Interaction between hippocampal-prefrontal plasticity and thalamic-prefrontal activity

The prefrontal cortex integrates a variety of cognition-related inputs, either unidirectional, e.g., from the hippocampal formation, or bidirectional, e.g., with the limbic thalamus. While the former is usually implicated in synaptic plasticity, the latter is better known for regulating ongoing activity. Interactions between these processes via prefrontal neurons are possibly important for linking mnemonic and executive functions. Our work further elucidates such dynamics using in vivo electrophysiology in rats. First, we report that electrical pulses into CA1/subiculum trigger late-onset (>400 ms) firing responses in the medial prefrontal cortex, which are increased after induction of long-term potentiation. Then, we show these responses to be attenuated by optogenetic control of the paraventricular/mediodorsal thalamic area. This suggests that recruitment and plasticity of the hippocampal-prefrontal pathway is partially related to the thalamic-prefrontal loop. When dysfunctional, this interaction may contribute to cognitive deficits, psychotic symptoms, and seizure generalization, which should motivate future studies combining behavioural paradigms and long-range circuit assessment.

,b depicts two representative neurons, one from each recorded site. The analysis includes perievent raster plots (Fig. 2a), and corresponding Z-scored smoothed histograms ( Fig. 2b; 10 ms bins) comparing the baseline vs. the post-HFS period. According to Fig. 2a,b, both units showed paired pulse-locked excitatory responses (<200 ms latency), followed by a transient suppression (~200-400 ms), and then a new excitatory response (~400-800 ms). This secondary excitation was stronger in the mPFC unit, and unlike the PV/MD it was potentiated by HFS. A closer look at paired pulse-locked responses is provided by Fig. 2c (180 ms perievent window), illustrating how spikes (black dots) were timed in relation to stimulus artefacts (grey vertical lines). Corresponding histograms ( Fig. 2d; 3 ms bins) show excitatory responses to each pulse (~20-40 ms latency), which were stronger in the thalamic unit, and indifferent to HFS in both units.  Micro-screw holes are omitted, except for the one used as ground. Electrode connectors and microscrews were covered together with acrylic cement, aiming at chronic recordings. (b) Coronal sections and Nissl histology. Drawings were made based on the Paxinos and Watson rat brain atlas 19 . Red circles situate the electrolytic lesions across rats. Photomicrographs from two rats represent each brain site, with arrows indicating subtle lesions. (c) Timeline of the chronic recording session, undertaken once per rat. Electrical paired pulses (80 ms separation) were delivered into CA1/sub every 10 s throughout the session, except during HFS. Perfusion was made within 30 min after the recording session. shows a gradient of response intensity, ranging from no response to the representative patterns of Fig. 2a,b. As indicated by red areas, late-onset (>400 ms) excitatory responses to CA1/sub pulses were more prevalent among mPFC units, especially during the initial post-HFS period. In more details, the heatplots of Fig. 3a show averaged data from three periods: baseline (its second half), initial 15 min post-HFS, and final 15 min of the session. The rows of each heatplot are perievent histograms from individual units (except putative FSI), and columns are time bins (10 ms). Red, blue, and green tones respectively represent excitation, suppression, and no change (Z-scores against the 400 ms pre-pulse period). Of note, single units were sorted from top to bottom according to the mean moduli of post-pulse Z scores during the initial post-HFS period. Thus, the stronger the response within the 15 min after LTP induction, the higher the row position across images. As can also be seen, the transient suppression and the re-excitation varied in their durations and latencies (<200 ms variation), which can reflect uncontrolled microwire positioning in different mPFC layers (see Methods; Surgery and electrodes subsection), not to mention between-subject factors.
