Blockade of GABAA Receptors Facilitates Induction of NMDA Receptor-Independent Long-Term Potentiation

Lawrence M. Grover and Chen Yan

Department of Physiology, Marshall University School of Medicine, Huntington, West Virginia 25755-9340


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Grover, Lawrence M. and Chen Yan. Blockade of GABAA receptors facilitates induction of NMDA receptor-independent long-term potentiation. An N-methyl-D-aspartate (NMDA)-independent form of long-term potentiation (LTP), which depends on postsynaptic, voltage-dependent calcium channels (VDCCs), has been demonstrated in area CA1 of hippocampus. GABA acting at GABAA receptors limits postsynaptic depolarization during LTP induction. Blockade of GABAA receptors should therefore enhance activation of postsynaptic VDCCs and facilitate the induction of this NMDA receptor-independent, VDCC-dependent LTP. In agreement with this hypothesis, pharmacological blockade of GABAA receptors in the in vitro rat hippocampal slice increased the magnitude of LTP resulting from a normally effective, high-frequency (200 Hz) tetanic stimulation protocol. In addition, GABAA receptor blockade allowed a lower frequency (25 Hz) and normally ineffective tetanic stimulation protocol to induce this form of LTP. Intracellular recordings from CA1 pyramidal cells revealed that blocking GABAA receptors during tetanic stimulation allowed greater postsynaptic depolarization, increased the number of postsynaptic action potentials fired during the tetanization, and also increased the duration of synaptically evoked action potentials. To mimic the increased action potential firing observed when GABAA receptors were blocked, we paired 25-Hz antidromic stimulation with 25-Hz orthodromic stimulation. Paired antidromic + orthodromic 25-Hz stimulation induced NMDA receptor-independent LTP, whereas neither antidromic nor orthodromic stimulation alone induced LTP. Increased action potential firing can therefore at least partially account for the facilitation of NMDA receptor-independent LTP caused by blockade of GABAA receptors. This conclusion is consistent with prior studies demonstrating that action potentials are particularly effective stimuli for the gating of VDCCs in CA1 pyramidal cell dendrites.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

High-frequency (200 Hz) tetanization can induce a slow onset, N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation (LTP) in hippocampal area CA1 (Cavus and Teyler 1996; Grover 1998; Grover and Teyler 1990, 1992, 1994, 1995). Previous studies (Cavus and Teyler 1996; Grover and Teyler 1990) indicated that induction of this NMDA receptor-independent LTP requires Ca2+ influx through postsynaptic voltage-dependent Ca2+ channels (VDCCs). During tetanization, GABAA receptor-mediated inhibition limits the degree of postsynaptic depolarization and postsynaptic action potential firing and thus limits the magnitude of Ca2+ influx into postsynaptic neurons (van der Linden et al. 1993). Relief from GABAA inhibition should therefore enhance postsynaptic Ca2+ entry and thereby facilitate induction of LTP. Although it was shown that LTP can be enhanced by blockade of GABAA receptors (Wigström and Gustafsson 1983), this enhancement was attributed to an increase in postsynaptic Ca2+ influx through NMDA receptors (Wigström and Gustafsson 1985) because the blockade of GABAA receptors allows greater postsynaptic depolarization and thus more complete relief from the voltage-dependent block of NMDA receptors by Mg2+. The first objective of this study was to determine if GABAA receptors regulate the induction of an NMDA receptor-independent LTP similar to the GABAergic regulation of NMDA receptor-dependent LTP.

A previous study (Grover 1998) found that firing of action potentials by the postsynaptic CA1 pyramidal neurons during tetanization was required for induction of NMDA receptor-independent LTP. Other studies have shown that action potential waveforms are effective stimuli for the gating of high-threshold Ca2+ currents (McCobb and Beam 1991; Scroggs and Fox 1992). In addition, propagation of action potentials into dendrites generates VDCC-dependent dendritic Ca2+ signals (Magee and Johnston 1995; Spruston et al. 1995). These observations suggest that postsynaptic action potential discharge during tetanization is critical for triggering Ca2+ entry into the postsynaptic neurons through VDCCs. A second objective of this study then was to determine if blockade of GABAA receptors would alter postsynaptic action potential discharge in a manner that would be expected to facilitate Ca2+ influx through VDCCs.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation

Hippocampal slices were prepared from 1- to 2-mo-old male and female Sprague-Dawley rats (Hilltop Laboratory Animals). Animals were sedated by inhalation of a CO2-air mixture and decapitated. The skull was opened, and the brain was removed and submerged in chilled, oxygenated (95% O2-5% CO2), low-Ca2+/high-Mg2+ artificial cerebrospinal fluid (ACSF) composed of (in mM) 124 NaCl, 26 NaHCO3, 3 KCl, 0.5 CaCl2, 5.0 MgSO4, and 10 glucose. After cooling in the low-Ca2+/high-Mg2+ ACSF, the brain was trimmed and glued to the stage of a vibratome and immersed in a bath of chilled, oxygenated, low-Ca2+/high-Mg2+ ACSF. Coronal sections, 400 µm thick, were then cut. Sections containing the hippocampus in transverse profile were selected and transferred to a small petri dish, where they were further dissected to free the hippocampus from surrounding tissue. In most slices, the CA3 region was also removed to prevent any spontaneous synaptic activity driven by the bursting of CA3 neurons in the presence of bicuculline. However, identical results were obtained in slices where area CA3 was left intact. After dissection, hippocampal slices were transferred to a holding chamber, where they were stored for later use.

