Reversal of LTP in the Hippocampal Afferent Fiber System to the Prefrontal Cortex In Vivo With Low-Frequency Patterns of Stimulation That Do Not Produce LTD

F. Burette, T. M. Jay, and S. Laroche

Laboratoire de Neurobiologie de l'Apprentissage et de la Mémoire, Centre National de la Recherche Scientifique URA 1491, Université Paris Sud, 91405 Orsay, France

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Burette, F., T. M. Jay, and S. Laroche. Reversal of LTP in the hippocampal afferent fiber system to the prefrontal cortex in vivo with low-frequency patterns of stimulation that do not produce LTD. J. Neurophysiol. 78: 1155-1160, 1997. We examined the efficacy of several patterns of low-frequency stimulation for producing long-term depression (LTD) or depotentiation in the hippocampal fiber pathway to the prefrontal cortex in the anesthetized rat. Field potentials elicited by stimulation of the CA1/subicular region of the ventral hippocampus were recorded in the prelimbic area of the prefrontal cortex. We found no evidence that low-frequency trains (0.5-1 Hz), consisting of either single pulses, paired pulses (35-ms interpulse interval), or two-pulse bursts (5-ms interval), produce LTD in the prefrontal cortex. In contrast, all three stimulus protocols were found to induce a small-amplitude, persistent potentiation of the amplitude of the negative wave of the field response recorded in the prefrontal cortex. We also examined the ability of patterns of low-frequency stimulation to produce depotentiation of previously established long-term potentiation (LTP). Although low-frequency stimulation with single pulses or paired pulses was ineffective, we found that the two-pulse burst protocol selectively produced a rapid reversal of LTP in the hippocampo-prefrontal cortex pathway. Depotentiation is reversible and can be induced >2 h after the induction of LTP. Repeated trains failed to decrease the prefrontal cortex response below the original, unpotentiated level. These findings demonstrate the existence of a depotentiation mechanism that is capable of exerting powerful control over ongoing or recently induced synaptic plasticity in hippocampocortical connections in vivo.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Electrophysiological and anatomic studies have shown a direct monosynaptic projection that originates in the CA1/subicular region and projects to medial orbital and prelimbic areas of the prefrontal cortex (PFC) (Jay and Witter 1991). This pathway uses glutamate as a transmitter (Jay et al. 1992) and is of particular importance because it connects two structures critical for memory function. We have previously shown that high-frequency stimulation of the hippocampus induces long-term potentiation (LTP) of stimulated synapses in the PFC in vivo (Laroche et al. 1990), and that LTP is prevented by blockade of the N-methyl-D-aspartate (NMDA) receptor (Jay et al. 1995). Although LTP in neural networks is accepted widely as a potential candidate for the cellular storage of information, it is also predicted the networks have the capacity to undergo a form of long-term depression (LTD) or depotentiation of previously potentiated synapses, and the question arises as to whether this pathway supports LTD. There is in fact experimental evidence to suggest that both synaptic potentiation and synaptic depression occur in hippocampo-PFC synapses during certain behavioral training conditions (Doyère et al. 1993), yet the type of neuronal activation required to produce LTD or depotentiation in this pathway remains undetermined. In the hippocampus, different patterns of synaptic activation can induce LTD or depotentiation in certain pathways and experimental conditions. For example, one form of LTD that has been widely studied in area CA1 is induced by delivery of prolonged low-frequency stimulation, typically at 1-5 Hz, in hippocampal slices from young animals (Dudek and Bear 1992; Mulkey and Malenka 1992). The efficacy of this protocol in inducing LTD in the adult rat in vivo (Heynen et al. 1996) remains controversial, however, because