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spike dissociation in the dentate gyrus: GABAergic and non-GABAergic components
Brain Research, 561 (1991) 27-34 (~) 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939117005H
27
BRES 17005
LTP-associated EPSP/spike dissociation in the dentate gyrus: GABAergic and non-GABAergic components Richard A. Tomasulo 1, William B. Levy e and Oswald Steward 1 Departments of ~Neuroscience and 2Neurosurgery, University of Virginia School of Medicine, Charlottesville, VA (U.S.A.) (Accepted 7 May 1991) Key words: Hippocampus; Dentate gyrus; y-Amino butyric acid; Long-term potentiation, Synaptic plasticity; Inhibition; Excitatory postsynaptic potential/spike dissociation
The induction of long-term potentiation (LTP) in the dentate gyrus (DG) leads to a change in the firing characteristics of the dentate granule cells. This phenomenon, termed EPSP/spike dissociation, is seen in field potential studies as a shift to the left of the E-S curve, in which population spike amplitude is plotted against pEPSP slope at various stimulus intensities. It has been suggested that EPSP/spike dissociation reflects a decrease in feed-forward inhibition. To test this hypothesis, we blocked GABA-A neurotransmission in a circumscribed area of the DG in urethane-anaesthetized rats by inserting a micropipette filled with 8 mM bibuculline methiodide in saline. We then recorded E-S curves from 9 such electrodes and from 8 control electrodes before and after inducing LTP in the perforant path. Bicuculline prevented the LTP-associated leftward shift of the E-S curves. Instead, the E-S curve showed a consistent shift to the right at the bicuculline sites after LTP, reflecting potentiation of the pEPSP without corresponding increases in the population spike amplitude. The results indicate that the EPSP/spike relationship is controlled largely by GABAergic input, and that potentiation of the population spike in the DG depends largely on a change in the EPSP/spike relationship. INTRODUCTION High-frequency activation of selected h i p p o c a m p a l afferents results in two long-lasting changes of neuronal responsiveness. First, a given presynaptic input p r o d u c e s a larger dendritic depolarization than before. This is called long-term p o t e n t i a t i o n 5 (LTP), or synaptic potentiation, because it reflects an increase in the efficacy of the activated synapses. Second, the postsynaptic cells become m o r e excitable: an action potential can result from a dendritic current which was previously insufficient for spike generation 2'1s. The latter effect, t e r m e d EPSP/spike dissociation, can be d e m o n s t r a t e d from field recordings by plotting cell discharge as a function of dendritic depolarization following stimulation at various intensities. Cell firing is rec o r d e d as p o p u l a t i o n spike amplitude, and dendritic depolarization is estimated by the p o p u l a t i o n E P S P (pEPSP). The resulting ' E - S curve' shifts to the left after high-frequency stimulation, indicating an increased spike-generating capacity for a given degree of dendritic depolarization 2°,21. Two hypotheses have b e e n p r o p o s e d to account for
the EPSP/spike dissociation: (1) the EPSP/spike dissociation reflects an internal alteration of the postsynaptic cells 18, or (2) the dissociation reflects a change in the balance b e t w e e n the excitatory and inhibitory synaptic currents activated by stimulation of the afferent population 2'2°'21. The latter hypothesis, first p r o p o s e d by Wilson et al. 2°'21, is based on current concepts of hippocampal feed-forward inhibition. In CA1 and the d e n t a t e gyrus ( D G ) , inhibitory interneurons receive innervation from the same excitatory afferents as do the projection cells 14. These inhibitory interneurons fire with extremely short latencies following activation of the shared afterents 6'17. Their feed-forward inhibitory action could therefore control the efficiency with which dendritic depolarization translates into action potentials following a given afferent volley. The EPSP/spike dissociation which follows the induction of LTP could result from a real decrease in feedforward inhibition l°'le or from a relative decrease due to an u n o p p o s e d increase in excitation 2'2°' Zl If the EPSP/spike dissociation does reflect a shift in the balance b e t w e e n excitation and inhibition, then eliminating feed-forward inhibition should prevent the E - S
Correspondence: O. Steward, Department of Neuroscience, Box 5148 MR4 Medical Center, University of Virginia School of Medicine, Charlottesville, VA 22908, U.S.A.
28 shift which normally follows high-frequency stimulation. This result has been obtained in the CA1 region in hippocampal slice preparations 2'7. We report here that blocking ),-aminobutyric acid (GABA)-A mediated inhibition in the DG in vivo, via a local infusion of bicuculline methiodide, prevented the leftward shift of the E - S curve which normally accompanies LTP in the perforant path. Instead, the induction of LTP under GABA receptor blockade resulted in a shift of the E - S curve to the right. Because of this unexpected shift, LTP induced during G A B A receptor blockade was not accompanied by consistent increases in population spike amplitude. The findings indicate that, in the DG, a substantial component of the increase in population spike amplitude following LTP results from a change in the balance between synaptic excitation and feed-forward inhibition.
MATERIALS A N D M E T H O D S
these single-electrode experiments 3 were with bicuculline and two with saline only. The total sample, therefore, consisted of data from 11 bicuculline and 10 saline electrodes.
Data analysis The pEPSP slope was measured on the rising phase, at a fixed latency from the stimulus, before the onset of the population spike. If LTP (defined as a 10% increase in pEPSP slope at two stimulus intensities) did not occur at either electrode, the experiment was excluded from the study. Spike amplitude was measured from the peak negativity to a line connecting the prespike maximum positivity to the postspike maximum positivity. We measured only the first of the multiple spikes evoked at the bicuculline site. For latency measurements, the spike onset was defined as the prespike maximum positivity, the point at which the pEPSP slope became zero in transition to the negative-going population spike. The measurements were made by an Apple computer employing 'Waveman' software. The E - S curves were constructed from the average values obtained for each stimulus intensity at each electrode. Initially, the average of each set of 4 sweeps was plotted as a single point, so that data from the two consecutive collection periods for each experimental condition could be compared. If these were judged sufficiently similar (the determination was subjective), then all the sweeps collected at a given stimulus intensity were averaged and
Experimental procedures Male Sprague-Dawley rats (250-450 g) were anaesthetized with urethane (1.5 g/kg), and prepared for acute physiological recording. A bipolar stimulating electrode was placed in the angular bundle (AB, -8.1 mm, 4.4 mm lateral to bregma), and two recording micropipettes were placed in the ipsilateral dentate gyrus (DG). The first micropipette, containing 0.9% NaCI, was placed 4 mm posterior and 2.5 mm lateral to bregma. The depth was adjusted for maximal positivity of the EPSP following AB stimulation. A second micropipette, containing 8 mM bicuculline methiodide in saline ~6, was inserted about 1.5 mm anteromedial to the first. The depth was again adjusted for maximal positivity of the EPSP. As described previously x6, the effectiveness of bicuculline in blocking inhibition could be confirmed by examination of the field responses. At the bicucuUine site, the spike threshold was lower, multiple population spikes were evoked by a single perforant path stimulus, and paired-pulse inhibition was either eliminated or markedly reduced (Fig. 1). Once the electrodes were placed and the G A B A blockade confirmed, the preparation was allowed to stabilize for at least 45 min before data were collected. In order to construct E - S curves, evoked responses were recorded simultaneously from both electrodes, digitized, and stored on floppy discs for subsequent analysis by microcomputer. Test stimuli were delivered every 15 s to the AB. The lowest intensity was just above spike threshold for the bicuculline electrode, and the highest was that which evoked a maximum-amplitude spike from the control (saline) electrode. Four stimuli were given at each intensity, and steps were chosen so that at least 5 different spike amplitudes were recorded from each electrode. Because spike threshold and maximum spike amplitude were reached at lower intensities at the bicuculline site, we used 6-10 intensities overall. After the first data set, the collection procedure was repeated. In some experiments, the number of stimulus intensities was reduced in the second data collection period, but 4 stimuli were still delivered at each intensity. Immediately after the baseline data collection, we delivered 8 400-Hz. 17.5-ms stimulus trains (i.e. 8 pulses per train) at 15-s intervals. Stimulus intensity was adjusted to evoke at least a 5 mV population spike at the saline electrode. After waiting at least 5 min, we repeated the data collection procedure. One or two additional stimulus intensities, below the initial minimum, were added to the routine, because the spike thresholds invariably decreased following high-frequency conditioning. We performed 8 double-electrode experiments, as described, and 5 additional experiments with only a single recording electrode. Of
A
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Fig. 1. Effect of bicuculline on evoked responses. A: upper trace is from a bicuculline-containing electrode. Lower is from control. The responses were obtained simultaneously, following paired angular bundle stimuli delivered at an interval of 20 ms. Note the absence of paired-pulse inhibition at the bicuculline site. Calibration 5 mV, 5 ms. B: same as A, but with a 50-ms interstimulus interval. Robust feedback inhibition is retained at the control site. Calibration 5 mV, 10 ms.
