Spike timing in CA3 pyramidal cells during behavior: implications for synaptic transmission.

Spike timing is thought to be an important mechanism for transmitting information in the CNS. Recent studies have emphasized millisecond precision in spike timing to allow temporal summation of rapid synaptic signals. However, spike timing over slower time scales could also be important, through mechanisms including activity-dependent synaptic plasticity or temporal summation of slow postsynaptic potentials (PSPs) such as those mediated by kainate receptors. To determine the extent to which these slower mechanisms contribute to information processing, it is first necessary to understand the properties of behaviorally relevant spike timing over this slow time scale. In this study, we examine the activity of CA3 pyramidal cells during the performance of a complex behavioral task in rats. Sustained firing rates vary over a wide range, and the firing rate of a cell is poorly correlated with the behavioral cues to which the cell responds. Nonrandom interactions between successive spikes can last for several seconds, but the nonrandom distribution of interspike intervals (ISIs) can account for the majority of nonrandom multi-spike patterns. During a stimulus, cellular responses are temporally complex, causing a shift in spike timing that favors intermediate ISIs over short and long ISIs. Response discrimination between related stimuli occurs through changes in both response time-course and response intensity. Precise synchrony between cells is limited, but loosely correlated firing between cells is common. This study indicates that spike timing is regulated over long time scales and suggests that slow synaptic mechanisms could play a substantial role in information processing in the CNS.

Arg3.1/Arc mRNA induction by Ca2+ and cAMP requires protein kinase A and mitogen-activated protein kinase/extracellular regulated kinase activation.

Long-term potentiation (LTP) is a cellular model for persistent synaptic plasticity in the mammalian brain. Like several forms of memory, long-lasting LTP requires cAMP-mediated activation of protein kinase A (PKA) and is dependent on gene transcription. Consequently, activity-dependent genes such as c-fos that contain cAMP response elements (CREs) in their 5' regulatory region have been studied intensely. More recently, arg3.1/arc became of interest, because after synaptic stimulation, arg3.1/arc mRNA is rapidly induced and distributed to dendritic processes and may be locally translated there to facilitate synapse-specific modifications. However, to date nothing is known about the signaling mechanisms involved in the induction of this gene. Here we report that arg3.1/arc is robustly induced with LTP stimulation even at intensities that are not sufficient to activate c-fos expression. Unlike c-fos, the 5' regulatory region of arg3.1/arc does not contain a CRE consensus sequence and arg3.1/arc is unresponsive to cAMP in NIH3T3 and Neuro2a cells. However, in PC12 cells and primary cultures of hippocampal neurons, arg3.1/arc can be induced by cAMP and calcium. This induction requires the activity of PKA and mitogen-activated protein kinase, suggesting a neuron-specific pathway for the activation of arg3.1/arc expression.

Recognition memory correlates of hippocampal theta cells.

Investigations of hippocampal theta cell activity have typically involved behavioral tasks with modest cognitive demands. Recordings in rats locomoting through space or engaged in simple stimulus discrimination or conditioning have revealed some place specificity and S(+)/S(-) selectivity in addition to the hippocampal EEG theta-related behavioral/motor correlates. However, little data exist regarding theta cell activity during performance of more cognitively demanding, hippocampal-dependent recognition memory tasks. Here, we examined the cognitive firing correlates of theta cells in rats that were performing an olfactory recognition memory task with distinct sample and test phases. Discriminant analysis revealed odor and match/nonmatch memory correlates in theta cell activity comparable in relative magnitude to that of the principal cells. Odor-specific theta cell responses in the sample phase were restricted primarily to CA1 and linked to task performance. In the test recognition phase, match/nonmatch theta cells were found primarily in the CA3 and CA1 fields, most of which exhibited greater activity on correct nonmatch trials in which recognition occurred than on error match trials in which recognition failed. Odor selectivity of the match/nonmatch signaling was greatest in the dentate gyrus (DG) and CA3 and least in CA1. This inverted pattern of stimulus specificity in the sample versus test phase was similar to that observed in principal cells but with a greater contrast between the CA1 and DG/CA3 fields. Together, these findings suggest that theta cells actively participate in hippocampal recognition memory processing and play a specific role in shaping the cognitive firing properties of the hippocampal principal cells.

Dynamic filtering of recognition memory codes in the hippocampus.