several studies have reported mainly depotentiation (Barrionuevo et al. 1980; Stäubli and Lynch 1990) or no effect (Errington et al. 1995). More recently, a stimulation pattern consisting of pairs of pulses at low frequency was shown to reliably induce LTD in area CA1 of the adult rat in vivo (Doyère et al. 1996; Thiels et al. 1994), and although this protocol appears ineffective in the dentate gyrus (Doyère et al. 1996), LTD in this region has been obtained with the use of pairs of two pulses at very short intervals (Thiels et al. 1996). In the neocortex, LTD has also been mostly studied with the use of the in vitro slice preparation (Artola et al. 1990; Bindman et al. 1988), including slices of the PFC (Hirsch and Crépel 1990). Although recent experiments have shown that low-frequency trains similar to those used in the hippocampal slice can produce LTD in cortical slices (Dudek and Friedlander 1996; Kirkwood et al. 1993), it is as yet uncertain to what extent this can be applied to synaptic plasticity in the neocortex in vivo. In view of the potential importance of hippocampocortical communication in memory processes and of mechanisms of downregulation of synaptic efficacy for the efficient storage of information, we examined the ability of several patterns of low-frequency stimulation to induce LTD and depotentiation in the hippocampo-PFC pathway in vivo.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Adult male Sprague-Dawley rats (n = 36), weighing 300-400 g, were anesthetized with pentobarbitone sodium (65 mg/kg ip, supplemented if necessary during surgery) and placed in a stereotaxic frame. Body temperature was maintained at 37°C. The procedures for implantation and recording extracellular field potentials in the prelimbic area of the PFC are described elsewhere (Laroche et al. 1990). Briefly, recording electrodes (62-µm nichrome wire) were positioned in the prelimbic cortex (coordinates: 3.2 mm anterior to bregma, 0.8 mm lateral to the midline) and a concentric bipolar stainless steel stimulating electrode was lowered ipsilaterally into the CA1/subicular region of the ventral hippocampus (coordinates: 6.4 mm posterior to bregma, 5.6 mm lateral to the midline). Stimulations of the CA1/subicular region evoked a characteristic monosynaptic negativegoing field potential with a peak latency of 18-21 ms. The depths of the recording and stimulating electrodes (approx 3.2 and approx 5.5 mm below the cortical surface, respectively) were adjusted to maximize the amplitude of the negativegoing field excitatory postsynaptic potential. Test pulses (120-200 µs) were delivered every 30 s via an isolated constant current unit at an intensity that evoked a response of 70% of its maximum (range: 150-500 µA). At this intensity, the field potential is most likely to reflect summated excitatory postsynaptic potentials(Laroche et al. 1990). High-frequency stimulation to induce LTP consisted of two series of 10 trains (250 Hz, 200 ms) at 0.1 Hz, 6 min apart, delivered at test intensity. Protocols for inducing LTD consisted of 1) 900 pulses at 1 Hz, 2) 450 pairs of pulses delivered at 0.5 Hz, with an interpulse interval of 35 ms (paired pulse), and 3) 900 bursts of 2 pulses, at 1 Hz, with 5 ms between pulses in a burst [2-pulse burst (TPB)]. The intensity was the same as that of the test pulse. Field potentials were amplified, filtered (band pass 0.1 Hz to 3 kHz), digitized at 20 kHz, and stored on disc as averages of four consecutive responses. The amplitude of the negativegoing component of the field potential was measured by dropping a line from a tangent drawn between the negative peak onset and offset and was analyzed off-line with the use of theA/Dvance software (Mckellar Design). Results were expressed for each animal as a percentage change of the baseline and analyzed by analysis of variance. Correct placement of the electrodes was verified post mortem on brain sections stained with cresyl violet.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Failure to induce LTD or depotentiation with single- or paired-pulse low-frequency trains