29 plotted as a single point. Points on an individual E - S curve, therefore, represented an average of either 4 or 8 sweeps. If the curves were not reproducible, the experiment was excluded from the study. The data from two double-electrode experiments were excluded from statistical analysis, one because of unstable responses and one because LTP did not occur at either electrode. We therefore report the results of 6 double-electrode and 5 single-electrode experiments (9 bicuculline and 8 saline electrodes total). The E-S curves from these experiments were normalized by defining the maximum pretetany pEPSP slopes and spike amplitudes as 100%. Normalized spike amplitudes at selected normalized pEPSP values were taken from the individual E-S curves, by interpolation when necessary, and averaged to produce composite curves (Fig. 3). The statistical significance of the change in mean spike amplitude for each pEPSP value was determined by the Wilcoxon signed rank test.
RESULTS
As reported previously 16, bicuculline methiodide produced stable alterations in the responses evoked by AB stimulation. At moderate and high stimulus currents, a single AB stimulus evoked multiple population spikes at the bicuculline sites. Paired-pulse inhibition was eliminated or sharply reduced. Neither of these effects occurred at the control site, indicating that the bicuculline blocked GABAergic neurotransmission in a limited volume of tissue only (Fig. 1). Before high-frequency conditioning, population spikes appeared at the bicuculline sites at lower stimulus intensities and reached their maxima at lower intensities than at the control sites (Fig. 2A). In individual experiments, the maximum spike amplitudes recorded at the two sites often differed considerably (e.g. Fig. 2). However, neither electrode consistently showed larger responses, and the mean values were similar (Table I). Before conditioning, therefore, bicuculline did not seem to alter the maximum amplitudes of the population spikes. In contrast to the spike amplitudes, the pEPSP slopes rose monotonically as a function of stimulus intensity through the same range of stimuli at both sites (Fig. 2B, Table I). Thus, the bicuculline had no demonstrable effect on pEPSP generation. As reported by other investigators 19, high-frequency stimulation induced more potentiation of the pEPSPs during G A B A receptor blockade than under control conditions. The average increase following conditioning was 37% (S.E.M. = 3.5) at the bicuculline sites and 25% (S.E.M. -- 5.6) at the saline sites. The E - S curves showed the expected left shift at all 8 control electrodes. This left shift was significant at all points on the normalized E - S curve (Fig. 3; P values between 0.025 and 0.006). At 6 of 9 bicuculline sites, the left shift was completely eliminated. At the remaining 3, slight left shifts occurred. These were much smaller than those seen at the control sites, however, and they oc-
curred only at high stimulus intensities. In these 3 cases, the average increase in spike amplitude at maximum pretetany pEPSP was 8%, compared to an average increase of 22% at the same point for the saline electrodes. The majority of post-LTP curves from the bicuculline electrodes were not identical to the baseline curves, however. Instead, 6 of the 9 curves showed an unexpected shift to the right, indicating a decrease in the spike-generating capacity of a given pEPSP. This phenomenon is illustrated in Fig. 4, where pre- and postLTP responses with identical pEPSP slopes are compared. The right shift was statistically significant only at high stimulus intensities (Fig. 3; P = 0.025, 0.038, 0.043 at the top three points by Wilcoxon rank test). As a resuit, the slopes of the E - S curves decreased in the 6 cases showing the right shift (e.g. Fig. 2C). The mean slope of the E-S curves, as estimated by linear regres-
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Fig. 2. Data from a sample experiment. In each pair of graphs, the left panel represents data from the saline electrode, and the right panel that from the bicuculline site. A: spike amplitude as a function of stimulus intensity (in mA). Before tetany, the spike threshold and maximum amplitude are reached at lower stimulus strength at the bicueulline site. Note that the spike amplitude did not change significantly at the bicuculline site following high-frequency activation. B: EPSP slope as a function of stimulus. Pre- and postconditioning EPSPs are similar at the two sites. C: E - S curves. At the saline site, the expected left shift occurs. At the bicuculline electrode, the shift is in the opposite direction.
30 TABLE I
sion, d e c r e a s e d by 20% after c o n d i t i o n i n g in the bicu-
Effects o f tetany on EPSP slopes and spike amplitudes
culline s a m p l e ( P = 0.03 by W i l c o x o n signed r a n k test). ( N o t e that the E - S curves are not linear. W e are using
S.E.M. in parentheses.
linear r e g r e s s i o n o n l y for a statistical c o m p a r i s o n be-
Saline Stimulus b
t w e e n pre- and p o s t c o n d i t i o n i n g c u r v e s . )
BicucuUine
Pre-tet
Post-tet
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C o n s i s t e n t with the c u r v e shift data, the p o p u l a t i o n spike a m p l i t u d e s ( r e c o r d e d using the s a m e stimulus in-
EPSP slopes normalized a
2 3 4 5
0.65 0.87 1.00 1.18
(0.08) (0.02) (0.02)
0.27 0.67 0.69 1.00 1.32
0.83 (0.11) 1.10 (0.07) 1.27 (0.04) 1.35 (0.07)
(0.07) (0.12) (0.06) -
0.43 (0.13) 0.90 (0.17) 1.03 (0.14) 1.44 (0.13)
(0.11)
1.62 (0.11)
tensity pre- and p o s t c o n d i t i o n i n g ) i n c r e a s e d to a m u c h larger d e g r e e at the saline e l e c t r o d e s t h a n at the bicuculline sites (Fig. 2 A , Table I). T h e a v e r a g e i n c r e a s e in p o p u l a t i o n spike a m p l i t u d e at the saline sites was 172% ( S . E . M . = 21.5), and m e a n p o p u l a t i o n spike a m p l i t u d e
Spike amplitudes (mV)
2 3 4 5
3.30 10.63 9.81 13.80
(0.77) (2.6) (3.8) (5.2)
2.94 9.42 11.20 13.41 16.51
13.75 (2.4) 18.78 (3.2) 18.33 (3.6) 19.15 (3.5)
(0.86) 6.75 (1.6) (2.5) 9.87 (2.6) (2.6) 12.80 (2.8) (1.9) 14.07 (2.1) (1.8) 15.92 (2.2)
i n c r e a s e d f r o m 6.8 to 16.1 m V ( P = 0.0001 by p a i r e d t-test, n = 27). U s i n g the data in Table I (a m e a n increase in E P S P o f 28% at low stimulus intensity and o f 14% at high intensity) and the p o o l e d E - S curve (Fig. 3B), we estimate that 45-75% of the increase in spike amplitude
a Normalization: the EPSP value at the tetanizing stimulus was defined as 1.0 for each experiment. Note that this is different from the normalization used for the composite E-S curves (Fig. 3). b Stimulus intensities are as follows: 1 = spike threshold for bicueulline electrode;2 = spike threshold for saline electrode; 4 = tetanizing intensity; 3 and 5 were one step below and above tetanizing intensity respectively.
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Fig. 3. Normalized E-S curves. The maximum preconditioning pEPSP and spike values were defined as 100%. A: data from 9 bicuculline electrodes. The right shift is significant at the highest 3 EPSP values only (P = 0.043, 0.038, and 0.025 at 100%, 85%, and 75% respectively, by Wilcoxon signed rank test. NS below 75%). B: data from 8 control electrodes. P values vary between 0.025 and 0.006.