Slices were maintained in the holding chamber at room temperature (18-22°C) at the ACSF-atmosphere (95% O2-5% CO2) interface. The holding chamber was filled with standard ACSF containing (in mM) 124 NaCl, 26 NaHCO3, 3.4 KCl, 1.2 NaH2PO4, 2.0 CaCl2, 2.0 MgSO4, and 10 glucose. Slices were incubated in the holding chamber for >=  1 h before use.

Slices were withdrawn from the holding chamber as needed and placed in a low-volume (~200 µl) interface recording chamber, where they were continuously perfused at a rate of 1-2 ml/min with standard ACSF. The recording chamber was kept at a temperature of 35 ± 0.15°C. After transferring slices from the holding to the recording chamber, a minimum 30-min period was allowed for recovery.

Electrophysiology

Extracellular potentials were recorded through low-impedance (1-2 MOmega ) glass micropipettes, filled with ACSF, and placed into the stratum radiatum of area CA1. Signals were amplified (gain 100-1000) and filtered (DC-3,000 Hz or 0.1-10,000 Hz). After processing, signals were digitized at 10-40 kHz and stored on a personal computer. Excitatory postsynaptic potentials (EPSPs) were evoked by delivery of constant current stimuli through a bipolar stimulating electrode placed into stratum radiatum. EPSPs were evoked at an interstimulus interval of 30 s. In some slices a second stimulating electrode was placed into the alveus, on the subicular side of the slice, to evoke antidromic population spikes (see Fig. 8, B and C, for examples). Electrode placements and stimulus intensities were adjusted to maximize the antidromic spike without evoking an orthodromic response, which can result if sufficient stimulus current flows into the adjacent stratum oriens to excite afferent fibers. Antidromic population spikes were typically 2-3 mV in amplitude. In some slices, an antidromic population spike could not be evoked without contamination by orthodromic responses; these slices were excluded from analysis.

Intracellular recordings were used to observe postsynaptic potentials evoked by single stimuli and by tetanic stimulation. Individual CA1 pyramidal cells were impaled with glass microelectrodes filled with 2-4 M potassium acetate (60-110 MOmega ), connected to the headstage of an Axoclamp 2B amplifier operating in bridge mode. Cell input resistances were estimated by injection of small hyperpolarizing and depolarizing currents. Output from the amplifier was low-pass filtered at 1-2 kHz, digitized at 2-5 kHz, and stored on a personal computer.

Most extracellular and intracellular records were stored and analyzed with custom routines written in the Axobasic (Axon Instruments) programming environment. These routines allowed the extraction of waveform maxima, minima, slopes, and areas. Some recordings were made with Axotape (Axon Instruments) or WCP (Strathclyde Electrophysiology Software, John Dempster, University of Strathclyde) programs. Supplemental data analysis was performed with Origin (Microcal Software) and Excel (Microsoft).

Slices were tetanized in the presence of 50-100 µM D,L-2-amino-5-phosphonopentanoic acid (AP5) or AP5 + MK-801 (dizocilpine, 20 µM) to test for induction of NMDA receptor-independent LTP (Grover 1998; Grover and Teyler 1990, 1992, 1994, 1995) and to observe postsynaptic responses to tetanic stimulation. Tetanic stimulation consisted of either a single 25-Hz, 1-s stimulus train or a series of four 200-Hz, 0.5-s stimulus trains presented at an intertrain interval of 5 s. In some slices, the GABAA antagonist bicuculline (10 µM) was applied with AP5 or AP5 + MK-801. The stimulus intensity used for tetanization was set to evoke a population spike, measured with an extracellular electrode in s. pyramidale, of 1-2 mV. In slices perfused with bicuculline, stimulus intensities were selected before application of the drug. Test stimuli, delivered before and after tetanic stimulation, were evoked with the stimulus intensity set to one-half that used for tetanization.

Drug application

AP5, MK-801, and bicuculline methiodide (obtained from Tocris and RBI) were first dissolved in water to make concentrated stock solutions (5-100 mM). Stock solutions were diluted 250- to 1,000-fold into ACSF for delivery to slices. Drugs were applied to the tissue by switching between reservoirs containing ACSF and ACSF + drug.