The effect of low-frequency stimulation on synaptic transmission in naive (unpotentiated) hippocampal PFC synapses was investigated in nine rats. After a 30-min baseline, stimulation at 1 Hz was delivered for 15 min. The amplitude of the response decreased during the 1-Hz train (-22.6 ± 4.3%, mean ± SE), recovering to baseline within 5 min after resumption of test frequency stimulation (Fig. 1A). There was no evidence of LTD during the post-1-Hz stimulation period. In contrast, a small but significant increase in the response occurred within 10 min after 1-Hz stimulation (Fig. 1, A and B). Values 30-60 min after 1-Hz stimulation were significantly above baseline [11.9 ± 3.8%; F(1,8) = 8.63;P < 0.05]. Thus low-frequency (1-Hz) stimulation induces a form of slow-onset, persistent potentiation in the hippocampo-PFC pathway. High-frequency stimulation delivered 60 min after 1-Hz stimulation resulted in a further increase in the response [30.7 ± 7.5%, compared with pretetanus values; F(1,8) = 16.8; P < 0.005]. No additional LTP was observed after the second tetanus, delivered 30 min later.


View larger version (26K):
[in this window]
[in a new window]
 
FIG. 1. Low-frequency single- or paired-pulse stimulation does not produce long-term depression (LTD) or depotentiation in hippocampoprefrontal cortex pathway of anesthetized rat. A: effect of 1-Hz stimulation (900 pulses, thick horizontal bar) on amplitude of postsynaptic potential evoked in prefrontal cortex (PFC) by stimulation of CA1/subiculum (n = 9). Each point in this and subsequent graphs represents group mean ± SE of averaged response to 4 test stimuli given at 30-s intervals. Values are expressed as % change relative to baseline. Stimulation at 1 Hz induced slow-onset potentiation, rather than LTD, and high-frequency stimulation (down-arrow ) delivered 60 min later induced long-term potentiation (LTP). Ba-Bc: examples of averaged responses recorded in PFC at times indicated by letters in A (calibration: 0.1 mV, 20 ms). C: low-frequency (0.5-Hz) paired-pulse stimulation (450 pairs, 35-ms interpulse interval, hatched horizontal bar) also induced potentiation of response (n = 9). Sample responses collected during paired-pulse train are shown in Bd. D: paired-pulse stimulation (hatched horizontal bar) and 1-Hz single-pulse stimulation (thick horizontal bar) given after induction of LTP (n = 8) produced transient depression of PFC response, but depotentiation was not observed.

The effect of paired-pulse stimulation (450 pairs at 0.5 Hz, 35-ms interpulse interval) was investigated in another group of nine rats. During the pairing there was a significant decrease in the response evoked by the second pulse of the pair (-26.4 ± 6.5%; Fig. 1Bd). This procedure, however, failed to induce LTD (Fig. 1C). There was a decrease in the amplitude of the response during the paired-pulse train (-13.0 ± 2.2%), but again, as in the preceding experiment, the response recovered rapidly and potentiation of the response occurred [10.4 ± 3.6%; F(1,8) = 8.4; P < 0,05; Fig. 1C]. Thirty to 60 min after the paired-pulse train, the amplitude of the response was still significantly above baseline [F(1,8) = 10.8; P < 0.05], and high-frequency stimulation resulted in a further potentiation of the response[30.9 ± 6.0%, compared with values after the paired-pulse train; F(1,8) = 26.3; P < 0.001; Fig. 1C]. The second tetanus, delivered 30 min later, produced no further LTP.

Eight of the animals presented in Fig. 1C were used to examine the effect of single-pulse and paired-pulse low-frequency stimulation on a potentiated response. As shown in Fig. 1D, both paired-pulse low-frequency stimulation, delivered 60 min after the LTP-inducing stimulus, and single-pulse stimulation, delivered 60 min later, produced substantial depression of the potentiated response (-29.8 ± 3.8% and -32.1 ± 3.8%, respectively), but in each case there was rapid recovery and the values 10-30 min after the end of the low-frequency trains were not significantly different from the potentiated level observed 30-60 min after the tetanus. During paired-pulse stimulation, a decrease in the response to the second pulse of the pair was observed in all animals (-46.8 ± 5.5%).