Fig, 4. Changes in spike-generating capacity and latency following the induction of LTP. A, B and C are from a saline electrode. D, E, and F are from the bicuculline electrode in the same animal. A: baseline response. B: postconditioning response evoked by a stimulus adjusted to produce an identical pEPSP slope. C: A and B superimposed. The interrupted line is the preconditioning response. The latency to spike onset decreased by 0.36 ms concurrent with the increase in spike amplitude. D and E: pre- and postconditioning responses, respectively, from the bicuculline site. The stimulus was adjusted to equalize the pre- and post-tetany pEPSP slopes. Note the decrease in spike amplitude. F: D and E superimposed. The interrupted line is the preconditioning response. The latency to spike onset decreased by 0.26 ms despite the decrease in spike amplitude. Digitized waveforms, slightly retouched. Calibration: 2.5 mV, 2 ms.
31 TABLE II
30
Effects of tetany on population spike latency S.E.M. in parentheses.
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Latency to population spike onset (ms) Saline electrodes Low 4.58 (0.27) 4.02 (0.31) High 3.79 (0.14) 3.56 (0.12)
-0.56 (0.12) -0.23 (0.06)
0.008 0.015
Bicuculline electrodes Low 4.89 (0.16) High 3.85 (0.18)
4.65 (0.26) 3.77 (0.16)
-0.23 (0.14) 4.08 (0.05)
0.146 0.229
Latency to population spike peak (ms) Saline electrodes Low 5.58 (0.54) 5.27 (0.68) High 4.67 (0.28) 4.46 (0.28)
-0.31 (0.17) -0.21 (0.07)
0.127 0.034
Bicuculline electrodes Low 6.59 (0.51) High 4.78 (0.36)
-0.42 (0.22) -0.09 (0.11)
0.119 0.417
30
SPIKE AMPLITUDE PRE-TETANY (mV)
6.17 (0.56) 4.69 (0.33)
Low stimulus intensity is just above spike threshold for the pretetany trials; high stimulus is that which evoked the maximum pre-tetany spike amplitude. Post-tetany latencies were taken from responses with pEPSP slopes equal to the pre-tetany low and high stimulus responses respectively. Interpolation was used when necessary. b Paired t-test. a
Fig. 5. Effect of tetany on spike amplitude. Each point represents spike amplitudes at a constant stimulus intensity before and after tetany. Each point is the average of 4 pre- and 4 post-tetany trials. Data are from all experiments. A change in spike amplitude is indicated by vertical displacement from the diagonal line, which represents the function x = y. At the bicuculline sites, granule cell output changed very little after tetany, despite significant potentiation of the EPSPs.
which followed conditioning could be attributed to the left shift. This is consistent with previous studies 1"9'2°'21. At the bicuculline sites, the amplitude of the population spike evoked by a given stimulus either did not change, or actually decreased, in the majority of trials (e.g. Fig. 2A). The average increase of population spike amplitude was just 20% (S.E.M. = 12.1), and the mean population spike amplitude increased only from 10.2 m V to 10.9 mV at the bicuculline sites following potentiation (P = 0.121, n = 43). Only at low stimulus intensities did LTP materially increase the mean population spike amplitude (Table I). These results are illustrated in a different way in Fig. 5. Here postconditioning spike amplitudes are plotted as a function of preconditioning amplitudes, evoked at the same stimulus intensity before and after conditioning. A diagonal line is drawn through the origin with a slope of 1. A change in spike amplitude with conditioning is indicated by vertical displacement from this line. All the points from the control electrodes are above the line (i.e. spike amplitude increased), while the majority of values from the bicuculline sites are on or below the line. Thus, high-frequency stimulation delivered during G A B A receptor blockade did not consistently increase spike amplitude despite its effectiveness in potentiating the EPSPs (see Table I).
Fig. 5 also explains why the mean increase of spike amplitude at the bicuculline sites was 20%, while the mean amplitude increased by only 0.7 mV. Within the bicuculline sample, substantial increases in population spike amplitude occurred only at low stimulus intensities, where the small baseline spike amplitudes guaranteed large percentage increases. These few responses contributed disproportionately to the average percentage increase. Previous studies have established that, in response to an unchanged excitatory drive (i.e. at a fixed pEPSP slope before and after conditioning), the population spike latency ordinarily decreases after the induction of LTP 1. Several investigators have suggested that this decrease in latency, like the left shift of the E - S curve, might reflect a change in the balance between excitation and inhibition 2,2°,21. If the decrease in population spike latency does indeed depend upon a decrease in feed-forward inhibition, then the latency change should be minimized when inhibitory neurotransmission is blocked. Consistent with this prediction, we found that, when measured at the same p E P S P slope before and after tetany, statistically significant decreases in spike latency (whether measured at onset or at peak negativity) occurred only in the control population (Table II). A t 3 of the bicuculline sites, no change in latency could be measured with high or low
32 intensity stimulation following high-frequency conditioning. At 3 other bicuculline sites, however, the spike latency for a given pEPSP slope actually decreased concurrent with a reduction of population spike amplitude (i.e the right shift) following conditioning (Fig. 4). At the control sites, in agreement with previous studies 1"2°" 21, a reduction in spike latency invariably accompanied an increase in population spike amplitude (Fig. 4). DISCUSSION Three points from the above data require elaboration: (1) In the DG, the LTP-associated leftward shift of the E - S function depends upon intact GABAergic neurotransmission. (2) G A B A receptor blockade reveals a second LTP-associated influence on the EPSP/spike relationship. This effect opposes the left shift. (3) Previous studies have established that the left shift contributes to the increase in granule cell output which follows tetanic conditioning. If the right shift we describe here occurs concurrently with, and is masked by, the G A B A dependent left shift, then GABAergic mechanisms must contribute more to altering granule cell output than the left shift alone would indicate.
The left shift is GABA-dependent The mechanism of the left shift was the initial subject of this project. We tested the hypothesis that the left shift reflects a decrease of GABAergic feed-forward inhibition relative to the increase in excitation which follows high-frequency stimulation. This hypothesis predicts that a blockade of GABAergic neurotransmission should prevent the LTP-associated leftward shift of the E - S curve. This is precisely the result we obtained. The findings are consistent with previous studies of LTP in the CA1 region in vitro, where G A B A receptor blockade also prevents the left shift of the E - S function 2'7. This mechanism therefore contributes to EPSP/spike dissociation in both regions of the hippocampus. Also consistent with a relative decrease in inhibition is the finding that the population spike latency (for a given pEPSP slope) decreased to a smaller extent at the bicuculline sites than at the control sites (Table II). Within this logical frame, the G A B A dependence of the left shift is compatible with two interpretations. Abraham et al. 2, following a suggestion made by Wilson et al. 2°'21, have proposed that the left shift reflects only an increase in excitatory drive (as a result of LTP) which is not matched by a compensatory increase in feed-forward inhibition. Thus, a given presynaptic volley would evoke an increased inward current following LTP without an accompanying increase in inhibitory outward current. The strength of this hypothesis is its avoidance of
experimentally unverified modifications in the inhibitory circuit. The hypothesis cannot, however, explain examples of EPSP/spike dissociation occurring in the apparent absence of EPSP potentiation 5'18. An alternate hypothesis is that the LTP-associated left shift of the E - S curve reflects a real decrease of synaptic efficacy in the inhibitory pathway. Because feed-forward inhibition in the hippocampus is effected largely through a disynaptic circuit 6"11, the left shift could reflect a reduction in synaptic driving of inhibitory interneurons, reduced efficacy of the GABAergic synapses on the projection cells, or alterations at both sites. According to two previous studies, however, driving of interneurons does not decrease with LTP; it actually inc r e a s e s 6'17. Increased interneuron discharges would presumably increase, not decrease, feed-foward inhibition, provided that the increased firing occurred at a short enough latency to affect the evoked granule cell discharge. In this case, GABAergic synapses would have to undergo significant activity-dependent depression after high-frequency activation to account for the net reduction of inhibition at the projection cells. This hypothesis receives support from a study by Steltzer et al. 15, who described depression of GABAergic neurotransmission following high-intensity 50 Hz conditioning of afferents to the CA1 region in hippocampal slices. Scharfman and Sarvey 13, however, failed to demonstrate alterations in G A B A sensitivity following more moderate 100 Hz stimulation.