Principal cells of the dentate gyrus (DG), CA3, and CA1 subfields of the hippocampus were recorded in rat during performance of an odor-guided delayed nonmatch-to-sample task with distinct sample and test phases. The hippocampus was found to possess multiple encoding modes. In the sample phase, odor-selective activity was restricted primarily to CA1 and, to a lesser extent, CA3. Odor representations in half of these cells were predictive of subsequent performance (i. e., correct vs error) in the test phase. Cells in each hippocampal subfield maintained elevated or suppressed activity in the delay interval relative to pre-odor baseline, but were indiscriminate with regard to sample odor identity. In the test phase, the regional distribution of odor-selective activity was inverse to that for the sample: maximal in DG and minimal in CA1. The inverted distribution of odor selectivity was also observed for cells that discriminated match/nonmatch trial types. Most match/nonmatch cells exhibited greater activity on correct nonmatch than error match trials, indicating the presence of a hippocampal recognition memory signal on trials where recognition occurred and its absence on trials where recognition failed. These findings reveal the hippocampus as a highly dynamic encoding device, restricting perceptual stimulus information to different subfields (or none, in the delay phase) depending on memory task contingencies. Moreover, the reduction in cue-specificity of match/nonmatch comparison signals as they pass through the hippocampal trisynaptic circuit may contribute to a generalized recognition signal for use in guiding behavior.

The polo-like protein kinases Fnk and Snk associate with a Ca(2+)- and integrin-binding protein and are regulated dynamically with synaptic plasticity.

In order to stabilize changes in synaptic strength, neurons activate a program of gene expression that results in alterations of their molecular composition and structure. Here we demonstrate that Fnk and Snk, two members of the polo family of cell cycle associated kinases, are co-opted by the brain to serve in this program. Stimuli that produce synaptic plasticity, including those that evoke long-term potentiation (LTP), dramatically increase levels of both kinase mRNAs. Induced Fnk and Snk proteins are targeted to the dendrites of activated neurons, suggesting that they mediate phosphorylation of proteins in this compartment. Moreover, a conserved C-terminal domain in these kinases is shown to interact specifically with Cib, a Ca(2+)- and integrin-binding protein. Together, these studies suggest a novel signal transduction mechanism in the stabilization of long-term synaptic plasticity.

Time-dependent reversal of long-term potentiation in area CA1 of the freely moving rat induced by theta pulse stimulation.

Previous studies in slices have shown that low-frequency stimulation at 5 Hz, i.e., theta pulse stimulation (TPS), completely reverses long-term potentiation (LTP) in area CA1 when delivered within 1-2 min after induction but produces progressively less depotentiation at longer delays, until it has no longer any impact at 30 min after induction. The present study examined whether LTP in the freely moving rat exhibits a similar time-dependent susceptibility to reversal. Adult male Long-Evans rats with bilateral stimulating electrodes activating collateral/commissural projections to area CA1 were used. A 1 min episode of TPS, ineffective when applied to naive pathways, was found to permanently erase LTP when delivered to the test pathway either 30 sec or 15 min after induction. Administered at a delay of 30 min, however, the same treatment no longer had any impact on established LTP. Additional experiments examined the ability of shorter TPS episodes to erase LTP and found that a 30 sec treatment was effective at 30 sec but not 15 min after induction. When the duration of TPS was further reduced to 15 sec, a reversal was no longer obtained at any delay. These results provide the first demonstration that the limited vulnerability of LTP to reversal by TPS, originally observed in vitro, also holds true for LTP in the awake animal and occurs along the same time frame, supporting the notion that LTP stabilization mechanisms take less than 30 min to be complete.

Pim kinase expression is induced by LTP stimulation and required for the consolidation of enduring LTP.

In animals and several cellular models of synaptic plasticity, long-lasting changes in synaptic strength are dependent on gene transcription and translation. Here we demonstrate that Pim-1, a serine/threonine kinase closely related to Pim-2 and Pim-3, is induced in hippocampus in response to stimuli that evoke long-term potentiation (LTP). Mice deficient for Pim-1 show normal synaptic transmission and short-term plasticity. However, they fail to consolidate enduring LTP even though Pim-2 and Pim-3 are constitutively expressed in the hippocampus and Pim-3 expression is similarly induced by synaptic activity. Thus, expression of Pim-1 is required for LTP. Its level of expression and, consequently, its capacity to phosphorylate target proteins in dendritic and nuclear compartments of stimulated neurons might be a determining factor for the establishment of long-lasting changes in synaptic strength.

