is related to the rate of rise of blood ethanol level and shows short-term tolerance. The maximum change in the PSI occurs after blood ethanol levels plateau suggesting a secondary process is necessary before neurophysiological effects are apparent.
INTRODUCTION Many studies have indicated an electrophysiological effect of ethanol on central neurc.ns 16,22. Both excitatory and inhibitory effects have been described. Most studies have utilized single unit or multi unit recordings of spontaneous rate as an index of excitability. However, we were also interested in possible effects of ethanol on specific synaptic events and on specific neurotransmitters. Our laboratory has, therefore, designed experiments to test the effects of ethanol in several well-defined brain regions.
114 We have recently published observations of the effects of acute and chronic ethanol on cerebellar Purkinje neurons 27. The hippocampus is also an excellent model for the study of neuronal actions of ethanol. Its synaptic connections are well-defined and documented 2,a, transmitters have been well defined in several of the pathways1,5,7, 25, identifiable test cells are spontaneously active 26. Furthermore, preliminary studies have shown that ethanol does have an effect on the neurophysiology of the hippocampus. Changes have been reported in the EEG and in multi-unit activity z3, in spontaneous activity of single units la, and in evoked population responses in vitro1°, 11,14. These changes were usually depressive. In order to locate specific synaptic changes which might underly those general activity changes we have studied the effect of ethanol on evoked single unit and population responses in area CA3 of the dorsal hippocampus. We have found increases both in excitation and inhibition. METHODS Male, Sprague-Dawley rats (Charles Rivers Breeding Company) between 250 and 350 g were used. The rats were anesthetized with halothane (3 ~ during surgery, 1 ~ during the experimental measures) via a tracheal cannula, and held in a stereotactic apparatus. Bone and dura over the dorsal hippocampus were removed. Recording electrodes were placed in area CA3 of the dorsal hippocampus (A 3500/~m, L 3500/~m, V 3300-4000/~m 24); placement was verified during the experiment by field potentials elicited by dentate pathway 2 or commissural pathway 6 stimulation. At the end of each experiment Pontamine sky blue dye was ejected with pulsed DC negative current (2.5 #A, 15 min), and placement was verified by histological examination. Recording electrodes were glass micropipettes filled with Pontamine sky blue in 3 M acetate solution (pH = 7.4) and broken to tip diameters of 1-2#m; resistances were 5-10 MfL Bipolar stimulating electrodes were constructed from two 100 #m teflon-coated stainless steel wires glued in parallel with Stoner-Mudge bond (Mobil Oil Corp.). Stimulating electrodes were placed in the contralateral hippocampus (A3500 /~m, L3500/zm, V3000/tm 24) for commissural pathway stimulation, or in the ipsilateral dentate gyrus (A3500 #m, L2200 #m, V3600 #m ~4)for mossy fiber pathway stimulation. At the end of each experiment a lesion was made by application of constant current (15/~A, 5 min) through the electrode tip, and electrode placement verified by histological examination. Simultaneous recording of evoked population responses and extracellular single unit activity (see Fig. 1) was done by branching the output of a high impedance preamplifier through different filtering systems. Population responses were displayed on a storage oscilloscope after 10 Hz low frequency filtering. The signal for single unit responses of 600--1200/zV amplitude was filtered by a series of filters (10, 300 and 600 Hz low frequency and 30, 10, and 10 kHz high frequency) to eliminate the population response from the signal, then fed through a window discriminator while monitoring
115 on an oscilloscope. The square wave pulse output from the discriminator integrated over 100 msec intervals was monitored by chart recorder as firing rate, and was fed to a Minc II (Digitimer)computer for on-line production of post-stimulus time histograms (PSTHs) (software by K. Liebold, DECUS). Stimulating currents were expressed as multiples of 'threshold' for all parameters measured, where 'threshold' current was the minimum current necessary to elicit a period of PSI. The range of threshold currents was 50-150/zA. The logn (test measure/control measure; In T/C) was computed for magnitude population responses, spike counts following stimulation, and the period of PSI following afferent pathway stimulation under control conditions; measures were then repeated in the same animal at 15, 30, 45, 60 and 120 min following the injection of ethanol or vehicle. Responses to stimulation were measured as follows: 'post-stimulus inhibition' was measured using post-stimulus time histograms as described in Fig. 2. Population EPSPs were measured in the stratum radiatum as described by Anderson et al.2 and Blackstad6 and population spikes were measured in the striatum