Data analysis

For each slice, EPSP slopes were averaged over a 10-min baseline period preceding tetanization. Each EPSP was then normalized relative to the averaged baseline response. EPSP measurements are reported as percentage change from the baseline.

Postsynaptic responses to tetanic stimulation were quantified in two ways. First, the response area (in mV · ms) of the intracellularly recorded membrane potential during tetanization was determined. For each cell, the response area was calculated by summing the area under each point in the digitized waveform, measured relative to the pretetanus resting membrane potential. This was used as an index of the overall magnitude of postsynaptic depolarization. Second, the number of action potentials occurring during a stimulus train was manually counted. Action potential duration was determined by measurements taken at the base of the action potential (defined as 10 mV positive to threshold).

For statistical analysis, responses were averaged over all the cells or slices in a group. Values are reported as means ± 1 SE. Statistical comparisons were made by use of independent or dependent samples t-tests, as appropriate, with P < 0.05 (two-tailed) considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Block of GABAA receptors enhances NMDA receptor-independent LTP

In previous studies (Grover 1998; Grover and Teyler 1990, 1992, 1994, 1995), NMDA receptor-independent LTP was induced by a series of four 0.5-s, 200-Hz stimulus trains delivered in the presence of the competitive NMDA receptor antagonist AP5. As in prior reports, slices tetanized with this protocol displayed an LTP requiring ~10-20 min to reach maximum amplitude (Fig. 1A). Some degree of potentiation was seen in all 11 slices examined (range: 15-38% increase in EPSP). Slices tetanized at 200 Hz in AP5 showed a mean increase in EPSP slope of 26 ± 2% (n = 11, averaged over a period from 40-45 min post-tetanus). Addition of bicuculline to the ACSF enhanced the degree of NMDA receptor-independent LTP obtained (Fig. 1A). For slices tetanized in AP5 + bicuculline, the mean increase in EPSP was 55 ± 12% (n = 10, range from 10-140% increase, 40-45 min post-tetanus). The magnitude of LTP obtained in AP5 + bicuculline was significantly greater than in AP5 alone (P < 0.02, Fig. 1C).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1. Bicuculline enhanced N-methyl-D-aspartate (NMDA) receptor-independent long-term potentiation (LTP) induced by 200-Hz tetanization. Slices received a series of 4 200-Hz. 0.5 s stimulus trains. For each slice, excitatory postsynaptic potentials (EPSPs) were expressed as percentage change from baseline. Results were averaged across slices, aligned by the time at which tetanic stimulation was delivered (0 min). A: slices were perfused with either D,L-2-amino-5-phosphonopentanoic acid (AP5; black-square) or AP5 + bicuculline (Bic; ). Slices tetanized in AP5 showed a typical delayed onset LTP. Slices tetanized in AP5 + bicuculline also showed a slow onset LTP, but the magnitude of potentiation was greater. B: to ensure that NMDA receptors were adequately blocked during tetanization, the preceding experiment was repeated in the presence of 2 NMDA receptor antagonists, AP5 and MK-801. Again, blockade of GABAA receptors by bicuculline led to a greater degree of LTP. C: summary of result from A and B. Bars show the magnitude of LTP (measured at 40-45 min post-tetanus) obtained under each of the 4 conditions shown in A and B. Bicuculline significantly increased the magnitude of LTP. On average, LTP was greater in slices that were tetanized in the presence of both NMDA receptor antagonists (AP5 and MK-801) when compared with slices tetanized in AP5 only, but these comparisons were not significant. A-C: error bars show ± 1 SE.

Although our previous studies (Grover 1998; Grover and Teyler 1990, 1994) found no contribution of NMDA receptors to the LTP induced by the same procedures used in this experiment, others (Pananceau and Gustafsson 1997), with similar procedures, reported that NMDA receptor activation could contribute to LTP induction, despite the presence of AP5. To determine whether residual, AP5-resistant, NMDA receptor activation might have contributed to the facilitation of LTP produced by bicuculline, we repeated the experiment of Fig. 1A, but this time we tetanized slices at 200 Hz in the presence of both AP5 (a competitive NMDA receptor antagonist) and MK-801 (20 µM, a noncompetitive antagonist). If some fraction of NMDA receptors was unblocked by AP5 alone, then the combination of AP5 + MK-801 should reduce the magnitude of LTP obtained. Again we found that bicuculline (10 µM) increased the magnitude of LTP (Fig. 1B). In AP5 + MK-801, EPSPs were potentiated by 39 ± 9% (n = 13, range from 7-105% increase, measured 40-45 min post-tetanus), whereas the addition of bicuculline increased the magnitude of LTP to 75 ± 14% (n = 11, range from 22-165% increase, 40-45 min post-tetanus). This difference was significant (P < 0.04, Fig. 1C). As previously noted (Grover 1998), MK-801 did not affect the magnitude of LTP obtained by 200-Hz tetanization in the presence of AP5; LTP resulting from tetanization in AP5 + MK-801 was on average slightly greater than LTP resulting from tetanization in AP5 alone, but this small difference (26 ± 2% vs. 39 ± 9%) was not significant (P > 0.17, see Fig. 1C). LTP resulting from tetanization in bicuculline was also unaffected by MK-801 (AP5 + bicuculline vs. AP5 + MK-801 + bicuculline, P > 0.27, see Fig. 1C). These observations are inconsistent with any contribution of AP5-resistant NMDA receptors to the LTP studied here.

