Abstract

Learning-related modifications in predisposition for long-term potentiation (LTP) and long-term depression (LTD) were studied in brain slices of the rat piriform cortex following olfactory learning. Rats were trained to discriminate between pairs of odors until they demonstrated rule learning. We have previously shown that such training is accompanied by enhanced neuronal excitability and increased synaptic transmission in the intrinsic synaptic pathway. Here we show that the susceptibility for further enhancing synaptic connectivity by inducing LTP in slices from trained rats is markedly reduced after training, compared with slices from pseudo-trained and naive rats. Accordingly, while 900 stimuli at 1 Hz did not induce LTD in slices from control rats, it induced significant LTD in slices from trained rats. Post-tetanic potentiation (PTP) was also reduced after training, indicating that synaptic release is enhanced after odor learning, as previously suggested. We suggest that learning-related cellular modifications and activity-dependent synaptic plasticity share a common mechanism in the primary olfactory cortex. Our data also support the prediction generated according to the sliding modification threshold theory that learning should be accompanied by reduced capability of inducing LTP and increased susceptibility for LTD induction.

Introduction

The idea that memory is manifested at the cellular level by enhancement of synaptic connections between simultaneously activated neurons was suggested half a century ago (Hebb, 1949), has been widely accepted since then, and has been the subject of several reviews (Hawkins et al., 1993; Bliss and Collingridge, 1993). During the last decade, behaviorally induced synaptic changes have been demonstrated, both in vivo (Ahissar et al., 1992; Wilson and McNaughton, 1994; Rioult-Pedotti et al., 1998) and in vitro (Mckernan and Shinnick-Gallagher, 1997; Power et al., 1997; Rioult-Pedotti et al., 1998; Saar et al., 1999). The in vitro studies have demonstrated that learning-related synaptic modifications can be preserved and detected in brain slices.

The long-term potentiation (LTP) and long-term depression (LTD) models have provided insights into cellular mechanisms of long-term activity-dependent changes in various brain areas (Grant et al., 1992; Bliss and Coolingridge 1993; Hawkins et al., 1993; Bear and Abraham, 1996; Lucher et al., 2000), and are the most analyzed models of activity-dependent synaptic enhance- ment in the mammalian brain.

According to the sliding modification threshold theory, the likelihood of inducing LTP and LTD in a synaptic pathway is considerably dependent on the previous activity of the synapses in this pathway (Bear 1996). Indeed, it was shown that LTP is less likely while LTD is more likely to occur in the visual cortex following exposure to light (Kirkwood et al., 1996) and that motor learning is accompanied by reduced LTP in the motor cortex (Rioult-Pedotti et al., 1998).

The rat olfactory modality offers significant advantages for the study of learning-related neuronal plasticity in the mammalian brain. Rats, for whom olfaction is the dominant sensory modality, can easily learn to associate odor with reward (Staubli et al., 1986; Saar et al., 1998; Sara et al., 1999). Activity- dependent plasticity in the piriform cortex has been reported in several studies. Synaptic activity evoked in the piriform cortex by stimulating the lateral olfactory tract is strongly enhanced by olfactory training (Roman et al., 1987, 1993; Litaudon et al., 1997). LTP can be readily induced in the piriform cortex in vitro (Jung et al., 1990; Kanter and Haberly, 1990, 1993; Jung and Larson 1994; Hasselmo and Barkai, 1995) and in vivo (Stripling et al., 1988, 1991). The piriform cortex has a simple and defined anatomical organization (Price, 1973). Pyramidal cell bodies are densely packed in a thin layer (layer II), with the inter-cortical association axons synapsing on the proximal zone of the apical dendrites (layer Ib). This laminar organization enables recording from a homogeneous population of neurons and stimulating specific synaptic pathways.

We have previously shown that odor learning results with the following cellular modifications in layer II pyramidal neurons: increased neuronal excitability, indicated by reduced after- hyperpolarization (Saar et al., 1998) and increased synaptic transmission, indicated by reduced paired-pulse facilitation (Saar et al., 1999). These learning-related modifications have been implicated with rule learning (e.g. enhanced learning capability) rather than in long-term memory for specific odors (Saar et al., 1998, 1999).

In the present study we show that odor learning is accompanied by modifications in the predisposition for inducing post-tetanic potentiation (PTP), LTP and LTD in this brain area.

Materials and Methods

Animal Training

Subjects and Apparatus

Young adult Sprague–Dawley male rats were used. Rats were 4 weeks old at the beginning of training, and 6–7 weeks old when sacrificed for brain slice experiments. Prior to training they were maintained on a 23.5 h water-deprivation schedule, with food available ad libitum. Olfactory discrimination training protocol was performed in a four-arm radial maze, with commercial odors that are regularly used in the cosmetics and food industry, as previously described (Staubli et al., 1987; Saar et al., 1998, 1999).