Black bars on the right of each heatplot (Fig. 3a) indicate which single units were responsive to CA1/sub paired pulses, irrespective of <200 ms variations. More specifically, we compared 10 ms-binned Z scores before (0.4 s) vs. after (1 s) pulses through t-tests, in a unit-wise manner. Z scores were converted to moduli in this particular analysis, thus capturing response magnitudes regardless of their directions. Figure 3a also shows the proportions of responsive units. According to a chi-square test, HFS modulated these proportions in the mPFC (χ 2 = 6.073; p = 0.048), but not PV/MD. Further comparisons between mPFC and PV/MD are represented by Fig. 3b-d. Data from each heatplot were converted to 20 ms-binned smoothed curves, with shaded areas delimiting standard errors. , showing spiking responses to CA1/sub paired pulses (y-axis: 180 baseline and 720 post-HFS sweeps, every 10 s). Bottom: color-coded arrays from the same raster plots. Spike counts (10 ms bins) were Z-scored against pre-event bins, and merged every 6 sweeps. Resulting arrays were then smoothed and plotted on a colour scale. Black and red arrowheads respectively indicate the paired pulses and HFS delivered into CA1/sub. (b) Perievent data from the smoothed raster plots of panel a. Sweeps were averaged from the final 15 min of the baseline, and initial 15 min of the post-HFS monitoring. Curves were then plotted on the Z-scored firing axis (y). The inset particularly compares post-HFS curves. (c) 180 ms-windowed raster plots from the same single units. Stimulus artefacts form grey vertical lines, indicating the timing between CA1/sub pulses and spiking responses. All other analyses are artefact-free.  In summary, these findings reveal that responses to CA1/sub paired pulses varied between mPFC and PV/ MD, especially at the 400-800 ms latency ( Fig. 3c grey areas), during which the mPFC excitation was transiently potentiated by HFS. mPFC putative FSI and principal cells responded in opposite manners to CA1/sub pulses.     Figure 4c describes the spontaneous firing rates of mPFC and PV/MD neurons (heatplots), and their mean ± standard errors (bottom graph) in 3 min bins. Heatplot rows consist of individual rate histograms, which were Z-scored against the baseline, smoothed, and sorted in descending order of the mean post-HFS Z score. This shows the diversity of SUA throughout the recording. Results show that a cumulative excitation began to emerge ~90 min after HFS (Fig. 4c). This net change was confirmed by effects of time (with baseline: F (49,3577) = 3.402, p < 1 × 10 −5 ; without baseline: F (39,2847) = 2.982, p < 1 × 10 −5 ). No differences were found between mPFC and PV/ MD.

Plasticity of fPSP responses and spontaneous firing rates.
Thus, both recorded areas underwent long-term changes after CA1/sub HFS: potentiation of field responses and net excitation of spontaneous firing. This contrasts with the evoked firing data (Fig. 3b-d), whose changes were confined to the mPFC and the initial post-HFS period. More generally, the effects of HFS showed distinct time courses depending on which dimension of neural activity was analysed: either evoked firing, or LTP and spontaneous firing.
Correlation between Zif268 expression and electrical recruitment. Immediate early genes are activated upon neural stimulation 23 . In particular, Zif268 expression is associated with NMDA receptor-mediated transmission, and synaptic plasticity 24,25 . The objective, here, was to examine Zif268 expression in relation to mPFC and PV/MD response evoking. Figure 5 shows a significant Spearman's correlation between prefrontal Zif268 immunopositivity and prefrontal fPSP amplitude, after a False Discovery Rate adjustment (Rho = 0.762, p = 0.006, adjusted p = 0.035). Thus, the stronger the prefrontal fPSP response to CA1/sub pulses (specifically after HFS), the stronger the prefrontal Zif268 expression (Fig. 5). Correlations were consistently absent when analysing thalamic fPSP, thalamic firing, and prefrontal firing. Therefore, we found a relationship between Zif268 expression and the CA1/sub-mPFC recruitment. These data show that stimulation effectiveness was commensurate with a non-electrophysiological assessment of the circuit.