Under conditions where GABAA inhibition is intact, high-frequency tetanic stimulation is required for the induction of NMDA receptor-independent LTP. Lower-frequency tetanic stimulation (25 Hz) can induce NMDA receptor-dependent LTP but ordinarily does not result in NMDA receptor-independent LTP (Grover and Teyler 1990, 1994). Consistent with these prior reports, a single 25-Hz, 1-s stimulus train delivered in the presence of AP5 failed to induce LTP (Figs. 2 and 9). However, when bicuculline was perfused with AP5, 25-Hz stimulation was sufficient to induce an NMDA receptor-independent LTP in ~50% of the slices examined (5/11 slices, Fig. 2). In this subset of slices, the mean increase in EPSP after 25-Hz tetanization in AP5 + bicuculline (31 ± 3%) was significantly greater (P < 0.01) than the change in EPSP seen in the slices tetanized at 25 Hz in AP5 only (-1 ± 7%). In 6 of 11 slices tetanized at 25 Hz in AP5 + bicuculline, the change in EPSP (-8 ± 5%) was not significantly different (P > 0.40) from slices tetanized at 25 Hz in AP5 only.



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 2. Bicuculline permitted induction of NMDA receptor-independent LTP in some slices tetanized at 25 Hz. A: single 25-Hz, 1-s stimulus train delivered in the presence of AP5 (black-square) did not result in LTP. However, blocking GABAA receptors with bicuculline allowed this normally ineffective stimulation protocol to induce NMDA receptor-independent LTP in ~50% of the slices examined (open circle ). The remaining slices tetanized in AP5 and bicuculline showed no change in EPSP (triangle ). Slices were assigned to "LTP" and "no LTP" groups initially on the basis of visual examination (graphic analysis). This visual method was verified by a more objective method. For each slice, the mean change in EPSP (pretetanus vs. post-tetanus) was compared with the variability in response amplitude from trial to trial; in all slices where visual inspection indicated that the EPSP was potentiated, the posttetanus EPSP was increased by at least 2 SDs relative to the pretetanus EPSP. B: summary of results from A. Measurements were averaged over the 25- to 30-min post-tetanus period. A and B: error bars show ± 1 SE.

In some slices, perfusion of AP5 + bicuculline was continued throughout the post-tetanus recording period (Fig. 3A), whereas in other slices AP5 and bicuculline were washed out (Fig. 3B). LTP was observed under both of these conditions, indicating that the maintenance of NMDA receptor-independent LTP does not require a change in the strength of GABAA inhibition.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. In some slices bicuculline was present throughout the recording period, whereas in other slices bicuculline was applied only around the time when tetanic stimulation was delivered. A: example of a slice that was exposed to AP5 + bicuculline (Bic) throughout the duration of the experiment. Tetanization at 200 Hz induced a sustained NMDA receptor-independent LTP in this slice. Because GABAA receptors were blocked throughout the experiment, a reduction in GABAA inhibitory postsynaptic potentials is not required for the maintenance of NMDA receptor-independent LTP. Sample waveforms at top show averaged EPSPs recorded over a 10-min period immediately before tetanization (1) and from 35 to 40 min after tetanization (2). B: example of a slice where AP5 and bicuculline (Bic) were applied from 10 min before tetanization until 8 min after tetanization. In this slice, NMDA receptor-independent LTP was induced by a single 25-Hz stimulus train. Sample responses at top show averaged EPSPs recorded over 5-min periods (1) before application of AP5 + bicuculline, (2) after application of AP5 + bicuculline but before tetanization, (3) after tetanization but before washout of the drugs, and (4) at the end of the 40-min recording period. Bicuculline disinhibited this slice, as seen by the broadening of the EPSP waveform and the appearance of multiple population spikes (top left). Normal GABAA inhibition recovered after the washout of bicuculline, as shown by the presence of only a single population spike in the potentiated EPSP (top right). Arrows indicate the presence of population spikes in the EPSP waveforms.

Basis for enhancement of NMDA receptor-independent LTP by GABAA receptor blockade

Intracellular recordings were obtained from CA1 pyramidal cells to gain insight into the mechanisms by which GABAA receptors might regulate NMDA receptor-independent LTP. The general procedure in these experiments was to record the postsynaptic membrane potential during tetanization (while perfusing AP5) and then apply bicuculline and repeat the previous tetanic stimulation. Cells were exposed to series of four 200-Hz stimulus trains or to single 25-Hz stimulus trains.