Training

Three groups of rats were used, a trained group and two control groups (pseudo-trained and naive). Olfactory training consisted of 20 trials per day for each rat with the following protocol: an electronic ‘start’ command opened randomly two valves, releasing pressured air-streams with positive-cue odor into one of the arms and negative-cue odor into another. Eight seconds later, the two corresponding guillotine doors were lifted to allow the rat to enter the selected arms. Upon reaching the far end of an arm (90 cm long), the rat's body interrupted an infrared beam and a drop of drinking water was released from a water hose into a small drinking well (for a trained rat – only if the arm contains the positive-cue odor; for a pseudo-trained rat – randomly). A trial ended when the rat interrupted a beam, or in 10 s if no beam was interrupted. A fan was operated for 15 s between trials, to remove odors.

The criterion for learning was performing at least 80% positive-cue choices in the last 10 trials of the day. The control rats were either exposed to the same protocol of training, but with random water rewarding (pseudo-trained) or were water-deprived, with no training (naive). Once all the rats in the trained group met the criterion for learning the first pair of odors, on the next day both trained and pseudo-trained groups resumed training with a new pair of unfamiliar odors. Thus, pseudo-trained rats were exposed to the same odors, for the same periods of time, as the trained rats. However, since they were rewarded randomly with no relation to any particular odor, they did not show a preference for any odor. Accordingly, the proportion of their entries into the arm containing the ‘correct’ odor was never above chance level (Saar et al., 1998).

As we have previously reported (Saar et al., 1998, 1999), our training study confirms the original report by Staubli et al. (1987) that once the rats reach good performance with the first pair of odors, their capability to distinguish between new odors is increased. Rats were trained with two pairs of odors, a procedure exposing them to a total of four odors, to ensure that rule learning indeed occurred.

Slice Preparation, Stimulation and Recording

Brain slices were taken from 16 naive, 10 pseudo-trained and 15 trained rats 1 day after training completion. Coronal brain slices, 400 mm thick, were cut as previously described (Saar et al., 1999) and kept in oxygenated (95% O2 + 5% CO2) Normal Slice Ringer's solution (NSR) containing NaCl 124 mM, KCl 5 mM, MgSO4 2 mM, NaH2PO4 1.25 mM, NaHCO3 26 mM, CaCl2 2 mM and glucose 10 mM.

Synaptic Activity

Stimulating electrodes were placed in layer Ib, to stimulate the intrinsic connections between pyramidal cells. To ensure that stimulation is restricted to this specific layer, fine-tipped tungsten electrodes were used to apply small currents with short duration. Extracellular recordings were performed with Ringer solution-filled glass electrodes. Stimulus intensity was adjusted to generate EPSPs with amplitudes that are 50% of the maximal responses before the conditioning stimuli.

LTP, LTD and PTP Induction and Measurements

LTP was induced by applying six cycles of theta bursts (5 Hz). Each cycle consisted of 10 such bursts, each entailing four stimuli at 50 or 100 Hz. Thus, a total of 240 stimuli were applied for LTP induction. LTD was attempted by stimulating repetitively at 1 Hz for 15 min (900 stimuli). The extent of potentiation and depression was defined as the ratio between the amplitude of the field post-synaptic potentials (fPSP) evoked every 15 s before and after the tetanic stimulation. PTP was determined by measuring the maximal fPSP value during the first 3 min after the tetanus stimuli. LTP and LTD values were calculated by averaging the 20 traces recorded 17.5–22.5 min after application of the tetanus stimuli. Different slices from the same rat were used to apply LTP or LTD at different frequencies. Slices from each rat were used only once for stimulating at a specific frequency. Thus, numbers noted below for each stimulating frequency, depict the number of slices, each taken from a different rat, that were used for each stimulating paradigm. The identity of rats (naive, trained or pseudo-trained) was not known to the person conducting the experiments and measurements.

Statistical Analysis

One-way ANOVA was used to evaluate significance of difference between three populations (e.g. trained, pseudo-trained and naive groups). When the ANOVA test indicated that significant differences existed, post hoc multiple Student's t-tests were performed for each pair of groups. Learning-related modifications were considered to occur when trained group differed from the pseudo-trained group and the naive groups and if the two control groups were not different.