G A B A receptor blockade revealed a second influence on the EPSP/spike relationship Our results demonstrate a second LTP-associated effect on the EPSP/spike relationship. This appears as a reduction of slope and a shift to the right of the E - S curve at the bicuculline sites after conditioning (Fig. 2C and 3). Though not explicitly linked to a blockade of inhibition, this right shift -- or a very similar effect -- is discernible in previous studies 4'9"2°. Kairiss et al. 9 evoked a slope decrease and slight right shift of the E - S curve via low-intensity conditioning trains to the perforant path in vivo. Following higher intensity tetanic stimulation, the usual left shift occurred. Their result supported the existence of two separable effects, which were apparently activated at different thresholds. In an early study of the E - S shift, Wilson et al. 2°'21 found that a crossed temperodentate pathway which had sprouted following a unilateral lesion of the entorhinal cortex could express LTP, but did not show the usual E - S shift to the left. Instead, the E - S curve shifted slightly to the right and decreased in slope, as did the curves in our bicuculline experiments. These investigators did not attempt to explain the right shift, but they
33 explained the absence of a leftward shift by a failure of the sprouted pathway to achieve effective innervation of inhibitory interneurons. If this view was correct, their experimental manipulation selectively reduced or eliminated feed-forward inhibition, and was therefore comparable to our infusion of bicuculline. Bliss et al. 4 reported that increasing extracellular calcium in the D G in vivo can induce potentiation of the EPSP, while reducing spike amplitude and latency. These effects, of course, are remarkably similar to the results we obtained via tetanic stimulation under G A B A receptor blockade. The meaning of this similarity is uncertain. The cause of the right shift/slope decrease is unclear. One possible mechanism is potentiation of a nonGABAergic, short-latency inhibitory synaptic potential. Such an increase of feed-forward inhibition should, however, produce an increase in population spike latency. Our failure to observe such latency increases at the bicuculline sites argues against this mechanism. In fact, we encountered decreases of spike latency in cases where spike amplitude also decreased following high-frequency conditioning (Fig. 4). Under control conditions, of course, changes in latency are inversely related to changes in spike amplitude. Our finding of parallel reductions in latency and amplitude -- even though the latency decreases were small -- suggests a mechanism entirely different from that which is responsible for the left shift. Another possible cause for the right shift is a change in tonic input from an extrahippocampal source. The absence of the right shift/slope decrease with LTP in picrotoxin-treated hippocampal slices 2'7 is consistent with this idea. An alternate interpretation is that the right shift/slope decrease is specific to the DG. We cannot even be sure that the right shift reflects the behavior of granule cells in general. The population spikes we recorded in the hilus may represent the summed activity of several cell types which respond differentially to tetanic stimulation. We think it improbable that the right shift is an artifact due entirely to the effects of bicuculline, but the possibility must be considered. One possibility is that the limited spike potentiation could represent a saturation effect, due to maximization of the pre-tetany population spike amplitude as a result of bicuculline. The term 'saturation' can be applied in two senses: (1) The maximum spike amplitude could have been achieved at a low stimulus intensity, and at a correspondingly submaximal pEPSP. Under these conditions, test stimuli might elicit maximal population spikes before the induction of LTP so that further increases in amplitude could not be detected. This seems unlikely because bicuculline limited spike potentiation throughout the range of spike ampli-
tudes (Fig. 5, Table I). (2) The maximum population spike amplitude for each stimulus intensity (a given set of afferent fibers) could have been reached before potentiation; i.e. increases in the efficacy of the activated synapses could not measurebly increase cell firing. This is an unlikely possibility because the activated synapses should have been distributed to the granule cells in nearly random proportions. With most submaximal stimuli, therefore, the postsynaptic population would include cells that could achieve firing threshold only after synaptic potentiation. In support of this interpretation, we note that paired pulse facilitation was observed at 5 of 9 bicuculline sites prior to tetanic stimulation (e.g. Fig. 1). In 3 of these cases, despite this demonstrated capacity for increased cell firing, bicucuUine prevented spike potentiation. Finally, this form of saturation cannot explain the reduction of population spike amplitude we often observed after conditioning (see Fig. 5). If the inhibitory interneurons behave like hippocampal projection cells, their excitatory synapses could show exaggerated potentiation in the presence of G A B A receptor blockade 19, due to release of tonic inhibition. (Hippocampal interneurons receive GABAergic innervation from the medial septal nucleus3'S.) This would increase feed-forward inhibition after potentiation if the G A B A blockade on the granule cells was incomplete. As discussed above, this increase in inhibition would be expected to increase spike latency following potentiation, and, since the latencies did not increase, this mechanism is unlikely. Another way bicuculline might produce misleading resuits is by depolarizing the granule cells. Tonic depolarization would reduce the amplitude of each cell's action potential. If this tonic depolarization increased for any reason following potentiation, then the action potential amplitude would decrease further. The reduction in population spike amplitude would not then reflect a reduction in cell firing, but only the sum of reduced action potential amplitudes. GABAergic mechanisms are an important source o f spike potentiation EPSP/spike dissociation (the left shift) contributes significantly to the alterations of granule cell output which follow high-frequency conditioning. In our saline sample, the left shift increased population spike amplitude by 20-120% above that predicted from EPSP potentiation alone (Fig. 3B). We estimated that 45-75% of the increase in spike amplitude which followed conditioning could be attributed to the left shift. Our results indicate that this very significant effect depends on GABAergic mechanisms. If the left shift reflects synaptic changes in the disynaptic inhibitory circuit, then the interneurons
34
are potent effectors of change in projection cell output. If, on the other hand, high-frequency conditioning does not alter synaptic transmission in the inhibitory circuit, then hippocampal interneurons must still enable or amplify the changes of projection cell output which accompany LTP. In either case, the inhibitory interneurons must play an important role in the integrative function of the hippocampus. The rightward shift of the E - S curve which occurred at our bicuculline sites does not alter the above conclusion. It merely complicates the attempt to quantify the
interneurons' contribution. If the mechanisms responsible for the right shift are activated concurrently with the left shift in the untreated DG, then the effect of the interneurons is even greater than the estimates provided above.
Acknowledgements. Supported by NSF Grant BNS8818766 to O.S.W.B.L. was supported by NIH Grant NS15488 and NIMH Career Development Award MH0622. R.T. was supported by NIH Training Grant NS07199 and by the Department of Neurological Surgery, Dr. John A. Jane, Chairman.
