Anti-Hebbian Long-Term Potentiation in the Hippocampal Feedback Inhibitory Circuit

Associative N-methyl-D-aspartate receptor (NMDAR)–dependent LTP is induced by coincident activity in afferent pathways sufficient to depolarize postsynaptic neurons (1). However, the voltage dependence of Ca²⁺-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) is opposite to that of NMDARs (2, 3). Because CP-AMPARs are blocked by cytoplasmic polyamines upon depolarization (4, 5), maximal Ca²⁺ influx occurs when the membrane potential is relatively negative. LTP dependent on CP-AMPARs occurs in interneurons of the spinal cord and amygdala (6, 7), but its postsynaptic voltage dependence has not been explored. In hippocampal interneurons, CP-AMPARs have been implicated in long-term depression (8–10), and contribute to synaptic Ca²⁺ transients, especially in the stratum oriens/alveus (11). Many interneurons in the oriens/alveus also show NMDAR-independent LTP (12). We therefore looked for associative LTP in these cells, while recording with the gramicidin perforated patch technique to preserve intracellular polyamines (13).

Stimulation of pyramidal cell axon collaterals in the alveus evoked monosynaptic excitatory postsynaptic potentials (EPSPs) subthreshold for evoking action potentials. After recording a baseline, we paired high-frequency burst (HFB) stimulation (five pulses at 100 Hz, repeated 20 times) with stimulation of a second, suprathreshold, alveus pathway. “In-phase” associative pairing (phase difference ΔΦ = 0°) failed to elicit associative LTP in either pathway (n =7; Fig. 1, A and B). In a further set of experiments, we alternately stimulated two weak pathways, and then delivered HFBs to both pathways antiphase (ΔΦ = 180°). This evoked a persistent increase in EPSP initial slope in one or both pathways in all cells (n = 7; Fig. 1, C and D). LTP was elicited even when HFB stimuli were delivered to only one weak pathway (n =7; Fig. 1, E and F). Thus, LTP at excitatory synapses on interneurons in the oriens/alveus is prevented by associative pairing, in direct contrast to NMDAR-dependent LTP (1).

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Can direct manipulation of the postsynaptic membrane potential similarly gate LTP induction? We delivered HFBs to one pathway coinciding with the trough (somatic voltage: –90 mV) of an imposed 4-Hz sinusoidal somatic membrane potential oscillation. HFBs were then delivered to the other pathway coinciding with the depolarizing phase. In 8 out of 11 cells, pairing with hyperpolarization, but not with depolarization, resulted in LTP (Fig. 2, A and B). One cell showed the opposite behavior, and the other two showed no effect of either pairing (fig. S1). Single alveus stimuli in phase with maximum hyperpolarization (100 times) also induced LTP (n = 10; Fig. 2C), but pairing with depolarization was ineffective (Fig. 2D). Thus, even low-frequency stimulation can trigger LTP if interneurons are hyperpolarized.

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Because the induction requirements for LTP in most interneurons in the oriens/alveus are diametrically opposite to Hebb's postulate (14, 15), we refer to it as “anti-Hebbian.” We tested the same LTP induction protocols in interneurons in the stratum radiatum. Hebbian LTP could be elicited in about half of these cells, many of which mediate feedforward inhibition (16), whereas pairing either HFB or low-frequency stimuli with hyperpolarization was uniformly unsuccessful (figs. S1 and S2). Anti-Hebbian LTP is thus characteristic of excitatory synapses made by local pyramidal cells on interneurons in the oriens/alveus but not of Schaffer collateral synapses on interneurons in the stratum radiatum.

Can differences in synaptic glutamate receptors explain whether Hebbian, anti-Hebbian, or no LTP is elicited? When interneurons in the oriens/alveus were recorded in whole-cell voltage clamp [with γ-aminobutyric acid (GABA) receptors blocked, and with spermine included in the pipette solution], synaptic AMPARs activated by alveus stimulation were generally strongly rectifying (Fig. 2E), consistent with expression of CP-AMPARs (11). Furthermore, only small NMDAR-mediated synaptic currents were detected at a positive holding potential, consistent with low synaptic expression of the NR1 subunit (17).

We tested interneurons in the oriens/alveus, recorded in perforated patch mode, with a further anti-Hebbian protocol High-frequency stimulation of one alveus pathway (100 Hz, 100 pulses, delivered twice) paired with hyperpolarization, with NMDARs blocked, elicited LTP in 25 out of 31 cells (Figs. 3A and 4C). We repatched 11 of these cells in whole-cell voltage-clamp mode and found pronounced synaptic AMPAR rectification in every cell where anti-Hebbian LTP was evoked. The rectification index did not differ detectably between control and potentiated pathways (Fig. 3B), yielding no evidence for an LTP-related change in the permeability of synaptic AMPARs to Ca²⁺ (18).

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In contrast, repatched interneurons in the stratum radiatum generally showed nonrectifying AMPARs and a large NMDAR-mediated component of Schaffer collateral-evoked synaptic currents (fig. S2) (9). The anti-Hebbian LTP induction protocol was successful in only 2 out of 20 cells in the stratum radiatum. AMPARs at Schaffer collateral synapses on 11 cells (none of which showed anti-Hebbian LTP) were nonrectifying (fig. S3).