Complementary optogenetics. Purpose and design. As a follow-up to the main experiment, we explored the thalamic role in the CA1/sub-mPFC recruitment. Rats were transfected with AAV5-hSyn-eArch3.0-eYFP in PV/MD for expression of archaerhodopsins (green-light gated outward proton pumps 26 ). A month later, rats were anesthetized with urethane and implanted as above, except for an optrode into PV/MD. This time, LTP was not induced, so we could focus on basal CA1/sub-mPFC-PV/MD interactions using optical and electrical co-stimulation (Fig. 6a). Briefly, the CA1/sub was electrically stimulated (every 10 s, 120 min) while recording from mPFC and PV/MD, similarly to the main experiment. This time, PV/MD optical pulses (3 s) randomly accompanied the CA1/sub pulses at the probability of 50%, and no HFS was applied. Data are from two rats, which represent the most successful recordings out of six attempts (see Methods for exclusion criteria). Validation. We initially report data from PV/MD, specifically. The raster plot of Fig. 6b shows unsorted multi-unit activity (MUA) from rat 1. Light pulses elicited a sustained inhibition in PV/MD, only disrupted by CA1/sub stimuli, as demonstrated by the central area of the raster plot. Also noticeable are the horizontal bands of the raster plot (Fig. 6b). They represent sweeps with low (light tones) or high (dark tones) thalamic firing rates, which coincide with changes in thalamic local field potentials (LFP), as shown by the aligned spectrogram (shared y-axis). These LFP changes reflect the sleep-like alternation between "activated" and "deactivated" patterns of urethane anaesthesia, as previously described based on delta oscillations 27  (a) Experimental design. Electrical paired pulses were delivered into CA1/sub every 10 s for 120 min, either with or without 3 s light pulses into PV/MD, randomly. (b) Rat 1 data. Top: perievent raster plot describing MUA reactions of PV/MD to light pulses (green bar) and CA1/sub electrical paired pulses (arrowheads). The raster plot is aligned to an LFP spectrogram (shared y-axis) demonstrating the spontaneous cycling between "activated" and "deactivated" oscillatory states 27 (here called weak and intermediate delta, respectively; colour scale: decibels). These oscillatory states correspond to the horizontal bands of the raster plot, whose sweeps were analysed through separate histograms (bottom graphs). Light-onset suppressions (<250 ms duration) and lightoffset rebounds (~50-250 ms latency) are indicated (green and black arrows, respectively). (c) Rat 2 data. Top: same organization of panel b. In this case we observed a state of persistently strong delta oscillations. Hence, all data were analysed as a single histogram (bottom). (d) Left: optrode positioning, illustrating the penetration of the green light cone (525 nm wavelength). Drawings were made based on the Paxinos and Watson rat brain atlas 19 . Centre: coronal section demonstrating the plasma membrane expression of archaerhodopsins, as evidenced by eYFP fluorescence (green tones). Right: closer views of strongly (top) and weakly (bottom) eYFP-marked cells depending on the distance from the injection/stimulation site. Cell nuclei were stained with Hoechst 33342 (blue tones). 3 V, third ventricle; DG, dentate gyrus; Hb, habenula. are crucially involved in these oscillatory patterns 28 , and therefore we decided to sort the sweeps based on their basal rates. In other words, each sweep was assigned a firing rate based on the 3 s period before light. Rates were then categorized using percentiles: high rates (above 55th) and low rates (below 45th), thus ruling out dubious transition states 27 .