One potential complication of these experiments was that the first tetanization (in AP5) might induce LTP, leading to an enhanced postsynaptic response to the second tetanic stimulation (in AP5 + bicuculline). Any enhancement caused by LTP might then be confused with changes caused by the blockade of GABAA receptors. Two approaches were taken to circumvent this problem.

The first approach took advantage of the fact that NMDA receptor-independent LTP is expressed with a delayed onset (Grover 1998; Grover and Teyler 1990, 1992, 1994, 1995). After an initial enhancement resembling post-tetanic potentiation, which typically lasts from 1 to 3 min, the response returns toward the baseline level (returning all the way to the baseline in ~1/2 of the slices examined previously). This is followed by the gradual emergence of a sustained potentiation. With the perfusion apparatus used here, solution exchanges could be completed within 3 min. It was therefore possible to tetanize a slice, wash bicuculline onto the slice, and tetanize the slice again at a time point after the decay of PTP and preceding the development of LTP. An example of this procedure is illustrated in Fig. 4. Single EPSPs were recorded immediately before the first series of tetanic stimuli (in AP5) and immediately before the second series of tetanic stimuli (in AP5 + bicuculline). As shown in Fig. 4A, the rising slopes of the EPSPs were identical, demonstrating an identical level of excitatory synaptic transmission for both series of tetanic stimuli. Figure 4A also shows that the fast inhibitory postsynaptic potential (IPSP) was adequately blocked by the GABAA antagonist at the time of the second set of tetanic stimuli. In addition, the cell input resistance was measured before tetanization in AP5 and again after tetanization in AP5 + bicuculline (Fig. 4B). There was no detectable change in input resistance.



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4. Intracellular recording from a CA1 pyramidal cell showing the effects of bicuculline on the postsynaptic response to tetanic stimulation. A: 3 superimposed EPSPs recorded in AP5 and 3 EPSPs recorded in AP5 + bicuculline (Bic). EPSPs were recorded immediately before the first tetanization (in AP5) and immediately before the second tetanization (in AP5 + bicuculline, ~3 min after the first series of 200-Hz stimulus trains). Responses of this cell to the 200-Hz stimulus trains are shown in C. Recordings shown in C, bottom, were obtained at a time point after the decay of the post-tetanic potentiation and preceding the development of NMDA receptor-independent LTP because the EPSP rising slopes shown in A are identical. B: responses to injection of small currents through the recording electrode (+0.1 to -0.3 nA). These recordings were taken before the first tetanization (in AP5) and shortly after the second tetanization (in AP5 + bicuculline). Input resistance was unchanged (47 MOmega in AP5 and 48 MOmega in AP5 + bicuculline). C: responses to 2 series of 200-Hz, 0.5-s stimulus trains (5-s intertrain interval). The 200-Hz stimulus trains shown here are identical to those used to induce LTP (Fig. 1). The first series was delivered in AP5 (top), and the second series was delivered in AP5 + bicuculline (bottom). Although bicuculline blocked both the hyperpolarizing (single arrow, Train 1) and depolarizing (double arrow, Train 1) (see also Grover et al. 1993) GABAA receptor-mediated responses, the net effect of bicuculline was to enhance postsynaptic depolarization during the tetanization. Enhanced postsynaptic depolarization produced by bicuculline caused an increase in the number of action potentials fired; this effect was observed during all 4 stimulus trains but was most pronounced during the first train. These effects of bicuculline on the postsynaptic response to tetanization cannot be attributed to a potentiation of the EPSP because the responses to tetanic stimulation were recorded at a time when no potentiation was evident (as shown in A) nor can these effects be attributed to any change in cell input resistance (B). All recordings (A-C) were obtained at the resting membrane potential (-63 mV) of this neuron.

A second approach was also used to ensure that the only difference between the first and second episodes of tetanic stimulation was the blockade of GABAA inhibition. In this second approach, responses to 25-Hz tetanization were examined. Because 25-Hz tetanization in AP5 alone fails to induce LTP (Figs. 2 and 8A), it was possible to record responses to 25-Hz, 1-s stimulus trains in AP5 and then add bicuculline to the ACSF and record a response to the same stimulus train with confidence that the level of excitatory synaptic transmission was unaltered.