REFERENCES 1 Abraham, W.C., Bliss, T.V.P. and Goddard, G.V., Heterosynaptic changes accompany long-term but not short-term potentiation of the perforant path in the anaesthetized rat, J. Physiol., 363 (1985) 335-349. 2 Abraham, W.C., Gustafsson, B. and WigstrOm, H., Long-term potentiation involves enhanced synaptic excitation relative to synaptic inhibition in guinea-pig hippocampus, J. Physiol., 394 (1987) 367-380. 3 Bilkey, D.K. and Goddard, G.V., Medial septal facilitation of hippocampal granule cell activity is mediated by inhibition of inhibitory interneurons, Brain Research, 361 (1985) 99-106. 4 Bliss, T.V.P., Errington, M.L. and Lynch, M.A., Calcium-induced long-term potentiation in the dentate gyrus is accompanied by a sustained increase in glutamate release. In T.P. Hicks, D. Lodge and H. McLennan (Eds.), Excitatory Amino Acid Transmission, Liss, New York, 1986, pp. 337-340. 5 Bliss, T.V.P. and L0mo, T., Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol., 232 (1973) 331-356. 6 Buzstiki, G. and Eidelberg, E., Direct afferent excitation and long-term potentiation of hippocampal interneurons, J. NeurophysioL, 48 (1982) 597-607. 7 Chavez-Noriega, L.E., Bliss, T.V.P. and Halliwell, J.V., The EPSP-spike (E-S) component of long-term potentiation in the rat hippocampal slice is modulated by GABAergic but not cholinergic mechanisms, Neurosci. Lett., 104 (1989) 58-64. 8 Freund, T.E and Antal, M., GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus, Nature, 336 (1988) 170-173. 9 Kairiss, E.W., Abraham, W.C., Bilkey, D.K. and Goddard, G.V., Field potential evidence for long-term potentiation of feed-forward inhibition in the rat dentate gyms, Brain Research, 401 (1987) 87-94. 10 Kanda, M., Maru, E., Ashida, H., Tatsuno, J. and Takatani, O., Effects of LTP-inducing tetanic stimulations of the perforant path on the commissural inhibition and facilitation of dentate granule cell discharge, Brain Research, 484 (1989) 325-332. 11 Lacaille, J.-C., Kunkel, D.D. and Schwartzkroin, P.A., Elec-
12
13
14
15
16
17
18
19
20
21
trophysiologic and morphological characterization of hippocampal interneurons. In V. Chan-Palay and C. Kohler (Eds.), The Hippocampus -- New Vistas, Liss, New York, 1989. Maru, E., Ashida, H. and Tatsuno, J., Long-lasting reduction of dentate paired-pulse depression following LTP-inducing stimulations of the perforant path, Brain Research, 478 (1989) 112120. Scharfman, H.E. and Sarvey, J.M., G A B A sensitivity does not change during long-term potentiation in rat hippocampal slices, Neuroscience, 15 (1985) 695-702. Seress, L. and Ribak, C.E., Direct commissural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-forward inhibition, J, Neurocytol., 13 (1984) 215-225. Steltzer, A., Slater, N.T. and Bruggencate, G., Activation of NMDA receptor blocks inhibition in an in vitro model of epilepsy, Nature, 326 (1987) 698-701. Steward, O., Tomasulo, R. and Levy, W.B., Blockade of inhibition in a pathway with dual excitatory and inhibitory action unmasks a capability for LTP that is otherwise not expressed, Brain Research, 516 (1990) 292-300. Taube, J.S. and Schwartzkroin, P.A., lntracellular recording from hippocampal CA1 interneurons before and after development of long-term potentiation, Brain Research, 419 (1987) 3238. Taube, J.S. and Schwartzkroin, P.A., Mechanisms of long-term potentiation: EPSP/spike dissociation, intradendritic recordings, and glutamate sensitivity, J. Neurosci., 8 (1988) 1632-1644. Wigstr6m, H. and Gustafsson, B., Facilitation of hippocampal long-lasting potentiation by G A B A antagonists, Acta Physiol. Scand., 125 (1985) 159-172. Wilson, R.C., Changes in translation of synaptic excitation to dentate granule cell discharge accompanying long-term potentiation. I: Differences between normal and reinnervated dentate gyrus, J. Neurophysiol., 46 (1981) 324-338. Wilson, R.C., Levy, W.B. and Steward, O., Changes in translation of synaptic excitation to dentate granule cell discharge accompanying long-term potentiation. 11: An evaluation of mechanisms utilizing dentate gyrus dually innervated by surviving ipsilateral afferents and sprouted crossed temperodentate inputs, J. Neurophysiol., 46 (1981) 339-355.
27
BRES 17005
LTP-associated EPSP/spike dissociation in the dentate gyrus: GABAergic and non-GABAergic components Richard A. Tomasulo 1, William B. Levy e and Oswald Steward 1 Departments of ~Neuroscience and 2Neurosurgery, University of Virginia School of Medicine, Charlottesville, VA (U.S.A.) (Accepted 7 May 1991) Key words: Hippocampus; Dentate gyrus; y-Amino butyric acid; Long-term potentiation, Synaptic plasticity; Inhibition; Excitatory postsynaptic potential/spike dissociation
The induction of long-term potentiation (LTP) in the dentate gyrus (DG) leads to a change in the firing characteristics of the dentate granule cells. This phenomenon, termed EPSP/spike dissociation, is seen in field potential studies as a shift to the left of the E-S curve, in which population spike amplitude is plotted against pEPSP slope at various stimulus intensities. It has been suggested that EPSP/spike dissociation reflects a decrease in feed-forward inhibition. To test this hypothesis, we blocked GABA-A neurotransmission in a circumscribed area of the DG in urethane-anaesthetized rats by inserting a micropipette filled with 8 mM bibuculline methiodide in saline. We then recorded E-S curves from 9 such electrodes and from 8 control electrodes before and after inducing LTP in the perforant path. Bicuculline prevented the LTP-associated leftward shift of the E-S curves. Instead, the E-S curve showed a consistent shift to the right at the bicuculline sites after LTP, reflecting potentiation of the pEPSP without corresponding increases in the population spike amplitude. The results indicate that the EPSP/spike relationship is controlled largely by GABAergic input, and that potentiation of the population spike in the DG depends largely on a change in the EPSP/spike relationship. INTRODUCTION High-frequency activation of selected h i p p o c a m p a l afferents results in two long-lasting changes of neuronal responsiveness. First, a given presynaptic input p r o d u c e s a larger dendritic depolarization than before. This is called long-term p o t e n t i a t i o n 5 (LTP), or synaptic potentiation, because it reflects an increase in the efficacy of the activated synapses. Second, the postsynaptic cells become m o r e excitable: an action potential can result from a dendritic current which was previously insufficient for spike generation 2'1s. The latter effect, t e r m e d EPSP/spike dissociation, can be d e m o n s t r a t e d from field recordings by plotting cell discharge as a function of dendritic depolarization following stimulation at various intensities. Cell firing is rec o r d e d as p o p u l a t i o n spike amplitude, and dendritic depolarization is estimated by the p o p u l a t i o n E P S P (pEPSP). The resulting ' E - S curve' shifts to the left after high-frequency stimulation, indicating an increased spike-generating capacity for a given degree of dendritic depolarization 2°,21. Two hypotheses have b e e n p r o p o s e d to account for
the EPSP/spike dissociation: (1) the EPSP/spike dissociation reflects an internal alteration of the postsynaptic cells 18, or (2) the dissociation reflects a change in the balance b e t w e e n the excitatory and inhibitory synaptic currents activated by stimulation of the afferent population 2'2°'21. The latter hypothesis, first p r o p o s e d by Wilson et al. 2°'21, is based on current concepts of hippocampal feed-forward inhibition. In CA1 and the d e n t a t e gyrus ( D G ) , inhibitory interneurons receive innervation from the same excitatory afferents as do the projection cells 14. These inhibitory interneurons fire with extremely short latencies following activation of the shared afterents 6'17. Their feed-forward inhibitory action could therefore control the efficiency with which dendritic depolarization translates into action potentials following a given afferent volley. The EPSP/spike dissociation which follows the induction of LTP could result from a real decrease in feedforward inhibition l°'le or from a relative decrease due to an u n o p p o s e d increase in excitation 2'2°' Zl If the EPSP/spike dissociation does reflect a shift in the balance b e t w e e n excitation and inhibition, then eliminating feed-forward inhibition should prevent the E - S
Correspondence: O. Steward, Department of Neuroscience, Box 5148 MR4 Medical Center, University of Virginia School of Medicine, Charlottesville, VA 22908, U.S.A.
28 shift which normally follows high-frequency stimulation. This result has been obtained in the CA1 region in hippocampal slice preparations 2'7. We report here that blocking ),-aminobutyric acid (GABA)-A mediated inhibition in the DG in vivo, via a local infusion of bicuculline methiodide, prevented the leftward shift of the E - S curve which normally accompanies LTP in the perforant path. Instead, the induction of LTP under GABA receptor blockade resulted in a shift of the E - S curve to the right. Because of this unexpected shift, LTP induced during G A B A receptor blockade was not accompanied by consistent increases in population spike amplitude. The findings indicate that, in the DG, a substantial component of the increase in population spike amplitude following LTP results from a change in the balance between synaptic excitation and feed-forward inhibition.
MATERIALS A N D M E T H O D S
these single-electrode experiments 3 were with bicuculline and two with saline only. The total sample, therefore, consisted of data from 11 bicuculline and 10 saline electrodes.