Results

Predisposition for LTP is Reduced after Training

When stimulating repetitively at a burst frequency of 50 Hz, LTP was less readily induced in slices from trained rats, compared with slices from pseudo-trained and naive rats. Reduced susceptibility for LTP induction was apparent both in the proportion of the slices in which LTP was induced and in the averaged LTP amplitude in slices where LTP induction was successful. LTP was induced successfully in seven out of nine slices taken from naive rats and in six out of seven slices taken form pseudo-trained rats. LTP was observed in only 6 out of 11 slices from trained rats. Furthermore, the averaged LTP amplitude for experiments in which it occurred was 18 ± 2% (n = 6) in slices from trained rats, compared with 36% ± 6 (n = 6) in slices from pseudo-trained rats and 41 ± 5% (n = 7) in slices from naive rats (P<0.02). Since LTP values were similar in slices from pseudo-trained and naive rats, they were joined together to form the control group. Figure 1A shows the development of LTP in slices from trained rats versus slices from control rats (only slices in which LTP was induced are represented in this graph). Notably, PTP induced by the same stimuli was also significantly lower in slices from trained rats (Fig. 1B).

When inducing LTP with 100 Hz stimuli, there was no difference between slices from trained rats and controls in LTP, as well as in PTP (Fig. 2). LTP was induced in all tested slices.

Predisposition for LTD is Enhanced after Training

Stimulating repetitively 900 times at 1 Hz did not induce LTD in most slices from pseudo-trained and naive rats. Notable LTD (of –8%) was induced in one out of five slices from naive rats. Also, one out of five slices from pseudo-trained rats showed LTD (of –18%). In sharp contrast, LTD was readily induced in seven out of nine slices taken from trained rats by the same stimulation protocol. The averaged fPSP reduction following application of 900 stimuli at 1 Hz was –20 ± 5% (n = 9) in slices form trained rats, compared with 2 ± 7% (n = 5) in slices from pseudo-trained, and 2 ± 4% (n = 5) in slices from naive rats (P < 0.001). As in the experiments intended for LTP induction, fPSP values in slices from pseudo-trained and naive rats were pooled together to form the control group, since they were similar. Figure 3 shows the development of LTD in slices from trained rats versus slices from control rats.

These results suggested that the threshold frequency in which repetitive stimuli will induce LTP in slices from trained rats should be at ~10 Hz. This was tested in two slices taken from two trained rats, and the averaged modification was +3.5%.

Figure 4 summarizes the effect of stimulating at different frequencies on long-term activity-dependent synaptic plasticity in slices from control and trained rats. A clear increase in the threshold frequency value for inducing synaptic enhancement is evident.

Discussion

Our results show that odor learning induces modifications in activity-dependent synaptic plasticity in the primary olfactory cortex. Three forms of synaptic plasticity, PTP, LTP and LTD, are affected by prior learning. The effects on each of these phenomena suggest that connectivity in the intrinsic pathway is enhanced after learning.

The Relation between Activity-dependent Synaptic Plasticity and Learning

Based on reduced paired pulse facilitation (PPF) in neurons from trained rats, we have previously suggested that olfactory learning results with increased release in the intrinsic synaptic pathway (Saar et al., 1999). PTP, like PPF, is thought to result from augmented synaptic release (Griffith, 1990; Zucker, 1993). Thus, reduced PTP after training further indicates that olfactory learning is accompanied by enhanced synaptic release in this pathway.

LTP and LTD are also modified after training, and in opposite directions. According to the sliding modification threshold theory, these two forms of synaptic plasticity are thought to be dependent on the previous activation of the synaptic pathway; LTP should be more easily induced in a less ‘experienced’ synapse, while LTD induction should be more difficult (Bear, 1996). Indeed, our data show that following learning LTP in the intrinsic pathway is significantly reduced. Accordingly, LTD that could not be induced in slices from control rats by applying 900 stimuli at 1 Hz becomes apparent under the same conditions in slices from trained rats.

The finding that LTP and PTP induced by 100 Hz stimuli do not differ between groups suggests that the maximal possible value of repetitive stimuli-induced synaptic strengthening is not modified after learning. However, olfactory learning is accompanied by shifting the averaged synaptic strength closer to this maximal value, revealed by the difference observed when stimulating at 1 Hz, at 10 Hz and at 50 Hz.

These data support the notion that cellular events underlying learning share a common mechanism with LTP and LTD. Further, it suggests that the prediction made according to the sliding modification threshold theory, that following learning the threshold frequency for LTP induction should increase, is indeed valid.

Specificity of Modifications in Synaptic Plasticity to Rule Learning

Changes in activity-dependent synaptic plasticity were observed only in slices from trained rats. The lack of difference between slices from pseudo-trained and naive rats suggests that modifications in PTP, LTP and LTD observed after training are not the result of exposure to the odors or to the training apparatus. Rather, they represent specific learning-related modifications. We have previously suggested that reduced after-hyperpolarization and enhanced neuronal transmission observed in slices from trained rats represent enhanced learning capability (rule learning), rather than long-term memory for specific odors (Saar et al., 1998, 1999). That the learning-related modifications in activity-dependent, short- and long-term synaptic plasticity occur after training for only four odors, suggests that these phenomena are also related to rule learning. It is unlikely that such pronounced modifications could represent long-term memory for four specific odors when rats are capable of memorizing at least 100 odors (Saar et al., 1999).