Sweep categorization resulted in the histograms of Fig. 6b (50 ms bins). Respectively, top and bottom histograms correspond to "activated" states (here called weak delta) and "deactivated" states (here called intermediate delta). As indicated by arrows, we observed transient suppressions at light onset (<250 ms duration), and thalamic rebounds 29 at light offset (~50-250 ms latency), regardless of the oscillatory state. The sustained inhibition, however, was specific to the weak delta background. Responses to CA1/sub pulses were similar to those of the main experiment, and were consistently observed across trials and oscillatory states (Fig. 6b). In turn, Fig. 6c shows PV/MD data from rat 2, which manifested a state of persistently strong delta oscillations. Light-onset suppressions (green arrow) and light-offset rebounds (black arrow) were again observed. In this rat, however, light pulses evoked a plateau excitatory response, only disrupted by CA1/sub stimuli (Fig. 6c). Figure 6d finally exemplifies the fluorescence-evidenced expression of archaerhodopsins in the optrode-implanted area (green tones). Blue tones correspond to Hoechst-stained nuclei. Figure 7 represents the main finding of this complementary experiment. It basically shows that PV/MD optical drive affects CA1/sub-driven activity in the mPFC, regardless of the activity background. Specifically, Fig. 7 depicts Z-scored 1.4 s-windowed data from mPFC, similarly to previous analyses (Fig. 3b,c), except that standard errors now reflect trial variability, i.e., subjects are their own controls. Figure 7a data are from rat 1, which manifested the spontaneous sleep-like cycling of oscillatory states. As can be seen, PV/MD light pulses were able to attenuate the >400 ms excitation in both activity states: weak delta (effect of perievent time: F ( Fig. 7b, the late-onset CA1/sub-mPFC response was again attenuated during light-on trials (effect of perievent time: F (49,3136) = 32.350, p < 1 × 10 −5 ; interaction: F (49,3136) = 2.424, p < 1 × 10 −5 ). Between-subject variations in long-latency responses can also be seen -i.e., rat 1 responses approximating the average pattern of the main experiment (Fig. 3b,c), and rat 2 responses starting ~200 ms later -consistently with the variability we reported earlier (Fig. 3a). Lastly, the insets show mPFC firing rates in the 400 ms period before electrical pulses in CA1, comparing light-on and light-off trials. Data were not Z scored in this analysis, so we could evaluate whether PV/MD light pulses were globally affecting the mPFC activity immediately prior to CA1 stimuli. According to t-tests, there were no significant differences (weak delta: t (294) = 1.176, p = 0.241; intermediate delta: t (294) = 1.544, p = 0.124; strong delta: t (658) = 0.893, p = 0.372). This reinforces the effects shown in the main graphs of Fig. 7, i.e., >400 ms Z-scored firing.

Disruption of CA1/sub-mPFC responses by thalamic optical drive.
Altogether, our two experiments demonstrate that late-onset mPFC responses are potentiated by HFS of CA1/sub, and attenuated by archaerhodopsin activation in PV/MD. This suggests a mutual relationship between hippocampal-prefrontal plasticity and thalamic-prefrontal activity, thus contributing to an emerging debate on intra-PFC amplification 9,10 , and long-range circuit interactions 30,31 . Future co-stimulation experiments with greater samples should expand on these observations.  Fig. 3b,c (400 ms pre-event, 20 ms bins), with standard errors representing trial variability (i.e., subject as its own control). Sweeps occurring in different oscillatory states were separately analysed (see also Fig. 6b). (b) Rat 2 data (see also Fig. 6c). Arrowheads indicate the paired pulses delivered into CA1/sub. Horizontal bars indicate Tukey's post-hoc differences (p < 0.05) after two-way repeated measures ANOVA. Insets show mPFC firing in the 400 ms period before CA1 stimuli. Inset data were not Z scored, thus clarifying whether PV/MD light pulses were affecting the mPFC activity immediately prior to CA1 stimuli. No significant differences were found (ns), which further validates the Z scored data.

Discussion
Exogenously induced synaptic plasticity has many more consequences than just the changes in afferent responding. There is probably a myriad of polysynaptic events between short-latency pathway recruitments and their brain-wide outcomes. Systems-level attempts like the present one are useful to map these intermediate events.