Both approaches yielded similar results (Figs. 4C and 6). The most salient effect of bicuculline on the response to tetanization was an increase in the number of action potentials fired during the stimulus train. In the experiments with 200-Hz tetanic stimulation, this effect was especially pronounced on the first train in each series of four (see Train 1, Fig. 4C; Fig. 5). Although this difference was maintained to some degree during later trains in the series, the effect became less prominent. This is most likely a reflection of the use-dependent depression of inhibition that follows high-frequency stimulation (McCarren and Alger 1985). In other words, by the third and fourth trains of a series delivered in AP5 alone, GABAA inhibition is already depressed so that the addition of the GABAA antagonist has considerably less effect.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Summary of the effects of bicuculline on the postsynaptic response to 200-Hz tetanization (n = 6 cells). A: bicuculline increased total response area during all 4 200-Hz, 0.5-s stimulus trains (*: significant comparisons, AP5 + bicuculline vs. AP5 alone, at P < 0.05). This measure includes both action potentials and the underlying envelope of depolarization (the summed postsynaptic potentials occurring during tetanization). B: bicuculline increased the number of action potentials fired in response to the first 3 200-Hz, 0.5-s stimulus trains (*: significant comparisons, AP5 + bicuculline vs. AP5 alone, at P < 0.05). Error bars show ± 1 SE.

Bicuculline increased the underlying envelope of depolarization during 200-Hz tetanization (Figs. 4C and 5); this effect presumably was responsible for the increased action potential discharge. Two hundred-hertz tetanization can elicit a depolarizing GABAA response (Grover et al. 1993), which itself can evoke action potential discharge. Because the depolarizing GABAA response is blocked by bicuculline, bicuculline potentially might have decreased the number of action potentials fired during 200-Hz tetanization. This turned out not to be the case, however, because the loss of the hyperpolarizing GABAA response component (Fig. 4C, Train 1, single arrows) had a greater overall impact on the postsynaptic membrane potential during tetanization than did the loss of the depolarizing GABAA response (Fig. 4C, Train 1, double arrows).

Bicuculline similarly affected the postsynaptic responses to 25-Hz tetanization and 200-Hz tetanization. The number of action potentials fired during 25-Hz stimulation was increased by bicuculline (Fig. 6, A-C). With the lower frequency of tetanization, individual EPSPs could be easily discerned. Bicuculline facilitated action potential firing by increasing the peak amplitude of the EPSPs (Fig. 6D), thereby allowing a greater proportion of the responses to reach spike threshold. Bicuculline did not affect the rising slope of the EPSPs (Figs. 4A and 6D).



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 6. Intracellular recording showing the effects of bicuculline on the postsynaptic response to 25-Hz tetanization. A-D: synaptic responses recorded during 25-Hz tetanization. A: response recorded when the stimulus train was delivered in AP5. B: tetanic response recorded after the addition of bicuculline (Bic) to the artificial cerebrospinal fluid. The number of action potentials evoked by the tetanus increased from 3 in AP5 to 11 in AP5 + bicuculline, but there was little other change in the overall membrane potential profile during the stimulation. C: response recorded after the washout of bicuculline. A partial recovery of the original response was obtained. D: first 7 EPSPs from the trains in A and B. The first train, delivered in AP5 alone, did not induce LTP, as indicated by the similar rising slopes of the EPSPs. All recordings (A-D) were obtained at the resting membrane potential (-65 mV) of this neuron.

In addition to changing the number of action potentials evoked during tetanic stimulation, blocking GABAA receptors also changed the shape of individual, synaptically evoked action potentials (Fig. 7). In the presence of bicuculline, action potential duration, measured at the base of the action potential, was increased from 1.25 ± 0.07 ms to 1.54 ± 0.09 ms (P < 0.02). This effect was reversed by washing bicuculline off the slice (Fig. 7).



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7. Bicuculline increased the duration of synaptically evoked action potentials. The examples shown in this figure are from the same cell shown in Fig. 7 (responses are from the first stimulus in each of the 25-Hz stimulus trains shown in A-C). A and B: same examples, shown at 2 different timescales. Responses were aligned in time relative to the action potential. In this cell, the action potential increased in duration from 1.37 ms in AP5 to 1.82 ms in AP5 + bicuculline (duration was measured at 10 mV positive to threshold). After washout of bicuculline, the original action potential duration was partially recovered (duration = 1.56 ms).