pyramidale 2,6 and measured from the first positivity to the following negativity (Fig. 1). Ethanol was diluted to 30~ (w/v) in distilled water and injected i.p. via an indwelling 21-gauge, 0.75 in. needle which had been inserted before experimentation. The injected dose was 3 g/kg in all cases; total volume injected varied from 2.5 to 3.5 ml. The total volume was injected over a period of 10 min to prevent pressure trauma to the gut. We measured 'time post ethanol' from the time point concluding injection. Blood samples were removed from the tail vein at 5 min post-injection and following each experimental measure (i.e. also at 15,30,45,60 and 120 min). Blood samples were spun in a microhematocrit centrifuge, and the plasma decanted for assay, using the Calbiochem-Behring Ethyl Alcohol Reagents Kit. Measures in our laboratory (Shoemaker and Bloom, pers. commun.) and by others 17 have shown that blood ethanol levels do not differ significantly from brain ethanol levels. RESULTS
Blood ethanol levels Blood ethanol levels rose to significant levels within 5 min (Table I). Rates of rise differed greatly between preparations, most reaching a plateau value by 15 min and some reaching a plateau value at 30 or 45 min; on the average maximum blood ethanol levels were reached by 15 min post-injection. Peak blood ethanol concentrations ranged from 110 to 440 mg~o; average peak concentration was 242 :k 30 m g ~ . Blood ethanol levels did not decline throughout the course of the experiments, usually 2-3 h. Increases in the period of post-stimulus inhibition Stimulation of the dentate (mossy fiber) pathway to area CA3 of the dorsal hippocampus produced an excitatory response followed by a period of inhibition, which can be monitored by extracellular single unit recording3 (see Fig. 1). The period of inhibition has been described as being due to a recurrent collateral and interneuron feedback4, but could also receive a contribution from a feedforward pathway1. In this
116 TABLE I Blood ethanol levels Blood was removed from the tail vein. Ethanol assay is described in Methods section, n = number of preparations in which assay was made. Errors are standard error of the mean. Time postinjection (min)
n
Blood ethanol concentration (mg%)
5 15 30 45 60 90 120
(20) (18) (15) (8) (17) (12) (11)
189 242 243 222 274 262 263
A
i 24 ± 30 4- 27 -4- 22 ~ 22 -4- 27 i 29
. III.. IILIII.I~I Ill llllr, llllJll II 11
200yV L I see
'II1",,[ Illrl,, I11111111 [1[ CONTROL
30 MIN POST EtOH 200~V
B t
c,_ !
STIMULUS
t IOOjlV L_. 200 msec
t
STIMULUS
Fig. 1. Recordings from stratum pyramidale of area CA3 of the dorsal hippocampus. A: spontaneous single unit activity. Stimulation of dentate gyrus produced an excitatory response (see B and D) followed by inhibition of spontaneous spiking. One sweep. Stimuli are 1 per 5 sec. See Methods for recording procedures. B: evoked single unit spike following stimulation of dentate gyrus: control and 30 rain post-ethanol injection (3 g/kg). C: recording of single unit activity following dentate stimulation: control and 30 rain post-ethanol injection. Each tracing is the sum of 30 sweeps. Recordings show inhibition following the stimulus artifact, with resumption of spontaneous activity. The length of the inhibitory period is increased in the presence of ethanol. D : population response elicited by stimulation of the dentate gyrus: control and 30 rain post-ethanol injection. The amplitude of the population spike (*) is increased in the presence of ethanol.
117
CONTROL ~C:3
z ~o
ETHANOL
TREATED
DCO D_N.
I0
stimulus
Fig. 2. Post-stimulus-time histograms. On-line, computer-generated post-stimulus-time histograms for one preparation are shown for control and 30 min post-ethanol injection (3 g/kg). Each histogram sums spike counts for 30 stimuli (12 Hz). The length of post-stimulus inhibition, taken as the number of msec following the stimulus artifact in which no spikes are counted, is seen to increase following ethanol injection. paper we will refer to the inhibitory period as 'post-stimulus inhibition' (PSI), and it is taken as the length of time, following 30 consecutive stimuli (Fig. 3), in which no spikes were observed. Post stimulus-time histograms (PSTHs) were used to sum the responses to 30 stimuli at each stimulating voltage. The length of PSI (measured in msec) increased with increasing stimulus voltage. For a given stimulus voltage, the period of PSI was consistently increased following the injection of ethanol (3 g/kg, i.p.), thus shifting the stimulus-response curve to the left (Fig. 2; Table II). The maximum increase in inhibition usually occurred at 30 minutes post ethanol injection. For statistical purposes, changes in the length of inhibition were analyzed in three ways. (1) The length of PSI (measured in msec) was measured for each stimulus intensity under control conditions and at 30 min following the administration of ethanol, and then averaged for all cells. By this measure, increases were observed at
* P < 0.025;
** P < 0.01;
9.4 (15) 18.6 (15) 26.4 (13) 46.4 (11)