GABAB receptor antagonism: facilitatory effects on memory parallel those on LTP induced by TBS but not HFS.

The present experiments used CGP 35348, a selective GABAB receptor antagonist with a significantly higher affinity for post- versus presynaptic receptors, to dissociate the role of antagonist concentration versus stimulation mode in determining whether GABAB receptor blockade facilitates or suppresses long-term potentiation (LTP). The antagonist was applied by pressure ejection to one of two recording sites in area CA1 of hippocampal slices before LTP was induced at both sites with either theta burst or high-frequency stimulation (TBS or HFS). TBS produced a dose-dependent facilitation of potentiation that turned into depression at the highest concentration tested, a result reflecting the dose-dependent balance between the drug's postsynaptic disinhibitory effect and its action on presynaptic autoreceptors regulating the release of GABA. In contrast, HFS-induced LTP increased monotonically with drug concentration, suggesting that blockade of postsynaptic GABAB receptors is the only factor contributing to HFS-induced LTP. To test the relevance of the two sets of LTP results, we performed behavioral studies examining the effect of different dosages of antagonist on spatial retention and found that memory was enhanced at intermediate dosages but not at very low and high concentrations, reminiscent of the bell-shaped dose-response curve obtained for TBS-induced LTP. These findings are consistent with the notion that LTP induced by electrical stimulation modeled after endogenous theta-modulated activity patterns bears more relevance to behavior than does potentiation induced by arbitrary tetanic trains.

Unilateral hippocampal ablation at birth causes a reduction in contralateral LTP.

Subcortical damage in neonates often has more severe consequences than in adults. Unilateral electrolytic hippocampal lesions in adult rats typically result in transient memory deficits, whereas neonatal lesions cause lasting memory impairments. We hypothesized that unilateral lesions made at birth may affect synaptic physiology in the contralateral hippocampus. Consequently, the ability to sustain long-term potentiation (LTP), a form of synaptic plasticity believed to underlie certain forms of memory, was compared between slices from the remaining hippocampus of rats lesioned as newborns and as adults. Initial studies showed that a train of 10 stimulation bursts patterned after the hippocampal theta rhythm produced robust and stable LTP both in slices from controls and rats lesioned at birth. However, a theta burst pattern of stimulation closer to intrinsic physiology (five burst pairs separated by 30 s each), induced significantly less LTP in slices from rats lesioned at birth compared to those from controls and rats lesioned as adults. To investigate possible mechanisms underlying the deficit, the degree of paired-pulse facilitation (PPF) as well as the amount of depolarization occurring between two successive theta bursts were analyzed. The lesion did not detectably change PPF characteristics, suggesting that presynaptic mechanisms are normal. However, the extent to which a burst response was increased by a prior burst was significantly diminished in slices from rats lesioned at birth compared to those from controls and rats lesioned as adults, indicating that postsynaptic factors involved in the initial triggering events of LTP are affected by the lesion. Reduced ability to sustain LTP in the remaining hippocampus may contribute to impaired memory function after unilateral neonatal hippocampal lesion.

Time-dependent reversal of long-term potentiation by an integrin antagonist.

The integrin antagonist Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) was applied by local ejection to one of two recording sites in hippocampal slices at various times before and after long-term potentiation (LTP) was induced at both sites with theta burst stimulation. Applications 10 min before, immediately after, and 10 min after induction caused LTP at the experimental site to decay steadily relative to that at the within-slice control site. However, application at 25 min or more after induction had no detectable effect on potentiation. Similar results were obtained when the integrin antagonist was perfused into the slice rather than applied locally. The time period after induction during which GRGDSP interfered with LTP consolidation corresponds to that during which LTP is susceptible to reversal by low-frequency afferent stimulation and newly formed memories are vulnerable to various disruptive treatments. Comparable experiments using a peptide that blocks an extracellular binding site of neural cell adhesion molecules (NCAMs) did not yield time-dependent reversal of LTP; i.e., an antagonist that interacts with the fourth immunoglobulin-like domain reduced LTP when applied before induction but not afterward. Moreover, LTP formation occurred normally in the presence of an antibody against the fibronectin repeat domain of NCAM. These results suggest that integrin activation and signaling occurring over several minutes after LTP induction are necessary for stabilizing synaptic potentiation and by inference may be required for the conversion of new memories into a not readily disrupted state.