In our study, they seem represented by the long-latency prefrontal responses to hippocampal electrical pulses. Because these responses were sensitive to both hippocampus-induced LTP and thalamic optogenetic control, they might consist of a network plasticity mechanism, which could partially underlie the executive functions, in line with recent evidences 9,10 . Similar mechanisms may also occur in other thalamocortical subsystems, like those involved in motor and sensory operations 32 .
A speculation is that the CA1/sub-mPFC recruitment could either: (1) enable a longer-lived resonance within the thalamic-prefrontal loop (~400 ms latency and duration); (2) be, on the contrary, regulated by thalamic activity; or (3) combine both. In any event, the local mPFC circuit would undergo temporary states of increased or decreased net excitation. For example, at transiently enhanced depolarization, CA1/sub-innervated pyramidal cells of the mPFC would be more prone to respond to other afferents, e.g., from basolateral amygdala 33 . In this case, CA1/sub-mPFC-PV/MD interactions could provide time frames of increased limbic connectivity. These depolarization events could also foster NMDA receptor-dependent plasticity processes, e.g., cytosolic Ca 2+ -mediated AMPA receptor trafficking/anchoring, and immediate early gene activation 23,34 , transforming millisecond-range input convergence into lasting modifications of mPFC synaptic efficacy. In addition, the plastic changes we observed followed different time courses depending on which aspect of neural activity was analysed: either perievent firing (changes were stronger in the initial post-HFS period), 3 min-binned firing (changes were stronger in the final post-HFS period), or fPSP amplitudes (LTP was rather constant). As recently discussed 35 , early and late LTP reflect a temporal sequence of biochemical mechanisms. They possibly range from ionic conductance changes in AMPA receptors that are already available in the postsynaptic density (seconds to minutes), to increases in synaptic size and quantal transmission based on neuromodulator-mediated protein synthesis (hours) 35 . Future studies should dissect LTP-like firing patterns -like the ones described here -in light of these cellular and neurochemical mechanisms.
In a broader neurophysiological sense, the afferent cooperation within the mPFC is possibly modulated by the oscillatory state. We have previously reported that the same activity background -with either urethane-driven slow or cholinergically induced rapid oscillations -differentially modulates the mPFC plasticity depending on the stimulated site: either CA1/sub 11,12 or PV/MD 13 . Further interpretation can be made based on the present data. Our recordings during sleep-like cycling indicated that optical pulses required rapid oscillations to induce sustained thalamic inhibition. This effect was indeed expected to be limited during slow oscillations, which reflect endogenous thalamic hyperpolarization 28 . The long-latency CA1/sub-mPFC response, on the other hand, was attenuated across oscillatory states. Intriguingly, this attenuation was replicated during strong delta oscillations, when light pulses paradoxically evoked plateau excitations. This suggests a partial dissociation between ongoing background activity (noise) and ephemeral polysynaptic interactions (signal), at least with respect to the exogenously manipulated CA1/sub-mPFC-PV/MD circuit. Thus, the balance between noise (ongoing thalamic activity) and signal (CA1/sub-evoked patterns) might fluctuate with the oscillatory context. It is anyway noticeable that the optical drive of PV/MD was unable to completely block long-latency mPFC responses, implying other subcortical resonators. Hence, we can also speculate that CA1/sub-mPFC information reverberates in multiple excitatory loops (e.g., between mPFC and ventral midline thalamus 36 ), and that preferentially resonating in this or that loop could depend on the oscillatory activity.