Data analysis The pEPSP slope was measured on the rising phase, at a fixed latency from the stimulus, before the onset of the population spike. If LTP (defined as a 10% increase in pEPSP slope at two stimulus intensities) did not occur at either electrode, the experiment was excluded from the study. Spike amplitude was measured from the peak negativity to a line connecting the prespike maximum positivity to the postspike maximum positivity. We measured only the first of the multiple spikes evoked at the bicuculline site. For latency measurements, the spike onset was defined as the prespike maximum positivity, the point at which the pEPSP slope became zero in transition to the negative-going population spike. The measurements were made by an Apple computer employing 'Waveman' software. The E - S curves were constructed from the average values obtained for each stimulus intensity at each electrode. Initially, the average of each set of 4 sweeps was plotted as a single point, so that data from the two consecutive collection periods for each experimental condition could be compared. If these were judged sufficiently similar (the determination was subjective), then all the sweeps collected at a given stimulus intensity were averaged and
Experimental procedures Male Sprague-Dawley rats (250-450 g) were anaesthetized with urethane (1.5 g/kg), and prepared for acute physiological recording. A bipolar stimulating electrode was placed in the angular bundle (AB, -8.1 mm, 4.4 mm lateral to bregma), and two recording micropipettes were placed in the ipsilateral dentate gyrus (DG). The first micropipette, containing 0.9% NaCI, was placed 4 mm posterior and 2.5 mm lateral to bregma. The depth was adjusted for maximal positivity of the EPSP following AB stimulation. A second micropipette, containing 8 mM bicuculline methiodide in saline ~6, was inserted about 1.5 mm anteromedial to the first. The depth was again adjusted for maximal positivity of the EPSP. As described previously x6, the effectiveness of bicuculline in blocking inhibition could be confirmed by examination of the field responses. At the bicucuUine site, the spike threshold was lower, multiple population spikes were evoked by a single perforant path stimulus, and paired-pulse inhibition was either eliminated or markedly reduced (Fig. 1). Once the electrodes were placed and the G A B A blockade confirmed, the preparation was allowed to stabilize for at least 45 min before data were collected. In order to construct E - S curves, evoked responses were recorded simultaneously from both electrodes, digitized, and stored on floppy discs for subsequent analysis by microcomputer. Test stimuli were delivered every 15 s to the AB. The lowest intensity was just above spike threshold for the bicuculline electrode, and the highest was that which evoked a maximum-amplitude spike from the control (saline) electrode. Four stimuli were given at each intensity, and steps were chosen so that at least 5 different spike amplitudes were recorded from each electrode. Because spike threshold and maximum spike amplitude were reached at lower intensities at the bicuculline site, we used 6-10 intensities overall. After the first data set, the collection procedure was repeated. In some experiments, the number of stimulus intensities was reduced in the second data collection period, but 4 stimuli were still delivered at each intensity. Immediately after the baseline data collection, we delivered 8 400-Hz. 17.5-ms stimulus trains (i.e. 8 pulses per train) at 15-s intervals. Stimulus intensity was adjusted to evoke at least a 5 mV population spike at the saline electrode. After waiting at least 5 min, we repeated the data collection procedure. One or two additional stimulus intensities, below the initial minimum, were added to the routine, because the spike thresholds invariably decreased following high-frequency conditioning. We performed 8 double-electrode experiments, as described, and 5 additional experiments with only a single recording electrode. Of
A
YL __
B
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•
Fig. 1. Effect of bicuculline on evoked responses. A: upper trace is from a bicuculline-containing electrode. Lower is from control. The responses were obtained simultaneously, following paired angular bundle stimuli delivered at an interval of 20 ms. Note the absence of paired-pulse inhibition at the bicuculline site. Calibration 5 mV, 5 ms. B: same as A, but with a 50-ms interstimulus interval. Robust feedback inhibition is retained at the control site. Calibration 5 mV, 10 ms.
29 plotted as a single point. Points on an individual E - S curve, therefore, represented an average of either 4 or 8 sweeps. If the curves were not reproducible, the experiment was excluded from the study. The data from two double-electrode experiments were excluded from statistical analysis, one because of unstable responses and one because LTP did not occur at either electrode. We therefore report the results of 6 double-electrode and 5 single-electrode experiments (9 bicuculline and 8 saline electrodes total). The E-S curves from these experiments were normalized by defining the maximum pretetany pEPSP slopes and spike amplitudes as 100%. Normalized spike amplitudes at selected normalized pEPSP values were taken from the individual E-S curves, by interpolation when necessary, and averaged to produce composite curves (Fig. 3). The statistical significance of the change in mean spike amplitude for each pEPSP value was determined by the Wilcoxon signed rank test.
RESULTS
As reported previously 16, bicuculline methiodide produced stable alterations in the responses evoked by AB stimulation. At moderate and high stimulus currents, a single AB stimulus evoked multiple population spikes at the bicuculline sites. Paired-pulse inhibition was eliminated or sharply reduced. Neither of these effects occurred at the control site, indicating that the bicuculline blocked GABAergic neurotransmission in a limited volume of tissue only (Fig. 1). Before high-frequency conditioning, population spikes appeared at the bicuculline sites at lower stimulus intensities and reached their maxima at lower intensities than at the control sites (Fig. 2A). In individual experiments, the maximum spike amplitudes recorded at the two sites often differed considerably (e.g. Fig. 2). However, neither electrode consistently showed larger responses, and the mean values were similar (Table I). Before conditioning, therefore, bicuculline did not seem to alter the maximum amplitudes of the population spikes. In contrast to the spike amplitudes, the pEPSP slopes rose monotonically as a function of stimulus intensity through the same range of stimuli at both sites (Fig. 2B, Table I). Thus, the bicuculline had no demonstrable effect on pEPSP generation. As reported by other investigators 19, high-frequency stimulation induced more potentiation of the pEPSPs during G A B A receptor blockade than under control conditions. The average increase following conditioning was 37% (S.E.M. = 3.5) at the bicuculline sites and 25% (S.E.M. -- 5.6) at the saline sites. The E - S curves showed the expected left shift at all 8 control electrodes. This left shift was significant at all points on the normalized E - S curve (Fig. 3; P values between 0.025 and 0.006). At 6 of 9 bicuculline sites, the left shift was completely eliminated. At the remaining 3, slight left shifts occurred. These were much smaller than those seen at the control sites, however, and they oc-
curred only at high stimulus intensities. In these 3 cases, the average increase in spike amplitude at maximum pretetany pEPSP was 8%, compared to an average increase of 22% at the same point for the saline electrodes. The majority of post-LTP curves from the bicuculline electrodes were not identical to the baseline curves, however. Instead, 6 of the 9 curves showed an unexpected shift to the right, indicating a decrease in the spike-generating capacity of a given pEPSP. This phenomenon is illustrated in Fig. 4, where pre- and postLTP responses with identical pEPSP slopes are compared. The right shift was statistically significant only at high stimulus intensities (Fig. 3; P = 0.025, 0.038, 0.043 at the top three points by Wilcoxon rank test). As a resuit, the slopes of the E - S curves decreased in the 6 cases showing the right shift (e.g. Fig. 2C). The mean slope of the E-S curves, as estimated by linear regres-
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Fig. 2. Data from a sample experiment. In each pair of graphs, the left panel represents data from the saline electrode, and the right panel that from the bicuculline site. A: spike amplitude as a function of stimulus intensity (in mA). Before tetany, the spike threshold and maximum amplitude are reached at lower stimulus strength at the bicueulline site. Note that the spike amplitude did not change significantly at the bicuculline site following high-frequency activation. B: EPSP slope as a function of stimulus. Pre- and postconditioning EPSPs are similar at the two sites. C: E - S curves. At the saline site, the expected left shift occurs. At the bicuculline electrode, the shift is in the opposite direction.
30 TABLE I
sion, d e c r e a s e d by 20% after c o n d i t i o n i n g in the bicu-
Effects o f tetany on EPSP slopes and spike amplitudes
culline s a m p l e ( P = 0.03 by W i l c o x o n signed r a n k test). ( N o t e that the E - S curves are not linear. W e are using
S.E.M. in parentheses.