Fear conditioning induces associative long-term potentiation in the amygdala.

Long-term potentiation (LTP) is an experience-dependent form of neural plasticity believed to involve mechanisms that underlie memory formation. LTP has been studied most extensively in the hippocampus, but the relation between hippocampal LTP and memory has been difficult to establish. Here we explore the relation between LTP and memory in fear conditioning, an amygdala-dependent form of learning in which an innocuous conditioned stimulus (CS) elicits fear responses after being associatively paired with an aversive unconditioned stimulus (US). We have previously shown that LTP induction in pathways that transmit auditory CS information to the lateral nucleus of the amygdala (LA) increases auditory-evoked field potentials in this nucleus. Now we show that fear conditioning alters auditory CS-evoked responses in LA in the same way as LTP induction. The changes parallel the acquisition of CS-elicited fear behaviour, are enduring, and do not occur if the CS and US remain unpaired. LTP-like associative processes thus occur during fear conditioning, and these may underlie the long-term associative plasticity that constitutes memory of the conditioning experience.

Neonatal vs. adult unilateral hippocampal lesions: differential alterations in contralateral hippocampal theta rhythm.

Subcortical damage often has more severe consequences in neonates than in adults. For example, unilateral hippocampal lesions in adult rats typically lead to transient memory deficits, whereas neonatal lesions cause lasting learning impairment. We hypothesized that the defects triggered by unilateral damage may include synaptic dysfunction in the contralateral hippocampus. Consequently, we examined the hippocampal theta rhythm, an EEG pattern thought to be associated with learning. Initial comparisons between intact and lesioned rats revealed no obvious differences in basal theta rhythm properties. However, manipulations of ascending brainstem projections to hippocampus with drugs specific for serotonergic, noradrenergic and cholinergic receptors uncovered differences. Antagonism of 5-HT3 receptors known to promote learning significantly increased theta frequency in controls and adult lesioned rats, but not after neonatal damage. In contrast, blockade of noradrenergic-alpha2 receptors had no effect. Antagonism of cholinergic receptors which typically impairs learning disrupted theta and caused irregular, high-amplitude activity that was significantly more pronounced in the lesioned groups. A final approach involved pharmacological facilitation of AMPA receptor-mediated currents, using a drug which enhances memory. This treatment significantly enhanced theta frequency in controls and animals lesioned as adults. In contrast, it failed to do so in rats lesioned at birth. These observations suggest that latent dysfunction in contralateral hippocampal physiology may contribute to the lasting memory deficits seen after unilateral hippocampal lesion in neonates.

AMPA receptor facilitation accelerates fear learning without altering the level of conditioned fear acquired.

Rats treated with the AMPA receptor-facilitating drug 1-(quinoxolin-6-ylcarbonyl)piperidine (BDP-12) during training acquired fear conditioning to a tone faster than vehicle-treated controls. The effect on acquisition was dependent on the dose given. BDP-12-treated rats and vehicle-treated controls reached the same level of conditioned fear and extinguished at the same rate. The dissociation of learning rate from these other normally covariant measures suggests that the drug had a specific and isolated effect on acquisition. Controls for drug effects on stimulus sensitivity, locomotor activity, generalized fearfulness, and other performance factors support this interpretation. The known action of BDP-12 on receptor dynamics suggests that its effect on acquisition may be attributed to specific modulation of an AMPA and NMDA receptor-dependent plasticity mechanism. The finding that the drug accelerates acquisition but does not affect the level of conditioned fear acquired parallels the effect of the drug on long-term potentiation (LTP) (increasing the rate but not the ceiling of potentiation) and suggests that common mechanisms may underlie fear conditioning and LTP.

Studies on long-term depression in area CA1 of the anesthetized and freely moving rat.