Collaterally to the attenuation of late-onset mPFC responses, we observed different PV/MD reactions to archaerhodopsin activation, similarly to a previous report on the monkey visual cortex 37 . Specifically, we found a consistent <250 ms activity drop at light onset. This transient event was then followed by plateaus of either strong suppression, weak suppression, or excitation, in an apparent relationship with weak, intermediate, and strong delta power, respectively. This could suggest that oscillatory states may determine the direction of green light-driven PV/MD responses. If this is the case, PV/MD responses could reflect distinct combinations between the direct hyperpolarization of thalamocortical cells, and their disinhibition via GABAergic terminals from the reticular thalamus, both of which supposedly transfected with eArch3.0. Reticular-mediated disinhibition is indeed critical for translating neurochemical states (e.g., cholinergic tone) into global rhythms 38,39 , which could explain the relationship we found between delta power and the direction of PV/MD responses. However, we also recognize that plateau responses could reflect biophysical constraints of sustained archaerhodopsin activation, including pH-dependent presynaptic calcium influx and the consequent increase in local neurotransmitter release 40 . These issues should motivate further evaluation of PV/MD activity with different optogenetic tools and during different oscillatory backgrounds.
Apart from this discussion on long-latency thalamic-prefrontal interactions, we also found shorter-latency mPFC responses to CA1/sub stimuli: paired pulse-locked sharp increases (<50 ms), and ensuing inhibitory components (~160-400 ms). This post-stimulus pattern is in agreement with a previous investigation 4 . Using pharmacological tools, the authors have attributed the sharp excitatory events to AMPA receptor-mediated neurotransmission, and the inhibitory components to GABAergic interneuronal processing. Our putative FSI data, together with previous reports 41,42 , corroborate this interneuronal participation, since perievent patterns from putative FSI and the main sample of mPFC units were opposite. Similar sequences of phasic excitation then inhibition possibly exist throughout the neocortex, with each neocortical area employing such pattern to a specific function. At least in the mPFC, the excitation-inhibition sequence is likely to organize its receptivity and refractoriness to limbic inputs, including thalamic ones 5,[43][44][45] .
Altogether, initial excitation, ensuing inhibition, and the slow re-excitation represent the temporal profile with which a proportion of mPFC cells react to CA1/sub stimuli. In addition, our work describes PV/MD reactions to CA1/sub pulses. As there are no known connections between CA1 pyramidal cells and the dorsal midline thalamus in rodents, the subicular recruitment could have been responsible for the PV/MD responses we observed 16,17,[46][47][48] . CA1/sub is commonly approached as an integrated source of hippocampal formation outputs 49 . Thus, nonspecific electrical stimulation of such area could explain the thalamic fPSP we observed, in addition to the proportion (>50%) of responsive PV/MD units. That being said, these PV/MD responses may reflect a thalamus-relayed pathway from hippocampal formation outputs to the mPFC, similarly to the circuit comprising CA1, mPFC, and the thalamic reuniens and rhomboid nuclei 33 . Other trans-thalamic routes are indeed found across the brain 32 . They contribute to efference copies, which participate in the self-monitoring of actions 50,51 , and are disturbed in schizophrenia 52 . Given the associative mPFC role, its connectivity with the CA1/sub and PV/MD areas could be important for the cognitive self-monitoring. Further investigation on the PV/MD recruitmente.g., using different paired-pulse protocols, or optical (rather than electrical) stimulation of CA1 or sub -could clarify these issues.
The limbic thalamic involvement in psychosis is evidenced by its response to locally infused MK-801 (non-competitive NMDA antagonist), which by itself replicates the drug's systemic effects on both spontaneous and subiculum-evoked mPFC activity 15 . Together with the post-mortem evidence of thalamic cell loss in schizophrenia 53 , this reinforces that the mPFC undergoes thalamus-related downstream alterations in psychosis. The midline thalamus is also increasingly implicated in the spread of limbic seizures 54 . Pharmacological inhibition of the MD has been shown to abbreviate subiculum-generated after-discharges in the mPFC, in addition to attenuating its subiculum-evoked responses 16,17 . Considering the thalamic participation in temporal lobe epilepsy 55,56 , the PV/MD might be important for the comorbidity between this neurological disease and psychotic symptoms 18 , a research topic that could benefit from approaches like those reported here.