linear r e g r e s s i o n o n l y for a statistical c o m p a r i s o n be-
Saline Stimulus b
t w e e n pre- and p o s t c o n d i t i o n i n g c u r v e s . )
BicucuUine
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Post-tet
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C o n s i s t e n t with the c u r v e shift data, the p o p u l a t i o n spike a m p l i t u d e s ( r e c o r d e d using the s a m e stimulus in-
EPSP slopes normalized a
2 3 4 5
0.65 0.87 1.00 1.18
(0.08) (0.02) (0.02)
0.27 0.67 0.69 1.00 1.32
0.83 (0.11) 1.10 (0.07) 1.27 (0.04) 1.35 (0.07)
(0.07) (0.12) (0.06) -
0.43 (0.13) 0.90 (0.17) 1.03 (0.14) 1.44 (0.13)
(0.11)
1.62 (0.11)
tensity pre- and p o s t c o n d i t i o n i n g ) i n c r e a s e d to a m u c h larger d e g r e e at the saline e l e c t r o d e s t h a n at the bicuculline sites (Fig. 2 A , Table I). T h e a v e r a g e i n c r e a s e in p o p u l a t i o n spike a m p l i t u d e at the saline sites was 172% ( S . E . M . = 21.5), and m e a n p o p u l a t i o n spike a m p l i t u d e
Spike amplitudes (mV)
2 3 4 5
3.30 10.63 9.81 13.80
(0.77) (2.6) (3.8) (5.2)
2.94 9.42 11.20 13.41 16.51
13.75 (2.4) 18.78 (3.2) 18.33 (3.6) 19.15 (3.5)
(0.86) 6.75 (1.6) (2.5) 9.87 (2.6) (2.6) 12.80 (2.8) (1.9) 14.07 (2.1) (1.8) 15.92 (2.2)
i n c r e a s e d f r o m 6.8 to 16.1 m V ( P = 0.0001 by p a i r e d t-test, n = 27). U s i n g the data in Table I (a m e a n increase in E P S P o f 28% at low stimulus intensity and o f 14% at high intensity) and the p o o l e d E - S curve (Fig. 3B), we estimate that 45-75% of the increase in spike amplitude
a Normalization: the EPSP value at the tetanizing stimulus was defined as 1.0 for each experiment. Note that this is different from the normalization used for the composite E-S curves (Fig. 3). b Stimulus intensities are as follows: 1 = spike threshold for bicueulline electrode;2 = spike threshold for saline electrode; 4 = tetanizing intensity; 3 and 5 were one step below and above tetanizing intensity respectively.
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Fig. 3. Normalized E-S curves. The maximum preconditioning pEPSP and spike values were defined as 100%. A: data from 9 bicuculline electrodes. The right shift is significant at the highest 3 EPSP values only (P = 0.043, 0.038, and 0.025 at 100%, 85%, and 75% respectively, by Wilcoxon signed rank test. NS below 75%). B: data from 8 control electrodes. P values vary between 0.025 and 0.006.
Fig, 4. Changes in spike-generating capacity and latency following the induction of LTP. A, B and C are from a saline electrode. D, E, and F are from the bicuculline electrode in the same animal. A: baseline response. B: postconditioning response evoked by a stimulus adjusted to produce an identical pEPSP slope. C: A and B superimposed. The interrupted line is the preconditioning response. The latency to spike onset decreased by 0.36 ms concurrent with the increase in spike amplitude. D and E: pre- and postconditioning responses, respectively, from the bicuculline site. The stimulus was adjusted to equalize the pre- and post-tetany pEPSP slopes. Note the decrease in spike amplitude. F: D and E superimposed. The interrupted line is the preconditioning response. The latency to spike onset decreased by 0.26 ms despite the decrease in spike amplitude. Digitized waveforms, slightly retouched. Calibration: 2.5 mV, 2 ms.
31 TABLE II
30
Effects of tetany on population spike latency S.E.M. in parentheses.
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•
•
SALINE
e~•
•
BICUCULLINE
•,'o
2o
Pre-tet
Post-tet
Change
lab
Latency to population spike onset (ms) Saline electrodes Low 4.58 (0.27) 4.02 (0.31) High 3.79 (0.14) 3.56 (0.12)
-0.56 (0.12) -0.23 (0.06)
0.008 0.015
Bicuculline electrodes Low 4.89 (0.16) High 3.85 (0.18)
4.65 (0.26) 3.77 (0.16)
-0.23 (0.14) 4.08 (0.05)
0.146 0.229
Latency to population spike peak (ms) Saline electrodes Low 5.58 (0.54) 5.27 (0.68) High 4.67 (0.28) 4.46 (0.28)
-0.31 (0.17) -0.21 (0.07)
0.127 0.034
Bicuculline electrodes Low 6.59 (0.51) High 4.78 (0.36)
-0.42 (0.22) -0.09 (0.11)
0.119 0.417
30
SPIKE AMPLITUDE PRE-TETANY (mV)
6.17 (0.56) 4.69 (0.33)
Low stimulus intensity is just above spike threshold for the pretetany trials; high stimulus is that which evoked the maximum pre-tetany spike amplitude. Post-tetany latencies were taken from responses with pEPSP slopes equal to the pre-tetany low and high stimulus responses respectively. Interpolation was used when necessary. b Paired t-test. a
Fig. 5. Effect of tetany on spike amplitude. Each point represents spike amplitudes at a constant stimulus intensity before and after tetany. Each point is the average of 4 pre- and 4 post-tetany trials. Data are from all experiments. A change in spike amplitude is indicated by vertical displacement from the diagonal line, which represents the function x = y. At the bicuculline sites, granule cell output changed very little after tetany, despite significant potentiation of the EPSPs.
which followed conditioning could be attributed to the left shift. This is consistent with previous studies 1"9'2°'21. At the bicuculline sites, the amplitude of the population spike evoked by a given stimulus either did not change, or actually decreased, in the majority of trials (e.g. Fig. 2A). The average increase of population spike amplitude was just 20% (S.E.M. = 12.1), and the mean population spike amplitude increased only from 10.2 m V to 10.9 mV at the bicuculline sites following potentiation (P = 0.121, n = 43). Only at low stimulus intensities did LTP materially increase the mean population spike amplitude (Table I). These results are illustrated in a different way in Fig. 5. Here postconditioning spike amplitudes are plotted as a function of preconditioning amplitudes, evoked at the same stimulus intensity before and after conditioning. A diagonal line is drawn through the origin with a slope of 1. A change in spike amplitude with conditioning is indicated by vertical displacement from this line. All the points from the control electrodes are above the line (i.e. spike amplitude increased), while the majority of values from the bicuculline sites are on or below the line. Thus, high-frequency stimulation delivered during G A B A receptor blockade did not consistently increase spike amplitude despite its effectiveness in potentiating the EPSPs (see Table I).
Fig. 5 also explains why the mean increase of spike amplitude at the bicuculline sites was 20%, while the mean amplitude increased by only 0.7 mV. Within the bicuculline sample, substantial increases in population spike amplitude occurred only at low stimulus intensities, where the small baseline spike amplitudes guaranteed large percentage increases. These few responses contributed disproportionately to the average percentage increase. Previous studies have established that, in response to an unchanged excitatory drive (i.e. at a fixed pEPSP slope before and after conditioning), the population spike latency ordinarily decreases after the induction of LTP 1. Several investigators have suggested that this decrease in latency, like the left shift of the E - S curve, might reflect a change in the balance between excitation and inhibition 2,2°,21. If the decrease in population spike latency does indeed depend upon a decrease in feed-forward inhibition, then the latency change should be minimized when inhibitory neurotransmission is blocked. Consistent with this prediction, we found that, when measured at the same p E P S P slope before and after tetany, statistically significant decreases in spike latency (whether measured at onset or at peak negativity) occurred only in the control population (Table II). A t 3 of the bicuculline sites, no change in latency could be measured with high or low
32 intensity stimulation following high-frequency conditioning. At 3 other bicuculline sites, however, the spike latency for a given pEPSP slope actually decreased concurrent with a reduction of population spike amplitude (i.e the right shift) following conditioning (Fig. 4). At the control sites, in agreement with previous studies 1"2°" 21, a reduction in spike latency invariably accompanied an increase in population spike amplitude (Fig. 4). DISCUSSION Three points from the above data require elaboration: (1) In the DG, the LTP-associated leftward shift of the E - S function depends upon intact GABAergic neurotransmission. (2) G A B A receptor blockade reveals a second LTP-associated influence on the EPSP/spike relationship. This effect opposes the left shift. (3) Previous studies have established that the left shift contributes to the increase in granule cell output which follows tetanic conditioning. If the right shift we describe here occurs concurrently with, and is masked by, the G A B A dependent left shift, then GABAergic mechanisms must contribute more to altering granule cell output than the left shift alone would indicate.