Homosynaptic long-term depression (LTD) is reported to occur in field CA1 of hippocampal slices collected from immature brains. Because the effect has been postulated to be a memory storage mechanism, it is of interest to test for its presence in adult, awake animals. Unfortunately, not only has hippocampal LTD proved difficult to obtain reliably in vivo, but the few successful studies vary with respect to protocols and evidence that the depression is input-specific. The present study tested for input-specific (homosynaptic) LTD in field CA1 after application of various stimulation protocols to the Schaffer collateral/commissural projections in freely moving, adult rats. The results indicate that although low-frequency trains do induce decrements in synaptic transmission lasting for hours to several days, the success rate of eliciting input-specific LTD in the awake rat is very modest compared with the ease with which stable potentiation is obtained in the same synapses. Moreover, it is questionable that the effective protocols represent patterns of activity likely to occur during behavior. The stronger the afferent activation during low-frequency stimulation, the greater was the probability of eliciting LTD accompanied by persistent heterosynaptic depression. Clear evidence for the occurrence of LTD, irrespective of stimulation protocol and current intensity, could not be obtained in rats under barbiturate anesthesia. In all, the results do not accord with the suggestion that LTD occurs routinely in the hippocampus in vivo as part of memory encoding.

Arg-Gly-Asp-Ser-selective adhesion and the stabilization of long-term potentiation: pharmacological studies and the characterization of a candidate matrix receptor.

Peptides known to block the extracellular interactions of adhesion receptors belonging to a subclass of the integrin family were tested for their effects on the stabilization of long-term potentiation (LTP) in hippocampal slices. Theta burst stimulation delivered after infusions of Gly-Ala-Val-Ser-Thr-Ala (GAVSTA) resulted in a potentiation effect that decayed steadily over a period of 40 min; LTP elicited in the presence of inactive control peptides remained stable over this time period. GAVSTA had no detectible influence on baseline responses, induction processes, or the initial degree of potentiation. Infusions of integrin antagonists after application of theta bursts also resulted in the occurrence of a decremental form of LTP. Affinity chromatography was then used in an effort to identify targets of the structurally dissimilar integrin blockers that disrupt LTP stabilization. Both integrin antagonists Gly-Arg-Gly-Asp-Ser-Pro and GAVSTA eluted a major species of 55 kDa (synaptegrin-1) from GRGDSP-affinity columns that had been loaded with solubilized synaptic membranes; lesser concentrations of three polypeptides of approximately 20, 27, and 30 kDa were also collected. Synaptegrin-1 was labeled by antibodies to the RGDS-binding integrin alpha5beta1. In addition, the synaptegrin, as well as the 27 kDa, protein was found to copurify with pre- and postsynaptic markers during the isolation of forebrain synaptosomes. These results indicate that a matrix recognition event occurring several minutes after induction of LTP is a necessary step in the stabilization of potentiated synapses; they also identify an integrin-like matrix receptor of 55 kDa that may contribute to this event.

Effects of 5-HT3 receptor antagonism on hippocampal cellular activity in the freely moving rat.

Recent physiological studies conducted in the hippocampi of freely moving rats have revealed that systemic injections of the selective serotonin-3 (5-HT3) receptor antagonist ondansetron facilitate induction of long-term potentiation (LTP), increase the frequency of the theta electroencephalogram rhythm, and enhance retention of memory in hippocampally dependent tasks. To gain insight into the cellular mechanisms underlying these observations, in the present study we examined the effects of intraperitoneal injections of ondansetron on the firing rate of CA1 interneurons and pyramidal cells in the dorsal hippocampi of freely moving rats. Mean firing rates of a substantial proportion (17 of 27) of isolated neurons were significantly different before and after ondansetron injection (500 and 1,000 micrograms/kg). Of the interneurons that exhibited an effect, all (11 of 11) significantly decreased their mean firing rate, with an average change of -22.4 +/- 3.9% (mean +/- SE) across cells. Eighty-three percent (5 of 6) of pyramidal cells showing a change in mean firing rate displayed a significant increase in activity, with an average change of 56.3 +/- 25.6% across cells. Ondansetron (1.0 mg/kg ip) had no detectable effect on spontaneous behavioral activity as measured by line crossings and rearings in an open-field apparatus. The present results show that pharmacological blockade of 5-HT3 receptors causes a reduction in firing activity of a subset of CA1 hippocampal interneurons, with concomitant increases in the firing rate of pyramidal cells. These changes may be directly related to the ondansetron-induced enhancement of LTP induction and memory formation observed in previous studies.

Short-term reverberant memory model of hippocampal field CA3.