Anti-Hebbian LTP thus typically occurs at synapses on interneurons in the oriens/alveus equipped with rectifying CP-AMPARs. Are these a uniform subgroup? Seven interneurons were regular-spiking oriens-lacunosum moleculare (O-LM) cells (Fig. 3C, fig. S4), which mediate feedback inhibition of the apical dendrites of pyramidal neurons (19). Twelve other interneurons had horizontal dendrites and electrophysiological properties typical of O-LM cells, but axon visualization was incomplete (fig. S5). Anti-Hebbian LTP, however, also occurred in 17 out of 24 fast-spiking interneurons in the strata oriens or pyramidale, including one anatomically confirmed axo-axonic and two basket cells, which are innervated by CA1 pyramidal cells and target their perisomatic area. Seven other cells could not be classified.

Anti-Hebbian LTP is, however, rare at Schaffer collateral synapses on interneurons in the stratum radiatum, which generally mediate feedforward inhibition and express nonrectifying receptors (Fig. 3, D and E). Synaptic responses evoked by stratum radiatum stimulation in fast-spiking interneurons in the stratum pyramidale, however, had strongly rectifying AMPARs and a small NMDA component, and the Hebbian LTP induction protocol was uniformly unsuccessful (n = 4; fig. S6).

Does rectification of CP-AMPARs fully explain the anti-Hebbian nature of LTP in interneurons in the oriens/alveus? We first verified that AMPA/kainate receptors are necessary for induction, by pairing HFS with postsynaptic hyperpolarization while AMPA/kainate receptors were blocked with 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX, 5 μM): After washout of the antagonist, EPSPs in the tetanized and control pathways recovered to the same extent (n = 7; Fig. 3F). We then explored systematically the voltage dependence of LTP. In five cells in the oriens/alveus where anti-Hebbian LTP was evoked in one alveus pathway, subsequent pairing of the other pathway with depolarization only evoked short-lived post-tetanic potentiation (Fig. 4A). In six other cells, pairing the second pathway with hyperpolarization elicited robust LTP in all cases (Fig. 4, B and C). We then adapted this experimental design to explore the effect of manipulating the rectification properties of CP-AMPARs. Having demonstrated anti-Hebbian LTP in one pathway, we repatched the interneuron in whole-cell mode either with or without spermine in the pipette solution. Following a short baseline recording (≤7 min from patch rupture), we then paired HFS of the second pathway either with depolarization (+20 mV) or with hyperpolarization (–90 mV). When spermine was omitted, pairing with depolarization evoked LTP in five out of five cells (Fig. 4D), consistent with Ca²⁺ influx via CP-AMPARs rendered nonrectifying by removal of polyamines (4). In contrast, HFS paired with depolarization failed to elicit LTP in five cells that were repatched with a spermine-containing pipette (Fig. 4E). In five other interneurons repatched with a spermine-containing solution, pairing HFS of the second pathway with hyperpolarization to –90 mV evoked LTP (Fig. 4F).

Polyamine-mediated rectification of AMPARs (and/or kainate receptors) thus explains the voltage dependence of LTP induction in these interneurons and reconciles our results with previous reports that a Hebbian protocol induces LTP in interneurons in the oriens/alveus when recorded with a polyamine-free whole-cell pipette solution (12). Also consistent with these reports, blockade of group I metabotropic glutamate receptors prevented LTP induction in interneurons with horizontal dendrites in the oriens/alveus (fig. S7). Finally, we looked for evidence that anti-Hebbian LTP is accompanied by an increase in glutamate-release probability (12), by applying extracellular polyamines, which also block CP-AMPARs in a use-dependent manner (20). After inducing anti-Hebbian LTP in one pathway, bath perfusion of N-(4-hydroxyphenylpropanoyl)-spermine (5 to 10 μM) caused a progressive decrease in EPSP initial slope, which was significantly faster in the paired than in the control pathway (n = 7; fig. S8). Given that anti-Hebbian LTP did not alter AMPAR rectification (Fig. 3B), this result is consistent with presynaptic expression.

Anti-Hebbian LTP may play distinct roles in neurons that show characteristic phase relationships in different network states (21, 22). During sharp-wave ripples, O-LM cells are typically silent, while many of their input pyramidal neurons fire at high frequency (21), possibly satisfying the induction conditions for anti-Hebbian LTP. Binding of pyramidal neurons to a spatial map may occur during periods of high-frequency firing (23), similar to sharp-wave ripples. In contrast, during theta activity, which is associated with exploratory behavior (24), O-LM cells fire in phase with pyramidal cells (21) and may contribute to this oscillation through phase-locked dendritic inhibition (25). Anti-Hebbian LTP induced during ripples may therefore result in a long-term alteration of pyramidal cell excitation of O-LM cells, which persists during theta activity, and may therefore contribute to spatial memory formation, the early stages of whichhavebeen shown to withstand NMDAR blockade (26).