Induction of depotentiation, but not LTD, with TPB low-frequency stimulation

Nine rats were used in this experiment. After a 30-min baseline recording period, the TPB low-frequency train was delivered (900 bursts at 1 Hz, 5-ms interpulse interval), followed by a 60-min recording period at test frequency and the induction of LTP. Each pair in the train evoked a single response in the prelimbic area of the PFC (Fig. 2Ba), which increased transiently during the early phase of the TPB train to return progressively toward baseline during the later phase (Fig. 2A). Again, a slow-onset but persistent potentiation of the response followed [10.8 ± 3.5%; F(1,8) = 9.4;P < 0.05; Fig. 2A], and two successive episodes of high-frequency stimulation resulted in a further potentiation of the response [50.3 ± 5.5%, compared with the values obtained after the TPB train; F(1,8) = 46.0; P < 0.0001].


View larger version (26K):
[in this window]
[in a new window]
 
FIG. 2. Two-pulse burst (TPB) low-frequency stimulation induces depotentiation, but not LTD, in hippocampo-PFC pathway of anesthetized rat. A: effect of TPB stimulation (900 bursts at 1 Hz, 5 ms between pulses in burst, open horizontal bar) on amplitude of postsynaptic potential evoked in PFC by stimulation of hippocampus (n = 9). TPB stimulation induced slow-onset potentiation, rather than LTD, and high-frequency stimulation (down-arrow ) delivered 60 min later induced LTP. Ba: example of response observed during TPB (calibration as in Fig. 1B). C: LTP was 1st induced with 2 episodes of tetanic stimulation (down-arrow ). Sixty min later, TPB stimulation (open horizontal bar) induced depotentiation of PFC response (n = 9). This was followed by slow-onset potentiation (see also Fig. 2A), and tetanus given 1 h later reinduced LTP. Sample responses collected at times indicated by letters are shown in Bb-Be. D: 3 successive TPB trains at 60-min intervals (open horizontal bars), given 136 min after LTP induction (n = 5), induced depotentiation, but failed to decrease PFC response below original, unpotentiated level.

A further group of nine rats was used in which maximal LTP of the hippocampo-PFC pathway was induced by delivering two episodes of tetanic stimulation, after which the TPB train was applied and LTP was reinduced (Fig. 2, B and C). Tetanic stimulation induced robust and stable LTP of the prelimbic cortex response [52.4 ± 6.1%; F(1,8) = 72.6; P < 0.0001]. Sixty minutes later, TPB stimulation then resulted in an immediate depression of the response. The amplitude of the response during the first 10 min after the burst train was significantly below the potentiated level [11.6 ± 5.8%; F(1,8) = 30.2; P < 0.01], but not significantly different from the pretetanus baseline [F(1,8) = 3.91, not significant]. This decrease in the response represented suppression of 79.2 ± 11.8% of LTP. The amplitude of the depotentiated response then started to increase again over a period of 20 min, to reach a stable level that was significantly above pretetanus baseline [F(1,8) = 15.48; P < 0.01; Fig. 2C]. This is a slow-onset potentiation similar to that shown to follow TPB stimulation in unpotentiated synapses (described in Fig. 2A), and there was no significant difference between the two groups [F(1,16) = 3.47, not significant]. At this point, however, the response was still significantly depotentiated [F(1,8) = 38.1; P < 0.001, compared with values after the tetanus]. Between-group comparisons also showed that the amplitude of the response after the depotentiating stimulus was significantly lower than that observed at corresponding times after the induction of LTP in the group that did not receive the TPB train [F(2,16) = 7.12; P < 0.05; group-by-period interaction: F(4,64) = 7.3;P < 0.0001], thus suggesting that the decrease in the response is not due to the decay of LTP over time. Attempts were then made to reinduce LTP after depotentiation. As shown in Fig. 2C, tetanic stimulation delivered 60 min after the end of the TPB train invariably reestablished LTP to a level (60.3 ± 7.8%) that was comparable with that obtained before the burst stimulus (52.4 ± 6.1%). This corresponds to an increase in the amplitude of the response of 35.6 ± 4.5%, compared with the values immediately preceding the tetanus [F(1,8) = 62.56; P < 0.0001]. The depression, therefore, is unlikely to be the result of deterioration of the synapses.