Synaptic plasticity mechanisms for associative learning require near-simultaneous pairs of inputs to target cells. Sensory cues encountered behaviorally, however, are typically staggered in time, implying the need for active short-term memory traces of antecedent cues. The dense recurrent connectivity within regions of hippocampal field CA3 is suggestive of the kind of re-entrant network that could subserve this kind of "holding" memory. Consequently, we have investigated whether an abstract model of this region incorporating its major anatomical and physiological features could function as a reverberatory memory network. The continuous-time model describes the behavior of highly connected groups of CA3 pyramidal cells, or "patches," in response to brief, rhythmic, sensory stimulation. Time constants for excitatory and inhibitory postsynaptic potentials and axonal transmission delays for local and distal connections were estimated from empirical data. When the inhibitory units in these patches were connected to an oscillator intended to model the theta wave activity of the medial septum, the network entered reverberatory states and maintained second-long memory traces of the cortical input, after which it lost its coherent behavior. Noise analysis indicated that the network's operation was moderately resistant to random fluctuations proportional to patch activity. These results suggest that field CA3 could function as a holding memory that assists the integration of disjoint stimuli found in innumerable associative tasks, and that the duration of its coherent operation might determine the temporal limits in their performance.

Remembrance of odors past: enhancement by central facilitation of AMPA receptors.

Pharmacological facilitation of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA)-type glutamate receptor has recently been demonstrated to enhance synaptic responses, promote long-term potentiation (LTP) induction in freely moving rats, and facilitate learning and retention of information. The present study verifies and extends the behavioral action of allosteric AMPA receptor modulation by showing that the benzoyl-piperidine compound BDP-12 promotes retention of olfactory and transient spatial memory in a dose-dependent fashion; is only effective when given before but not after training, consistent with the hypothesis that glutamatergic facilitation enhances information encoding by means of action on the machinery involved in LTP induction; and, following suboptimal training in a paradigm of enduring memory, prolongs the ability of rats to retain odors by extending the decay of weak memory traces.

Proactive and retrograde effects on LTP produced by theta pulse stimulation: mechanisms and characteristics of LTP reversal in vitro.

Previous studies have established that (1) a 1-min episode of theta pulse stimulation (TPS) is sufficient to reverse potentiation during the early phases of LTP in area CA1 without causing depression when administered to nonpotentiated pathways; (2) the magnitude of depotentiation is inversely related to the delay between LTP induction and reversal attempts; and (3) pharmacological facilitation of AMPA receptor-mediated currents significantly enhances the strength of the reversal mechanism. The present experiments confirm and extend these results by showing that the depotentiating action of TPS on prior LTP is antagonized by inhibitors of protein phosphatases and adenosine A1 receptors but is not affected by NMDA receptor blockade, and, moreover, that TPS interferes with subsequent LTP induction by triggering an inhibitory mechanism that is active for a few minutes and is blocked by phosphatase inhibition. The possible implications of these results are discussed.

The induction of homo- vs. heterosynaptic LTD in area CA1 of hippocampal slices from adult rats.

The induction of long-term depression (LTD) was investigated in area CA1 of hippocampal slices from adult rats. To produce LTD, prolonged low-frequency stimulation (LFS, 900 stimuli at 1 Hz) was delivered to one of two independent Schaffer-collateral/commissural projections, while the second input served as a control to monitor heterosynaptic effects. The depression was calculated as percent decrease in the slope of the dendritic field EPSP relative to baseline values, and LTD was considered established if the response decrement was at least 15% in magnitude and stable for 30-60 min. By delivering LFS in conditions of different relative baseline response magnitudes, it was revealed that the intensity of afferent low-frequency activity has a significant impact on the induction frequency, magnitude and input-specificity of the depression: the rate of LTD occurrence and the effect of LFS on the absolute response decrement increased successively as the stimulation strength was raised, but the impact of LFS on the relative LTD magnitude decreased at higher stimulation intensities; the depression was specific to the stimulated input (homosynaptic LTD) when baseline responses were spike-free, but spread to the pathway which was silent during LFS (heterosynaptic LTD) in experiments conducted above spiking threshold. The results indicate that in the adult rat (i) the induction of input-specific LTD is dependent on the level of synaptic activation during LFS, and (ii) LTD can easily be obtained in strongly stimulated pathways but may be the result of a generalized decrease in the postsynaptic response.