24.7(15)** 44.0 (15)*** 58.7 (13)* 59.9 (I1)***
0.599 0.361 0.332 0.328
i 4± 4-
15 min
In TIC
0.070(13) 0.144 (12)* 0.127 (10)* 0.113 (10)*
*** P < 0.005, paired t-test.
± ± 44-
111.5 252.3 339.8 519.3
± ± 4±
T 2T 3T 4T
42.0 115.2 227.6 328.9
30 min Post-EtOH ( msec )
Stimulus Control (msec) intensity
0.861 1.329 0.514 0.427
q- 0.252(15)*** ± 0.841 (15)* d_ 0.111 (13)*** -4- 0.116 (11)***
30 min 0.384 0.484 0.481 0.299
± 0.206(15)* -+ 0.146 (14)*** 4- 0.104 (13)*** 5_ 0.088 (11)***
60 min
0.525 0.380 0.491 0.271
~± i ±
120 min 0.209 0.153 0.073 0.174
(10)* (9)* (9)*** (9)
Length of post-stimulus inhibition is the period of time (msec) following the stimulus in which no spike activity occurred. Measurements were taken from post-stimulus-time histograms (see Fig. 2); 30 stimuli were given at a rate of 12 Hz. Stimulus intensities were expressed as multiples of threshold (T), where threshold was the minimum current necessary to produce inhibition. Length of post-stimulus inhibition is represented in msec for control measures and for 30 min post-injection of 3 0 ~ ethanol (3 g/kg, i.p.), and also as a normalized value. Normalized values were computed as In (test msec/control msec or (In T/C) for each preparation at 4 different times post-ethanol injection, and then averaged. Numbers in parentheses indicate numbers of preparation in which measurements were taken.
Length o f post-stimulus inhibition dentate to C.43 pathway
TABLE II
Oo
119 each stimulus intensity (T through 4T) and were significantly different from control measures (Table II). (2) Normalized changes were computed by the formula ln[test msec/control msec], (ln T/C); this computation gives equal arithmetical weight to increases and to decreases in the length of PSI. Such normalized computations were made for each stimulus voltage for each testing time; the averages are reported in Table II and show increased PSI after alcohol. There is an increase in the time of inhibition for each stimulus voltage tested. (3) The average normalized increase (In T/C) in PSI for all stimulus intensities, measured 15-45 min post-ethanol injection, for 18 cells, was 0.553 4- 0.084, n = 153. As a control measure a similar volume of vehicle (distilled water) was injected in 5 preparations. The normalized increase in PSI was 0.089 ~: 0.042, n ---- 52. Measures for the ethanol injected preparations differed significantly from that for the water-injected preparations (P < 0.001, Student's t-test). To test for pathway specificity, similar measurements were made for the commissural to CA3 pathway, where stimulation also produces an excitation in CA3 neurons, followed by a period of inhibition. By all statistical measures, the period of PSI was significantly increased for stimulus voltages of threshold and three times threshold (Fig. 3 and Table III) and differed significantly from water-injected controls (P < g
CONTROL G
m-,
.... I ...... h,~ ........... °o'. oo 7~.]q [~6.29 ~ 2~9.U3
/
,,=,
292.57
3~5.71
~8.86 ---
0-
_~-
ID (_)
ETHANOL
TREATED
"-t-
III
~:oo
.
.
.
.
.
.
7~.1q
.
.....
IJ..L
t~s.29 t 2'19.~3 /
stimulus
2b;.s7
3'6s.TJ
,~.~s
TIME - MSEC
Fig. 3. Expanded time scale post-stimulus-time histograms, control and 30 min post-ethanol injection (3 g/kg, i.p.). Each histogram sums the response to 30 stimuli (12 Hz). Spontaneous activity can be observed for 200 msec prior to the stimulus artifact; spikes can be counted following the stimulus artifact (0.2 msec), followed by the period of inhibition. A greater number of stimulus-evoked spikes occur in the ethanol-treated animal.
120 TABLE III Length of post-stimulus inhibition, commissural to CA3 pathway Length of post-stimulus inhibition was measured as described in caption to Table II and is expressed as raw data (msec) and as averaged, normalized data (In T/C) at 30 rain post-injection of 30 ~ ethanol (3 g/kg ip.). Numbers in parentheses indicate numbers of preparations. Intensity
Control (msec)
30 min post-EtOH ( msec)
In TIC 30 min
T 3T 5T
84.6 :k 26.4 (4) 283.8 i 77.0 (4) 508.6 ::k 138.8 (4)
182.3 ± 22.9 (4)** 462.2 -- 102.8 (4)* 506.6 ± 113.9 (4)
1.293 ± 0.461 (4)** 0.540 ± 0.157 (4)*** 0.096 ± 0.189 (4)
* P < 0.10;