We next examined whether LTD could be obtained if TPB trains are repeated after the induction of LTP and depotentiation. Five of the rats presented in Fig. 2A were given three successive TPB trains, at 30-min intervals, starting 136 min after the induction of LTP. As illustrated in Fig. 2D, the first train was sufficient to reverse LTP almost completely when delivered >2 h after induction. After the second and third TPB stimulation, however, the amplitude of the response did not decrease below the pretetanus level, and the values were not significantly different from the initial baseline [F(1,4) = 4.41 and F(1,4) = 2.46; P > 0.05]. Thus depotentiation is saturable and the TPB stimulation pattern selectively induces depotentiation, and not LTD, in the PFC.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

These experiments demonstrate, first, that low-frequency stimulus protocols that have been reported to elicit LTD or depotentiation in specific hippocampal and neocortical pathways under certain circumstances are not effective in inducing LTD in the hippocampal fiber system to the PFC in vivo. Prolonged low-frequency stimulation (1-5 Hz) has been shown to induce LTD in CA1 (Dudek and Bear 1992; Mulkey and Malenka 1992) and in the visual cortex (Kirkwood et al. 1993) in in vitro slice preparations. In the hippocampus, the efficacy of this protocol in inducing LTD in the adult rat in vitro or in vivo has been met with mixed success. For example, Heynen et al. (1996) have shown induction of LTD with these parameters, whereas others reported either depotentiation (Fujii et al. 1991; O'Dell and Kandel 1994; Stäubli and Lynch 1990) or no effect (Errington et al. 1995). In the PFC in vivo, our results show that a similar prolonged low-frequency (1-Hz) stimulation induces potentiation rather than depression. Another stimulation pattern, low-frequency pairs of pulses, which has been previously shown to produce LTD in area CA1 in the adult anesthetized (Thiels et al. 1994) and freely moving rat (Doyère et al. 1996), also failed to induce LTD in the PFC. In contrast, we repeatedly found that these low-frequency stimulation patterns induced a small amount of potentiation in this pathway, and a similar effect was observed with TPB stimulation. Our results differ from those obtained in slices of the visual cortex where low-frequency stimulation at 1 Hz was found to induce depression, whereas a TPB protocol (10-ms interpulse interval) had no effect (Kirkwood and Bear 1994). Whether this difference reflects regional specificity or a difference between in vitro and in vivo preparations has yet to be determined. The similarity in the amplitude and time course of potentiation observed with all three low-frequency stimulus protocols tested here (single pulse, paired pulse, and TPB) strongly suggests that repeated low-frequency stimulation is responsible for the effect in the PFC. This form of low-amplitude and persistent potentiation was observed both on unpotentiated and depotentiated synapses, but not on synapses that have been maximally potentiated, and it did not increase the amount of LTP that can be induced by strong tetanic stimulation. These results suggest that potentiation induced by low-frequency stimulation occludes LTP at least partially, and thus may share some common mechanisms with LTP. One possibility is a reduction in GABAergic inhibition during low-frequency stimulation (Davies and Collingridge 1993; McCarren and Alger 1985; Thompson and Gähwiler 1989), which may provide the conditions for increased NMDA receptor activation during electrical stimulation of this pathway.