Bicuculline increased both the number and duration of action potentials, and either of these changes could facilitate induction of NMDA receptor-independent LTP. We conducted a final experiment to test the importance of the increase in number of action potentials fired. Three groups of slices were examined in this experiment. In all three groups, tetanic stimulation was delivered in AP5, and GABAA inhibition was intact (bicuculline was not administered). One group was tetanized with a single 25-Hz, 1-s stimulus train delivered through a stimulating electrode placed in s. radiatum (orthodromic tetanization, identical to the tetanization protocol used in Fig. 2). A second group was simultaneously tetanized with 25-Hz, 1-s stimulation of the Schaffer collaterals and 25-Hz, 1-s stimulation of the alveus (orthodromic + antidromic tetanization) to mimic the increased postsynaptic action potential firing observed when GABAA inhibition was blocked. A third group received only the 25-Hz, 1-s antidromic (alvear) stimulation. The results of this final experiment are shown in Fig. 8. As in the previous experiment (Fig. 2), 25-Hz tetanization in the presence of AP5 failed to induce LTP (Fig. 8A, mean EPSP change measured 30 min post-tetanus was 2 ± 5%). Orthodromic tetanization paired with antidromic tetanization, however, was effective for inducing LTP (Fig. 8B, mean EPSP change = 17 ± 3%, P < 0.02 compared with results with orthodromic tetanization alone). Antidromic tetanization by itself (Fig. 8B) failed to induce LTP and instead resulted in a persistent decrease in EPSPs (mean change = -12 ± 7%) as previously reported (Christofi et al. 1993; Pockett et al. 1990). These results are consistent with the previous finding (Grover 1998) that postsynaptic action potential firing is required for induction of NMDA receptor-independent LTP. These results also indicate an important role for GABAA inhibition in regulating NMDA receptor-independent LTP by controlling the number of postsynaptic action potentials fired during tetanization but do not rule out a further contribution through control of action potential duration.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 8. Paired 25-Hz orthodromic + antidromic tetanization can induce NMDA receptor-independent LTP. A: afferent fibers (stratum radiatum) were tetanized for 1 s at 25 Hz (stimulation protocol identical to that used in Fig. 2). As was shown earlier (Fig. 2) (Grover and Teyler 1990, 1994), this stimulation protocol does not induce LTP. Right inset: averaged EPSPs recorded over the last 5 min before tetanization (1) and over the final 5 min of the post-tetanus period (2). B: afferent fibers (s. radiatum) and CA1 pyramidal cell axons (alveus) were simultaneously tetanized for 1 s at 25 Hz. NMDA receptor-independent LTP was induced by this orthodromic + antidromic tetanic stimulation protocol. Left inset: antidromic population spike evoked in 1 slice by single-pulse stimulation of the alveus (the recording electrode was initially placed in s. pyramidale to record this response and was then moved to s. radiatum). Right inset: EPSPs from the same slice, averaged over 5-min periods at the end of the pretetanus baseline (1) and at the end of the posttetanus recording period (2). C: 25-Hz, 1-s tetanization of CA1 pyramidal cell axons (alveus) only did not induce LTP and instead led to a small but persistent decrease in EPSPs. Left inset: antidromic population spike evoked in 1 slice by single pulse stimulation of the alveus (the recording electrode was initially placed in s. pyramidale to record this response and was then moved to s. radiatum). Right inset: EPSPs from the same slice, averaged over 5-min periods at the end of the pretetanus baseline (1) and at the end of the post-tetanus recording period (2). A-C: all data shown are from slices that were tetanized in the presence of AP5, but not bicuculline (GABAA inhibition was intact). Calibration bars indicate 1 mV, 5 ms.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented here demonstrate that blockade of GABAA receptor-mediated inhibition increases the magnitude of NMDA receptor-independent LTP obtained with a normally effective stimulation protocol (200-Hz tetanization). Furthermore, a normally ineffective stimulation protocol (a single 25-Hz tetanus) can become effective if GABAA-mediated inhibition is blocked. Observations of postsynaptic responses to tetanic stimulation under control conditions (AP5 alone) and with GABAA receptors blocked (AP5 + bicuculline) revealed an increase in the number of action potentials fired during tetanization and an increase in the duration of individual action potentials.

We conducted an experiment to test whether an increase in the number of action potentials fired during tetanic stimulation was sufficient to change the normally ineffective 25-Hz tetanization protocol to an effective protocol for inducing NMDA receptor-independent LTP. In this experiment, we paired simultaneous 25-Hz, 1-s orthodromic stimulation of the Schaffer collateral/commissural fibers with 25-Hz, 1-s antidromic stimulation of alvear fibers. The orthodromic + antidromic pairing protocol was effective for LTP induction, confirming the critical role of postsynaptic action potential firing in NMDA receptor-independent LTP (Grover 1998). Although the orthodromic + antidromic tetanization protocol mimicked the increased number of postsynaptic action potentials fired in bicuculline, this protocol did not mimic the increased action potential duration observed in bicuculline. For this reason, the importance of action potential duration for the induction of NMDA receptor-independent LTP will need to be assessed by further experimentation. Nonetheless, this experiment does support the conclusion GABAA receptors can regulate the induction of NMDA receptor-independent LTP by controlling action potential discharge in postsynaptic neurons.