** P < 0.05;
*** P < 0.025, paired t-test.
0.025, Student's t-test). We were able to test higher stimulus intensities (5 times threshold) with the stimulating electrode in the contralateral hippocampus, without significant current spread to the site of the recording electrode. N o significant increase in the length of PSI was noted at this higher stimulus intensity. Increases in excitability Excitation of pyramidal cell n e u r o n s was measured in two different ways: (1) single unit responses a n d (2) p o p u l a t i o n responses. In 4 of the 18 cells studied we could observe single unit spikes in response to dentate stimulation. Spikes could be counted with the aid of expanded-time P S T H s such as seen in Fig. 3. In all cells where such excitatory responses were obtained, we observed, after 30 stimuli, a n increase in the total n u m b e r of spikes for any given stimulus voltage following the injection of ethanol (Table IV). TABLE 1V Increases in excitability following ethanol administration dentate to CA3 path way All measures are represented as the average normalized increase (In T/C). Spike counts were read by the computer from expanded time post stimulus time histograms (such as shown in Fig. 4). Spike counts were summed for 30 stimuli at each stimulus intensity for 6 different preparations. Average counts were: T, 70.5 ± 11.5; 2T, 128.3 i 20.8; 3T, 134.0 ± 15. Measures were then taken at 30 and 45 min post-ethanol injection;normalized increases (In T/C) were computed and then averaged. The population spike (see Fig. 1) was measured at the pyramidal cell layer of area CA3 of the dorsal hippocampus. Responses were recorded as the average of three measures at each of 3 different stimulus intensities. Average control amplitudes were: T 0.22 ± 0.79 mV; 2T, 1.29 ± 0.08 mV; 3T, 1.67 ± 0.18 mV. Measures were then taken at 30 and 45 rain post-ethanol in 12 different preparations, normalized increases (In T/C) were computed and then averaged. The population EPSP was measured in the stratum radiatum of area CA3 in 5 different preparations. Average control amplitudes were: T, 2.31 ± 0.93 mV; 2T, 2.99 ± 1.07 mV; 3T, 3.83 ± 1.49 inV. Stimulus intensities were in multiples of threshold (T), which was the minimum intensity necessary to elicit a period of PSI in each preparation. Response measured
In T/C
P (paired t-test)
Spike counts Population spike Population EPSP
0.25 ± 0.11 (28)** 0.09 ± 0.07 (69)* 0.15 ± 0.10 (28)
<0.025 < 0.05 <0.20
121 Population responses were observed in all preparations following dentate pathway stimulation. Population responses were measured with stimulus frequencies of 1/5 sec and 1/20 sec. No facilitation of responses was noted at either stimulus frequency. The 'population EPSP' was measured with the recording electrode in the stratum radiatum of area CA3, and the 'population spike' with the recording electrode in the stratum pyramidaleL In 8 of 11 preparations the population spike increased following ethanol administration, in 2 preparations the population spike decreased, and there was no change in one preparation. The average change in the size of the population spike was a small, but significant, increase (Table IV). The size of the population EPSP showed no consistent trend across or within preparations. On the average it showed a small and insignificant, increase. Preparations receiving water injections showed no significant increase in any measure of excitability. Similar increases in excitability were noted with comlnissural pathway stimulation in the presence of ethanol, but were not formally measured. Changes in excitatory responses do not correlate with changes in the inhibitory response Since the increases in both excitation and inhibition were noted, one might hypothesize that the effect of ethanol was to increase pyramidal cell responsiveness to afterent excitatory drive, and thus increase the amount of recurrent inhibitory interneuron activation, or, alternatively, that the effect of ethanol was to increase pyramidal cell responsiveness to all synaptic input simultaneously. In either case, one might then expect a correlation between the change in amount of excitation and the amount of inhibition. However, such a correlation was not observed. There was no statistical correlation for the amount of change between either measure of increased excitability (population spike or single unit counts) and the increase in length of PSI. The correlation factor for In T/C for the length of PSI vs In T/C for population spike amplitude, measured at the same voltages, was 0.119 (n -- 16); and the correlation factor for the length of PSI vs spike counts (In test/control) was 0.129 (n =: 27). Furthermore, we noted that the peak increase in measures of excitability did not necessarily occur at the same time post ethanol injection as the increase in inhibition and in some preparations the maximum change in population spike occurred at a later time. Time course of effects The duration of PSI was noticeably increased at the first measurement after ethanol injection (15 min) and continued to increase, usually reaching a maximum length at 30 min post-injection. Fig. 4 shows a time plot of the average of changes in PSI (expressed as In T/C). Individual preparations typically followed the same pattern. The peak change in population spike amplitude (whether increase or decrease) occurred between 30 and 60 min, and the same was true for increases in spike counts. Increases in excitability and the increase in length of PSI both occurred at a time point later than the time where the plateau in blood ethanol levels was reached (Table I and Fig. 4). We considered the possibility that stimulus history might be important for the expression of ethanol effect or in the development of tolerance, as has been shown for other preparationsa2,a6,a7. In several preparations we did not stimulate the dentate
122 BLOOD ETHANOL LEVEL ,,~ 300 F o
/
~ 200 II50 . ' / ' " o, i" , E
J
1.2
F- 0.8 z
~.O 0.6
PERIOD OF RECURRENT INHIBITION
-":. :: /"
1.0
0
J
• '. ..
~-
~
T'-
STIMULUS
'... ~
INTENSITY
".