In summary, this study depicts sub-second-range prefrontal cortical and midline thalamic responses upon electrical stimulation of the hippocampal formation in vivo. Specifically in the prefrontal cortex, we observed a secondary excitatory response that was potentiated after hippocampal HFS, and attenuated during thalamic optical drive. Thus, our findings further describe how hippocampal-prefrontal-thalamic interactions are timed, including interneuronal processing within the prefrontal cortex. Although exogenously induced, these patterns give further idea of what occurs in limbic circuits during cognitive operations and their dysfunctions. Based on our contribution, future studies combining behavioural monitoring, multiple-site recording, and co-stimulation designs may advance the dissection of prefrontal-mediated flows of information.

Methods
Subjects. Adult male Wistar rats were housed in bedded cages under 12 h light/dark cycle (lights on at 7 AM), with food/water ad lib and standard temperature. Procedures followed the Brazilian Council for Control of Animal Experimentation guidelines. The local bioethics committee (Ribeirão Preto School of Medicine) approved our procedures (128/2014).
Both the mPFC and PV/MD received handmade eight-channel microwire bundles for recording (teflon-coated tungsten, 50 μm). These probes allow multichannel recordings with simplicity of electrode making, but at the expense of lower anatomical precision (precluding the ability to assign channels to cortical layers, for example). In turn, the CA1/sub area received a bipolar electrode for monophasic stimulation (teflon-coated tungsten, 60 μm, ~500 μm inter-pole). For optimizing the CA1/sub coordinate, paired pulses were delivered through the bipolar electrode during its dorsal-ventral trajectory until fPSPs were consistently evoked.
Six microscrews, including a ground reference on the contralateral cerebellum, were fastened to the bone around the electrodes. The resulting miniature system was enclosed together on the skull with dental cement. Rats were then allowed to recover for 5-7 days.
Recording and electrical stimulation. For surgery recordings, mPFC and PV/MD electrodes were connected to an analog-digital converter (ADInstruments) via a battery-operated preamplifier, aiming at fPSP acquisition (1 kHz low-pass, 1000x gain, 2 kHz digitization) upon CA1/sub stimulation. Stimuli consisted of pairs of square pulses (200 μs, 300 μA, 80 ms interpulse interval) delivered every 10 s. Pulses were generated from a stimulator and photoelectrically isolated (Grass Technologies). This system was merely used for optimizing the CA1/ sub dorsal-ventral coordinate, as explained above. Reasons for probing the circuit with paired pulses were: (1) to ensure efficient afferent stimulation without increasing the current intensity; and (2) to differentiate between paired pulse-locked responses (dozens of milliseconds) and longer-latency responses.
For chronic sessions, rats were plugged into the stimulation/recording cables (including unity-gain headstages), and allowed to move freely in a soundproof box. Cables were connected to their devices (photoelectric isolator and preamplifier) on the outside of the box without using a commutator relay. We employed a multichannel acquisition processor (Plexon) with the following parameters. LFP: 0.7-500 Hz band-pass, 1000x gain, and 2 kHz digitization. MUA: 250-8000 Hz band-pass, 1000x gain, and 40 kHz digitization. CA1/sub stimulation was made as above (Fig. 1c), with intensity based on an input-output curve prior to recording (~200-400 μA, i.e., ~70% of maximum fPSP amplitude). LTP was induced by CA1/sub HFS: two series (10 min apart) of 10 trains (every 10 s), each train with 50 pulses at 250 Hz [11][12][13] .
For each paired pulse, a timestamp from the stimulator was sent to the multichannel system at 40 kHz digitization, allowing perievent analysis. Pulse artefacts expectedly contaminated the spike signal, but they could be removed through principal component analysis. Only then we proceeded to semiautomatic spike sorting, area percentage. Percentages were then examined for Spearman's rank correlations with electrophysiological values from the post-HFS period: (1) amplitudes of fPSP2; and (2) moduli of MUA responses. False positives were minimized through the False Discovery Rate adjustment 62 .
Data availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.