The conclusions reached above are consistent with previous studies where Ca2+ influx into CA1 pyramidal neurons was directly examined. Action potentials propagate into the dendritic tree (Spruston et al. 1995) where they open VDCCs (Christie et al. 1995; Magee and Johnston 1995; Miyakawa et al. 1992; Spruston et al. 1995). The entry of Ca2+ into the dendrites of CA1 pyramidal cells during orthodromic stimulation is largely dependent on propagation of action potentials into dendritic processes (Christie et al. 1995; Miyakawa et al. 1992). The influx of Ca2+ through VDCCs depends not only on the number of action potentials fired but also on the shape of the action potential, with longer action potentials being more effective for gating VDCCs, especially high-threshold VDCCs (McCobb and Beam 1991; Scroggs and Fox 1992). Because bicuculline increased both the duration and the number of action potentials during tetanization, it is almost certain that the block of GABAA receptors led to a greater VDCC-mediated Ca2+ influx into the postsynaptic neurons. This postulated increase in Ca2+ influx provides a reasonable explanation for the facilitation of NMDA receptor-independent LTP produced by treating slices with bicuculline. This explanation is consistent with the observations of Tsubokawa and Ross (1996), who found that GABAA receptors limit action potential propagation and the associated increase in intracellular Ca2+ concentration in dendrites of CA1 pyramidal neurons.

The increased duration of synaptically evoked action potentials during block of fast, GABAA IPSPs may have additional consequences for VDCC-dependent forms of synaptic plasticity. As noted previously, increasing the duration of action potentials promotes the opening of high-threshold VDCCs. Because VDCCs differ in activation and inactivation kinetics and voltage dependency (Bertolino and Llinás 1992; Perez-Reyes and Schneider 1994; Tsien et al. 1991), an increase in the duration of action potentials might result in the opening of additional types of VDCCs. This could allow other types of VDCCs, in addition to the dihydropyridine sensitive (L-type) VDCCs previously implicated (Cavus and Teyler 1996; Grover and Teyler 1990), to participate in the induction of NMDA receptor-independent LTP. In agreement with this suggestion, it was shown (Ito et al. 1995) that T- and P-type VDCCs participate in the induction of LTP during theta burst stimulation, a type of patterned tetanic stimulation that is particularly effective in promoting disinhibition (Pacelli et al. 1989) and that might therefore increase the duration of postsynaptic action potentials similarly to pharmacological disinhibition.

The findings reported here may shed some light on the control over induction of NMDA receptor-independent LTP in situ. The stimulation protocols used in previous in vitro studies (Cavus and Teyler 1996; Grover 1998; Grover and Teyler 1990, 1992, 1994, 1995) to induce NMDA receptor-independent LTP relied on high-frequency (200 Hz) tetanic stimulation. However, as shown in this study, reduction of fast, GABAA receptor-mediated inhibition can allow lower stimulation frequencies to induce NMDA receptor-independent LTP. The hippocampus is innervated by noradrenergic, serotonergic, cholinergic, and GABAergic systems (Amaral and Kurz 1985; Eckenstein et al. 1988; Frotscher and Léránth 1985; Houser et al. 1985; Köhler et al. 1984; Lysakowski et al. 1989; Molliver 1987; Oleskevich et al. 1989; Steinbusch 1981; Wainer et al. 1985; Woolf et al. 1984), and GABAergic interneurons are targeted by at least some of these inputs (Freund and Antal 1988; Freund et al. 1990; Milner and Bacon 1989). Because evoked inhibition in the hippocampus is modulated by these transmitters (Marty and Llano 1995; Thompson 1994), the regulation of hippocampal interneurons by extrinsic neuromodulatory inputs might serve a gating function, either facilitating or suppressing the induction of NMDA receptor-independent LTP.

Although the results of this and previous (Grover 1998; Grover and Teyler 1990) investigations have highlighted the role of postsynaptic VDCCs in the induction of NMDA receptor-independent LTP, at least one additional process is also required, the activation of metabotropic glutamate receptors (mGluRs) (Grover 1998; Little et al. 1995). Although the specific role of mGluRs is not yet known, we have suggested that mGluR activation, and not postsynaptic VDCC activation, is responsible for the synapse specificity of NMDA receptor-independent LTP (Grover 1998; Grover and Teyler 1992). It is possible that blockade of GABAA receptors could, under some conditions, enhance mGluR activation and thereby facilitate NMDA receptor-independent LTP. For example, blocking GABAA receptors enhances the excitatory interactions between CA3 pyramidal neurons (Colom and Saggau 1994; Miles and Wong 1987); this could lead to greater recruitment of presynaptic neurons during tetanic stimulation, greater release of glutamate during tetanization, and enhanced activation of mGluRs. Although this scenario is possible, it most likely did not occur during our experiments because we removed the CA3 area from most slices exposed to the GABAA antagonist, thereby preventing this possibility. Furthermore, there were no differences in LTP between slices with and without an intact CA3 region, suggesting that the enhanced activation of mGluRs is not a major mechanism allowing facilitation of NMDA receptor-independent LTP by GABAA receptor antagonism.


    ACKNOWLEDGMENTS

We thank Drs. Idil Cavus and Pascal DiScenna for comments and suggestions.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-34650.


    FOOTNOTES

Address for reprint requests: L. M. Grover, Dept. of Physiology, Marshall University School of Medicine, 1542 Spring Valley Dr., Huntington, WV 25755-9340.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 June 1998; accepted in final form 1 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/99 $5.00 Copyright © 1999 The American Physiological Society