/T
(THRESHOLD)
-' ,'~. . . . . \-a, ..... = . = - - - - 7 " ~ - - 3 T 0.4
ZT~
............X-- 2T
3TX" / /
0.2 0 0
"~'-o
.......
o--4T
/ aT o/ i 15
i 30
i 60
TIME POST ETHANOL INJECTION
i 120 (min)
Fig. 4. "lime plot of increase in period of PSI compared to blood ethanol levels. Period of PSI induced by stimulation of the dentate to CA3 pathway was measured from post-stimulus-time histograms at the indicated times for 4 different stimulus intensities, expressed as multiples of threshold (T) intensity. The increases were normalized (In T/C). A value of In T/C = 0 would indicate no change. The peak average increase in the period of PSI was seen at 30 rain and began to reverse by 60 min. Blood ethanol levels, however,rose to near maximum values by 15 min and remained high throughout the experiment.
pathway at 15 rain, but took measurements at 30 and 45 min or at 30 and 60 rain. The maximum change in the period of PSI was still noted at 30 min post-ethanol injection. Therefore, we conclude that the amount of prior stimulation had no effect on the measures.
Recovery The increase in the duration of PSI and the increases in excitability declined to at least 50 ~ of peak values by 60 rain post-injection. In most preparations recovery was complete by 120 min post injection. Recovery time was, however, highly dependent upon the levels of blood ethanol at 15 min after ethanol injection (Fig. 5). When the data from individual preparations were analyzed it was found that the more rapid recovery occurred in preparations in which blood ethanol levels reached 200 mg ~ or more during the first 15 min post-injection. Recovery in the length of PSI occurred even though blood ethanol levels remained high (Table II; Fig. 4). Preparations with low blood ethanol levels (i.e. < 200 mg ~ ) at 15 min post-injection did not recover significantly during the time we were able to follow the cell, contributing to a deceptively high average PSI duration at 120 min (see Table II).
Dose-response relationships The observed change in PSI duration did not follow the traditional S-shaped dose-response curve. Examination of Fig. 6 shows that there was no relationship be-
123 >120
~
o,
~: 1 2 0 w
•
•
N
N,,,o
,,o
z
@
o81
§ 9D w
o
6D
0
~
•
•
•
w
~
n ~
3c
_W
~'0
,$0
,~0 2~0
2&o 3~0
BLOOD E T H A N O L
AT
,~o
~0
,~o
(mg%)
15 MINUTES
Fig. 5. Time to 50% reversal of the increase in the length of post-stimulus inhibition as a function of 15 rain blood ethanol levels. Measurements of PSI induced by stimulation of the dentate to CA3 pathway were made prior to and at 30, 45, 60, 90 and 120 rain post-ethanol injection (3 g/kg i.p.). Data points represent individual measures in 15 different preparations. A value of one-half the maximum value measured was taken as a 50 % recovery of the ethanol-induced response. Measures were made for 4 different stimulus voltages (T to 4T) in 16 different preparations. Preparations in which blood ethanol levels reached values of greater than 200 mg % by 15 min were seen to recover from the increase in poststimulus inhibition much sooner than those preparations which achieved lower blood ethanol levels within 15 min.
tween the magnitudes of increase in inhibition measured at 15, 30, and 45 min and the different blood ethanol levels measured at the same time (open circles). However, if the blood ethanol level at 15 min was plotted against the maximum observed increase in PSI for any given preparation, then a convex function was noted; the magnitude of DOSE-RESPONSE MEASUREMENTS FOR LENGTH OF RECURRENT INHIBITION 28 J5 _~
24 20
"6
•
BLOOD ETHANOL CONCENTRATION VS O-LENGTH OF RECURRENT INHIBITION,
0
BLOOD ETHANOL CONCENTRATION OD
12
•
o8
o
•
VS @--PEAK LENGTH OF RECURRENT INHIBITION
o
eo
•
o
0.4 o 0
af 15 mln
•
i
# -&
o
o
.
.
.
.
.
.
.
.
• .
.
o .
.
.
.
.
-04
-:
.~o
,~o
,~o
'
266
'
z~o
360
. . 3 5.0 . . .400 . .
450
rncj % BLOOD EIOH
Fig. 6. Dose-response measurements for increases in the period of inhibition. The normalized increase (In T/C) in the period of post-stimulus inhibition induced by stimulation of the dentate to CA3 pathway is plotted as a function of two different measures of blood ethanol concentration. Data represent individual measures in 15 different preparations as indicated: Open circles ( © ) plot the length of PSI vs blood ethanol concentration at the time the measurement was made. There is no dose-response correlation. Closed circles (O) plot the peak length of inhibition (which usually occurred at 30 min - - see text) vs the blood ethanol concentration at 15 min. This plot shows a convex function. The length of inhibition is seen to rise as the 15 min blood ethanol levels rise between values of 50 mg % to 200 mg %. At higher values of 15-rain blood ethanol concentration the increase in inhibition is seen to decline as a function of blood ethanol concentration.