The Relations between Learning-related and Development-related Modifications

It was previously shown that LTP can be induced in the visual cortex of dark-reared rats at lower stimulus frequency than in the cortex of normal rats (Kirkwood et al., 1996). Furthermore, the effect of normal development on activity-dependent synaptic plasticity is very similar to the effect of olfactory learning in terms of susceptibility for LTP and LTD induction. This is to the point where interfering in development creates a mirror image of learning, as reflected in the amplitude versus frequency curves [see Fig. 4 here and Fig. 7 in (Bear, 1996)]. This suggests that cellular modifications that occur during development and during learning may share common cellular pathways.

In summary, our findings suggest that rule learning-related cellular modifications and activity-dependent synaptic plasticity share a common mechanism in the primary olfactory cortex. They also support the prediction which stems from the sliding modification threshold theory (Bear, 1996), that learning should be accompanied by reduced capability of inducing LTP and increased susceptibility for LTD induction. Although learning- related enhanced synaptic release indicated by reduced PTP and PPF (Saar et al., 1999) may reduce the likelihood for LTP induction while creating favorable conditions for LTD induction (Stevens and Wang, 1994; Choi and Lovinger, 1997), further study is needed to determine the cellular mechanisms underlying learning-related modifications in LTP and LTD.

Notes

This research was supported by a grant from the United States–Israel Binational Science Foundation.

Address correspondence to Edi Barkai, Department of Morphology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer- Sheva 84105, Israel. Email: edi@bgumail.bgu.ac.il.

Figure 1.

Predisposition for LTP and PTP is reduced after learning. (A) LTP following 50 Hz stimuli. Top: representative recordings of fPSP prior to LTP induction and 20 min after tetanus stimuli application. Scale bars: 1 mV, 5 ms. Bottom: averaged fPSPs recorded in slices from trained and control rats. Tetanic stimuli were applied at time 0. Values represent means ± SEs. (B) PTP is reduced after learning. Bars represent the maximal averaged value ± SE for each group. PTP was measured from the same traces represented in (A).

Figure 1.

Predisposition for LTP and PTP is reduced after learning. (A) LTP following 50 Hz stimuli. Top: representative recordings of fPSP prior to LTP induction and 20 min after tetanus stimuli application. Scale bars: 1 mV, 5 ms. Bottom: averaged fPSPs recorded in slices from trained and control rats. Tetanic stimuli were applied at time 0. Values represent means ± SEs. (B) PTP is reduced after learning. Bars represent the maximal averaged value ± SE for each group. PTP was measured from the same traces represented in (A).

Figure 2.

LTP and PTP induced by 100Hz stimuli is similar in all groups. (A) LTP following 100 Hz stimuli. Averaged fPSPs recorded in slices from trained and control rats. Tetanus stimuli were applied at time 0. Values represent means ± SEs. Data recorded in five slices from trained rats, three slices from pseudo-trained rats and four slices from naive rats. (B) PTP in response to 100 Hz stimuli does not differ between groups. Bars represent the maximal averaged value ± SE for each group.

Figure 2.

LTP and PTP induced by 100Hz stimuli is similar in all groups. (A) LTP following 100 Hz stimuli. Averaged fPSPs recorded in slices from trained and control rats. Tetanus stimuli were applied at time 0. Values represent means ± SEs. Data recorded in five slices from trained rats, three slices from pseudo-trained rats and four slices from naive rats. (B) PTP in response to 100 Hz stimuli does not differ between groups. Bars represent the maximal averaged value ± SE for each group.

Figure 3.

Predisposition for LTD is increased after learning. LTD following 900 stimuli at 1 Hz. Top: representative recordings of fPSP prior to, and 20 min after, stimuli application in a slice from a trained rat. Scale bars: 1 mV, 5 ms. Bottom: averaged fPSPs recorded in slices from trained and control rats. Repetitive stimuli were applied at time 0 for 15 min. Values represent means ± SEs.

Figure 3.

Predisposition for LTD is increased after learning. LTD following 900 stimuli at 1 Hz. Top: representative recordings of fPSP prior to, and 20 min after, stimuli application in a slice from a trained rat. Scale bars: 1 mV, 5 ms. Bottom: averaged fPSPs recorded in slices from trained and control rats. Repetitive stimuli were applied at time 0 for 15 min. Values represent means ± SEs.

Figure 4.

Frequency response curve is modified after learning. Each point represents mean ± SE. Data for stimulation frequencies of 1, 50 and 100 Hz are the same as represented in Figures 1–3.

Frequency response curve is modified after learning. Each point represents mean ± SE. Data for stimulation frequencies of 1, 50 and 100 Hz are the same as represented in Figures 1–3.

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