The other finding in the present experiments is that LTP in the hippocampo-PFC pathway can be depotentiated in vivo by application of series of TPBs repeated at low frequency. This form of depotentiation is long lasting, reversible, and saturable. Moreover, depotentiation does not require that the stimulus be given within a short temporal window, because it was observed when delivered >2 h after the induction of LTP. The repotentiation experiment provides clear support for the conclusion that the rapid reversal of LTP in the PFC reflects true depotentiation and not the induction of LTD, superimposed on LTP, because repotentiation would not have been observed. Further evidence in support of this is that the same pattern of stimulation did not induce LTD in unpotentiated synapses, thus showing that the effect is restricted to potentiated synapses. Our study also shows that, after the induction of LTP, repeated trains fail to decrease the PFC response below the original, unpotentiated level, suggesting that this effect in the PFC is not due to some form of priming lowering the threshold for LTD after the induction of LTP (Wagner and Alger 1995; Wexler and Stanton 1993). Thus TPB stimulation selectively induces depotentiation, but not LTD, in the hippocampo-PFC pathway in vivo. After TPB stimulation, however, the response slightly increased, before reaching a stable depotentiated level. Although this may reflect incomplete depotentiation, we believe TPB stimulation induced slow-onset potentiation in depotentiated synapses, as previously observed in unpotentiated synapses. The same stimulus pattern, therefore, would produce opposite and, in the case of LTP reversal, summated effects. This is similar to the effect found in guinea pig hippocampal slices, where 5-Hz stimulation can induce a small amount of potentiation at naive synapses and reverse LTP (O'Dell and Kandel 1994), and to that of theta-burst stimulation in area CA1 of the rat hippocampal slice, although in this case different intensities were required for the selective induction of LTP or depotentiation (Barr et al. 1995).

In the present experiments, depotentiation was selectively observed with the TPB stimulus pattern, and not with similar low-frequency trains using single or paired pulses. These results suggest that low-frequency activation is in itself not sufficient for producing the effect, but that repeated stimulation with TPBs at a short interpulse interval is required. Thiels et al. (1996) reported that a low-frequency stimulus pattern consisting of pairs (25-ms intervals) of TPBs at short intervals (2.5-5 ms) induces LTD in the perforant path-dentate gyrus synapses in vivo, a system in which neither single-pulse nor paired-pulse low-frequency stimulation was found to induce LTD or depotentiation (Errington et al. 1995). In this pathway, the efficacy of the former protocol was attributed in part to the ability of the two pulses at very short intervals to increase the NMDA-receptor-mediated component of the excitatory postsynaptic potential (Blanpied and Berger 1992; Thiels et al. 1996). Although the profile observed in the hippocampo-PFC pathway is different from that in the dentate gyrus, because we observed depotentiation but not LTD, we suggest that the selective efficacy of this protocol in inducing depotentiation in the PFC is due to the requirement for enhanced NMDA receptor activation provided by the TPB paradigm. The relative contribution of NMDA receptor activation and of the level of inhibition in mediating depotentiation in the PFC remains to be investigated. It is interesting to note, however, that depotentiation in the PFC does not require patterns of stimulation associated with substantial inhibition, such as with pairs of either single or burst pulses at 25- to 35-ms intervals. Thus depotentiation in the PFC may primarily rely on a moderate NMDA receptor activation leading to a rise in intracellular Ca2+ that is below the threshold for inducing LTP (Artola and Singer 1993; Bear et al. 1987; Lisman 1989).

In summary, our findings establish that LTP at synapses of the hippocampal-PFC fiber pathway can be depotentiated homosynaptically in vivo by patterns of activation that do not induce LTD. The existence of a depotentiation mechanism capable of exerting powerful control over ongoing or recently induced synaptic plasticity in the pathway that connects two structures crucially involved in several forms of memory has important implications both for studying the mechanisms of depotentiation in a cortical area in vivo and for investigating the functional role of synaptic plasticity and of LTP reversal at hippocampocortical connections in cognitive functions.

    FOOTNOTES

  Address for reprint requests: F. Burette, Laboratoire de Neurobiologie de l'Apprentissage et de la Mémoire, CNRS URA 1491, Université Paris Sud, Bât. 446, 91405 Orsay Cedex, France.

  Received 11 March 1997; accepted in final form 29 April 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society