124 increase in inhibition rose at lower doses and fell at higher doses:i.e, the amount of increase in the length of PSI increased as a function of blood ethanol levels up to a blood ethanol level of 175 mg ~ ; at higher blood levels the function decreased with increasing ethanol concentrations. No relationship was found between measures of excitability (population spike or single unit spike counts) and blood ethanol levels. DISCUSSION Cellular actions o f ethanol on CA3 synaptic responses Injections of ethanol (3 g/kg, i.p.) increased the duration of inhibition in area CA3 after dentate stimulation in a dose-dependent manner. In contrast, the same treatments led to less consistent increases in excitability, whether measured by the population spike or by single unit spike frequencies, and changes in excitation were not dose-dependent: evoked single unit spikes were increased after ethanol in every preparation, and small increases in the population spike were observed occasionally. Our observed changes in population spike are opposite to those reported for CA1 by others10,11,14,15; these differences may be attributed to our in vivo preparation or the existence there of biochemical or functional processes not active in the slice preparation. The difference in area (CA3 as opposed to CA1) might also account for the different observations. These results indicate that parenteral treatment with ethanol influences synaptic transmission in hippocampus, but does not affect all pathways equally. Similar increases in both excitatory and inhibitory responses were noted after ethanol with stimulation of the commissural-CA3 pathway. Therefore, the effects of ethanol were not either pathway specific or transmitter specific. The transmitter of the commissural pathway is reported to be glutamate or aspartate25; the transmitter of the mossy fiber pathway remains unknown. The transmitter mediating recurrent inhibition for both pathways is reported to be GABA1, 5,7, as is the transmitter which is thought to mediate feedforward inhibition1, 5. A few measures (not reported here) were also made from the commissural to CA 1 pathway, the CA3 to CA 1 (Schaeffer collateral) pathway, and the commissural to dentate pathway. In each of these cases, increases of similar magnitudes in the length of PSI as well as increases in population spike amplitude were observed following ethanol, suggesting that these responses may be generalized throughout the hippocampus. The increases in excitability showed no correlation with the increase in PSI - - the magnitudes of change varied independently, and their time courses were also quite different. We conclude, therefore, that the increases in excitation and the increases in inhibition are functionally separable, and cannot, for example, be explained as a generalized increase in pyramidal cell responsiveness to all synaptic input. Both the increases in excitability and the increases in the period of PSI returned towards control levels within 2 h; however, blood ethanol levels remained high throughout the course of the experiments. Furthermore, we observed that the higher the blood ethanol levels at 15 rain post-injection, the sooner the length of PSI returned
125 towards control measures. These observations suggest that the whole animal preparations may have acutely adapted to ethanol; a similar adaptation with loss of response to an ethanol effect was observed on synaptic post-tetanic potentiation in Aplysia3z. Recent studies in our laboratory by Rogers et al. 27 have shown that in rats chronically exposed to ethanol, cerebellar Purkinje neurons clearly exhibit compensatory changes in firing patterns which may reflect adaptive changes in response to ethanol. Animals exposed to intoxicating doses of ethanol over a period of several weeks 2s and then withdrawn, showed a considerably shortened period of PSI in CA3 in response to dentate stimulation. Thus the return of the duration of PSI towards control values which is noted within two hours may be an early manifestation of this compensatory adjustment.
Time relationships Although blood ethanol levels reached a plateau at 15 min, the maximum increases in PSI did not occur until 30 min post-ethanol injection. The lag in time between maximum blood ethanol concentrations and peak response suggests the possibility that some biochemical by-product or physiological processg,12,~6,37 is necessary to elicit the maximum effect. Whole brain ethanol levels closely follow blood ethanol levels31. However, uptake of ethanol into some critical synaptic or lipid components13,18,21,29,35-37 may not parallel blood ethanol levels and could also account for the lag time in effect onset. There also remains the possibility that either an ethanol metabolite or an ethanol-induced novel metabolite of some endogenous factor must be produced 9,12, and that its concentration is the critical determinant in the magnitude of effect measured.
Dose-response relationships The levels of blood ethanol achieved in individual preparations varied among subjects even though a similar dose was injected. Study of dose-response relationships was therefore possible. When plotting the peak increase in PSI against the level of blood ethanol at 15 min post-injection, we observed a convex function ; the amount of change increased with blood ethanol concentrations up to 200 mg ~, and then declined with higher blood ethanol levels. There was no relationship between the increase in inhibition and the blood ethanol levels at the time the measurement of inhibition was made. Investigators using rat performance on a tilting plane as a measure of intoxication have also found that their behavioral measure (presumably mediated by GABA) is similarly related to the levels of blood ethanol reached shortly after injection19. Additional experiments will be required to explore the changes in response when lower doses yield lower blood levels at longer post-injection periods. Grupp and Perlanski TM have reported that lower doses of ethanol produce excitatory effects (i.e. speeding of single unit spontaneous firing) in the hippocampus, while higher doses produce inhibitory effects (i.e. slowing of firing rate). Our observations do not follow this trend. The excitatory effects which we observed showed no dose relationship; the inhibitory effects are greatest at a middle range of 15-min blood ethanol concentration, and smaller at both low and high 15 min blood ethanol levels.