The left shift is GABA-dependent The mechanism of the left shift was the initial subject of this project. We tested the hypothesis that the left shift reflects a decrease of GABAergic feed-forward inhibition relative to the increase in excitation which follows high-frequency stimulation. This hypothesis predicts that a blockade of GABAergic neurotransmission should prevent the LTP-associated leftward shift of the E - S curve. This is precisely the result we obtained. The findings are consistent with previous studies of LTP in the CA1 region in vitro, where G A B A receptor blockade also prevents the left shift of the E - S function 2'7. This mechanism therefore contributes to EPSP/spike dissociation in both regions of the hippocampus. Also consistent with a relative decrease in inhibition is the finding that the population spike latency (for a given pEPSP slope) decreased to a smaller extent at the bicuculline sites than at the control sites (Table II). Within this logical frame, the G A B A dependence of the left shift is compatible with two interpretations. Abraham et al. 2, following a suggestion made by Wilson et al. 2°'21, have proposed that the left shift reflects only an increase in excitatory drive (as a result of LTP) which is not matched by a compensatory increase in feed-forward inhibition. Thus, a given presynaptic volley would evoke an increased inward current following LTP without an accompanying increase in inhibitory outward current. The strength of this hypothesis is its avoidance of
experimentally unverified modifications in the inhibitory circuit. The hypothesis cannot, however, explain examples of EPSP/spike dissociation occurring in the apparent absence of EPSP potentiation 5'18. An alternate hypothesis is that the LTP-associated left shift of the E - S curve reflects a real decrease of synaptic efficacy in the inhibitory pathway. Because feed-forward inhibition in the hippocampus is effected largely through a disynaptic circuit 6"11, the left shift could reflect a reduction in synaptic driving of inhibitory interneurons, reduced efficacy of the GABAergic synapses on the projection cells, or alterations at both sites. According to two previous studies, however, driving of interneurons does not decrease with LTP; it actually inc r e a s e s 6'17. Increased interneuron discharges would presumably increase, not decrease, feed-foward inhibition, provided that the increased firing occurred at a short enough latency to affect the evoked granule cell discharge. In this case, GABAergic synapses would have to undergo significant activity-dependent depression after high-frequency activation to account for the net reduction of inhibition at the projection cells. This hypothesis receives support from a study by Steltzer et al. 15, who described depression of GABAergic neurotransmission following high-intensity 50 Hz conditioning of afferents to the CA1 region in hippocampal slices. Scharfman and Sarvey 13, however, failed to demonstrate alterations in G A B A sensitivity following more moderate 100 Hz stimulation.
G A B A receptor blockade revealed a second influence on the EPSP/spike relationship Our results demonstrate a second LTP-associated effect on the EPSP/spike relationship. This appears as a reduction of slope and a shift to the right of the E - S curve at the bicuculline sites after conditioning (Fig. 2C and 3). Though not explicitly linked to a blockade of inhibition, this right shift -- or a very similar effect -- is discernible in previous studies 4'9"2°. Kairiss et al. 9 evoked a slope decrease and slight right shift of the E - S curve via low-intensity conditioning trains to the perforant path in vivo. Following higher intensity tetanic stimulation, the usual left shift occurred. Their result supported the existence of two separable effects, which were apparently activated at different thresholds. In an early study of the E - S shift, Wilson et al. 2°'21 found that a crossed temperodentate pathway which had sprouted following a unilateral lesion of the entorhinal cortex could express LTP, but did not show the usual E - S shift to the left. Instead, the E - S curve shifted slightly to the right and decreased in slope, as did the curves in our bicuculline experiments. These investigators did not attempt to explain the right shift, but they
33 explained the absence of a leftward shift by a failure of the sprouted pathway to achieve effective innervation of inhibitory interneurons. If this view was correct, their experimental manipulation selectively reduced or eliminated feed-forward inhibition, and was therefore comparable to our infusion of bicuculline. Bliss et al. 4 reported that increasing extracellular calcium in the D G in vivo can induce potentiation of the EPSP, while reducing spike amplitude and latency. These effects, of course, are remarkably similar to the results we obtained via tetanic stimulation under G A B A receptor blockade. The meaning of this similarity is uncertain. The cause of the right shift/slope decrease is unclear. One possible mechanism is potentiation of a nonGABAergic, short-latency inhibitory synaptic potential. Such an increase of feed-forward inhibition should, however, produce an increase in population spike latency. Our failure to observe such latency increases at the bicuculline sites argues against this mechanism. In fact, we encountered decreases of spike latency in cases where spike amplitude also decreased following high-frequency conditioning (Fig. 4). Under control conditions, of course, changes in latency are inversely related to changes in spike amplitude. Our finding of parallel reductions in latency and amplitude -- even though the latency decreases were small -- suggests a mechanism entirely different from that which is responsible for the left shift. Another possible cause for the right shift is a change in tonic input from an extrahippocampal source. The absence of the right shift/slope decrease with LTP in picrotoxin-treated hippocampal slices 2'7 is consistent with this idea. An alternate interpretation is that the right shift/slope decrease is specific to the DG. We cannot even be sure that the right shift reflects the behavior of granule cells in general. The population spikes we recorded in the hilus may represent the summed activity of several cell types which respond differentially to tetanic stimulation. We think it improbable that the right shift is an artifact due entirely to the effects of bicuculline, but the possibility must be considered. One possibility is that the limited spike potentiation could represent a saturation effect, due to maximization of the pre-tetany population spike amplitude as a result of bicuculline. The term 'saturation' can be applied in two senses: (1) The maximum spike amplitude could have been achieved at a low stimulus intensity, and at a correspondingly submaximal pEPSP. Under these conditions, test stimuli might elicit maximal population spikes before the induction of LTP so that further increases in amplitude could not be detected. This seems unlikely because bicuculline limited spike potentiation throughout the range of spike ampli-
tudes (Fig. 5, Table I). (2) The maximum population spike amplitude for each stimulus intensity (a given set of afferent fibers) could have been reached before potentiation; i.e. increases in the efficacy of the activated synapses could not measurebly increase cell firing. This is an unlikely possibility because the activated synapses should have been distributed to the granule cells in nearly random proportions. With most submaximal stimuli, therefore, the postsynaptic population would include cells that could achieve firing threshold only after synaptic potentiation. In support of this interpretation, we note that paired pulse facilitation was observed at 5 of 9 bicuculline sites prior to tetanic stimulation (e.g. Fig. 1). In 3 of these cases, despite this demonstrated capacity for increased cell firing, bicucuUine prevented spike potentiation. Finally, this form of saturation cannot explain the reduction of population spike amplitude we often observed after conditioning (see Fig. 5). If the inhibitory interneurons behave like hippocampal projection cells, their excitatory synapses could show exaggerated potentiation in the presence of G A B A receptor blockade 19, due to release of tonic inhibition. (Hippocampal interneurons receive GABAergic innervation from the medial septal nucleus3'S.) This would increase feed-forward inhibition after potentiation if the G A B A blockade on the granule cells was incomplete. As discussed above, this increase in inhibition would be expected to increase spike latency following potentiation, and, since the latencies did not increase, this mechanism is unlikely. Another way bicuculline might produce misleading resuits is by depolarizing the granule cells. Tonic depolarization would reduce the amplitude of each cell's action potential. If this tonic depolarization increased for any reason following potentiation, then the action potential amplitude would decrease further. The reduction in population spike amplitude would not then reflect a reduction in cell firing, but only the sum of reduced action potential amplitudes. GABAergic mechanisms are an important source o f spike potentiation EPSP/spike dissociation (the left shift) contributes significantly to the alterations of granule cell output which follow high-frequency conditioning. In our saline sample, the left shift increased population spike amplitude by 20-120% above that predicted from EPSP potentiation alone (Fig. 3B). We estimated that 45-75% of the increase in spike amplitude which followed conditioning could be attributed to the left shift. Our results indicate that this very significant effect depends on GABAergic mechanisms. If the left shift reflects synaptic changes in the disynaptic inhibitory circuit, then the interneurons
34
are potent effectors of change in projection cell output. If, on the other hand, high-frequency conditioning does not alter synaptic transmission in the inhibitory circuit, then hippocampal interneurons must still enable or amplify the changes of projection cell output which accompany LTP. In either case, the inhibitory interneurons must play an important role in the integrative function of the hippocampus. The rightward shift of the E - S curve which occurred at our bicuculline sites does not alter the above conclusion. It merely complicates the attempt to quantify the
interneurons' contribution. If the mechanisms responsible for the right shift are activated concurrently with the left shift in the untreated DG, then the effect of the interneurons is even greater than the estimates provided above.
Acknowledgements. Supported by NSF Grant BNS8818766 to O.S.W.B.L. was supported by NIH Grant NS15488 and NIMH Career Development Award MH0622. R.T. was supported by NIH Training Grant NS07199 and by the Department of Neurological Surgery, Dr. John A. Jane, Chairman.
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