126 Possible interactions o f halothane and ethanol Previous reports have indicated pharmacological and biochemical interactions between halothane and ethanol and that halothane may lower blood ethanol concentrations over time~0, 84. Such effects are unlikely to alter our experiments, however, since we studied only acute ethanol administration and blood ethanol levels thereafter remained high. Furthermore, we observed that the level of halothane anesthesia (between 0.75 ~ and 3.0 Y/o)had no bearing on the magnitude of the evoked responses we report. Further testing in unanesthetized preparations is, of course, necessary to verify the conclusion that halothane does not modify the effects of ethanol.
CONCLUSIONS Ethanol can increase the length of inhibition following afferent pathway stimulation. The effect shows distinct dose relationships and acute tolerance. This increase in PSI could be due to increases in the function of either of two GABA-mediated pathways which have been described for the dorsal hippocampus 1,4,5. However, other transmitters are known to have an effect on PSI. For example, Zieglgansberger et al. 3s have shown that enkephalin can shorten and block the period of PSI. In preliminary studies (not published) we f o u n d that serotonin may produce a similar effect. Norepinephrine is also known to be active in the hippocampus3°; and changes in norepinephrine sensitivity have been implicated in ethanol effects in the cerebellum 27. Bloom has suggested that any of these transmitter systems can heterosynaptically modulate primary synaptic events a. Therefore, any of the 'modulating' transmitter systems could also be implicated in the effect of ethanol on PSI. The effects could also be due to general changes in membrane resistance caused by ethanol zl. Pharmacological studies with antagonists and neurotransmitter analogues will be necessary in order to determine if a specific neurotransmitter system is responsible for the effects reported here. Intracellular recording will help determine possible membrane actions of ethanol and any contribution of an after hyperpolarization to the response. The hippocampal model could be a useful tool in testing for specific transmitter interactions. ACKNOWLEDGEMENTS We wish to thank Sam Madamba for excellent technical assistance, Drs. Steven Henriksen, George Siggins, Ed French and Ted Berger for technical advice and comments on the manuscript, Klaus Liebold for computer programming and Nancy Callahan for typing the manuscript. S.A.N. was supported by a grant from the National Institute on Alcohol Abuse and Alcoholism and the Ruth Harvey Foundation.
REFERENCES 1 Alger, B. E. and Nicoll, R. A., Biphasic IPSPs in rat hippocampal slice, Neurosci..4bstr., 5 (1979) 2472. 2 Andersen, P., Blackstad, T. W. and Lomo, T., Location and identification of excitatory synapses on hippocampal pyramidal ceils, Ex. Brain Res., 1 (1966) 236-248.
127 3 Andersen, P., Bliss, T. V. P. and Skred¢, K. K., Unit analysis of hippocampal population spikes, Exp. Brain Res., 13 (1971) 208-221. 4 Andersen, P., Eccles, J. C. and Loyning, Y., Pathway of postsynaptic inhibitionin the hippocampus, J. Neurophysiol., 27 (1964) 608-619. 5 Ayala, G. F. and Thalmann, R. H., A biphasic IPSP in pyramidal neurons of hippocampal slices in the presence of phenobarbital, Neurosci. Abstr., 5 (1979) 2476. 6 Blackstad, T. W., Commissural connections of the hippocampal region of the rat, with special reference to their mode of termination, J. comp. Neural., 105 (1956) 417-536. 7 Bloom, F. E. and iversen, L. L., Localizing 3H-GABA in nerve terminals of rat cerebral cortex by electron microscopic autoradiography, Nature (Lond.), 229 (1971) 628-630. 8 Bloom, F. E., Is there a neurotransmitter code in the brain? Advanc. Pharmacol. Ther., 2 (1979) 205-213. 9 Cohen, G., Alkaloid products in the metabolism of alcohol and biogenic amines, Biochem. Pharmacol., 25 (1976) 1123. 10 Corrigal, W. A., Carlen, P. L. and Stainman, A. L., Acute effects of ethanol on hippocampal CA1 field potentials recorded in vitro, Neurosci. Abstr., 3 (1977) 288. 11 Corrigai, W. A. and Carlen, P. L., Ethanol: electrophysiological evidence of tolerance remains after isolation of the hippocampus in vitro, Neurosci. Abstr., 4 (1978) 1329. 12 Deitrich, R. A. and Erwin, V. G., Involvement of biogenic amine metabolism in ethanol addiction, Fed. Proc., 34 (1975) 1962-1967. 13 Denovo, E. C., Gruber, B. and Noble, E. P., Alterations of fast-reacting sulfhydryl groups of rat brain microsomes by ethanol, Biochem. biophys. Res. Commun., 68 (1976) 975-981. 14 Doller, H. J., Eckhardt, M. J. and Weight, F. F., Effects of ethanol on the hippocampal slice preparation, Fed. Proc., (1980) No. 80. 15 Durand, D. and Carlen, P., Slow potentiation of CA1 hippocampal slice field potentials and acute effects of low-dose ethanol, Neurosci. Abstr., 5 (1979) 1875. 16 Faber, D. S. and Klee, M. R., Actions of ethanol on neuronal membrane properties and synaptic transmission. In Alcohol and Opiates: Neurochemical and Behavioral Mechanisms, Academic Press, New York, 1977, pp. 41-63. 17 Fischer, E. and Wallgrew, H., Cerebral blood flow and cerebral alcohol uptake of anesthetized dogs, Physiologist, 1 (1957) 27. 18 Grupp, L. A. and Perlanski, E., Ethanol-inducedchanges in the spontaneous activity of single units in the hippocampus of the awake rat: a dose-response study, Neuropharmacology, 18 (1979) 63-70. 19 Hakkinen, H.-M. and Kulonen, E., Ethanol intoxication and ~'-aminobutyric acid, J. Neurochem., 27 (1976) 631-633. 20 Han, Y. H., Why do chronic alcoholics require more anesthesia? Anesthesiology, 30 (1969) 341-2. 21 Israel, Y. and Kuriyama, K., Effects of in vivo ethanol administration on adenosine triphosphatase activity of subcellular fraction of mouse brain and liver, Life Sci., 10 (1971) 591-599. 22 Kalant, H., Ethanol and the nervous system, Exp. neurophysiol., Asp., 9 (1974) 111-124. 23 Klemm, W. R. and Stevens, R. E., IlI, Alcohol effects on EEG and multiunit activity in various brain regions of rats, Brain Research, 70 (1974) 361-368. 24 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas of the Forebrain andLower Parts of the brain Stem, Williams and Wilkins, 1963. 25 Nadler, J. V., Vaea, K. W., White, W. F., Lynch, G. S. and Cotman, C. W., Aspartate and glutamate as possible transmitter of excitatory hippocampal afferents, Nature (Lond.), 260 (1976) 538-540. 26 Ranck, J. B., Jr., Behavioral correlates and firing repertoires of neurons in the dorsal hippocampal formation and septum of unrestrained rats. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. 2, Plenum Press, New York, 1975, pp. 207-244. 27 Rogers, J., Siggins, G. R., Schulman, J. A. and Bloom, F. E., Physiological correlates of ethanol intoxication, tolerance, and dependence in rat cerebellar Purkinje cells, Brain Research, in press. 28 Rogers, J., Weiner, S. G. and Bloom, F. E., Long term ethanol administration methods for rats: advantages of inhalation over intubation or liquid diets, Behav. neur. Biol., 27 (1979) 466-486. 29 Sceman, P., The membrane actions of anesthetics and tranquilizers, Pharmacol. Rev., 24 (1972) 583-655. 30 Segal,M. and Bloom, F. E., The action of norepinephrine in the rat hippocampus. IV. The effects
of locus coeruleus stimulation on evoked hippocampal unit activity, Brain Research, 107 (1976) 513-525. 31 Siggins, G. R. and Bloom, F. E., Alcohol-related electrophysiology. In Proc. 5th Biennial Int. Syrup. Alcoholism, Drug Alcohol Dep., in press.
128 32 Traynor, A. E., Schlapfer, W. T. and Barondes, S. H., Stimulation is necessary for the development of tolerance to a neuronal effect of ethanol, in preparation. 33 Traynor, M. E., Woodson, P. B. J., Schlapfer, W. T. and Barondes, S. H., Sustained tolerance to a specific effect of ethanol on post-tetanic potentiation in Aplysia, Science, 193 (1976) 510-511. 34 Ugarte, G., Pino, M. E., Peroda, T. and Iturriagia, H., Increased blood ethanol elimination in rats treated with halothane, Pharmacology, 9 (1973) 275-280. 35 Volicer, L. and Hurter, B. P., Effects of acute and chronic ethanol administration and withdrawal on adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate levels in the rat brain, J. Pharmacol. exp. Ther., 200 (1977) 298-305. 36 Wallgreen, H., Nikander, P., von Boguslawsky, P. and Linbola, J., Effects of ethanol, butanol and clomethiazole on net movements of sodium and potassium in electrically stimulated cerebral tissue, Actaphysiol. scand., 91 (1974) 83-93. 37 Wallgreen, I-I., Nikander, P. and Virtanero, P., Ethanol-induced changes in cation-stimulation adenosine triphosphatase activity and lipid-proteolipid labeling in brain microsomes, Advanc. exp. Med. BioL, 59 (1975) 23-36. 38 Zieglg~nsberger, W., French, E. D., Siggins, G. R. and Bloom, F. E., Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory neurons, Science, 205 (1979) 415--417.