VinnikpersName, AntopolskiypersName, ItskovpersName, DiamondpersName (2012date). “Auditory stimuli elicit hippocampal neuronal responses during sleep.” Frontiers in Systems Neurosciencetitle·j.
Object of study — how hippocampal CA1term·key neurons respond to sounds that predict rewardterm·key, and whether they still respond outside the task
Research question — whether hippocampal sound responses survive when the animal is not engaged in a discrimination task, including during sleepterm
Contribution — distinguishes two kinds of hippocampal sound response - a short-latency onset responseterm present even in sleepterm, and a longer sound-specific responseterm·def tied to task engagement
phenomenon · claim-type · cross-domain-transfer · mechanism · claim-relation · replication-status · limitation — grounded in the full text| phenomenon | hippocampal neuronal responses to sounds during sleep — “In the current experiment we characterized responses of hippocampal neurons to behaviorally relevant sounds which were played during sleep and to awake passively listening rats.” | |
| claim-type | existence — “In this study we show that a significant subset of hippocampal neurons responds to sounds presented passively to a rat during both quiet wakefulness and during sleep.” | |
| method — evidence | — “To investigate how hippocampal neurons code behaviorally salient stimuli, we recorded from neurons in the CA1 region of hippocampus in rats while they learned to associate the presence of sound with water reward.” | |
| object of study — evidence | — “To investigate how hippocampal neurons code behaviorally salient stimuli, we recorded from neurons in the CA1 region of hippocampus in rats while they learned to associate the presence of sound with water reward.” | |
| research question — evidence | — “In the current experiment we characterized responses of hippocampal neurons to behaviorally relevant sounds which were played during sleep and to awake passively listening rats. In the behavioral task, the sounds cued the upcoming release of a reward. Outside the task, the sounds were unrelated to the animal's behavior.” | |
| contribution — evidence | — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | |
| transfer | from auditory psychophysics and speech research: artificial vowels with formant structure, imported as a more naturalistic stimulus class than pure tones — “We chose artificial vowels as a more “naturalistic” class of stimuli than pure tones.” | transfers[0] |
| mechanism | Thirst plus placement in a completely novel dim environment raises the animal's attention to the sounds, which is why hippocampal neurons responded here although they did not in the earlier over-trained, satiated condition — “In the present study the animals were thirsty during passive presentation and were placed in a completely novel dim environment, which could have facilitated attention to the auditory stimuli.” | mechanisms[0] |
| mechanism | Short-latency hippocampal responses are suppressed once the animal comes to expect the stimulus, so over-training or satiation abolishes them — “These responses are not present in the over-trained and/or satiated animals suggesting that they might disappear when the animal expects the stimuli.” | mechanisms[1] |
| mechanism | Sound-evoked CA1 responses are driven anatomically by perforant path terminals from entorhinal cortex, or polysynaptically through the Schaffer collaterals — “The evoked responses in the CA1 region may be generated by perforant path terminals, originating in the entorhinal cortex or polisynaptically via the Schaffer collaterals.” | mechanisms[2] |
| mechanism | Persistent sustained firing, of the kind described in entorhinal cortex, holds the stimulus trace after stimulus offset and thereby produces the long-lasting responses — “This memory trace could be stored by sustained activity, previously described in entorhinal cortex (Egorov et al., 2002 ), or by some other cellular or network mechanisms in higher order associative cortices or hippocampus itself.” | mechanisms[3] |
| mechanism | the lottery design holds position and motivational state constant between sound and no-sound trials, since both begin with the same nosepoke — “Trials with and without sound presentation were compared, assuring that the animal was in exactly the same position and motivational state in both types of trials.” | mechanisms[4] |
| mechanism | the 50% schedule was introduced specifically to raise reward salience and make the animal attend to the sounds — “On the fifth day of the training, the reinforcement schedule was changed to increase the salience of the reward and, especially, to make the rat attend to novel sounds associated with reward.” | mechanisms[5] |
| mechanism | long randomised inter-stimulus intervals of 25-30 s were used to reduce adaptation to repetition — “The long inter-stimulus interval was intended to reduce the animal's adaptation to stimulus repetitions” | mechanisms[6] |
| mechanism | responses are tested at four a-priori bin sizes and a neuron counts as responsive if any bin survives FDR correction — “To take into account the variability of the responses (e.g., fast transient or sustained responses), we used four a-priori defined bin sizes (50 ms, 500 ms, 1 and 2 s).” | mechanisms[7] |
| mechanism | only sleep trials that produced no visible behavioural response were analysed — “Only the trials where sounds did not cause any visible behavioral response from the sleeping animal were analyzed further.” | mechanisms[8] |
| contradicts | Itskov et al. (2012), which found no hippocampal neuronal responses to sounds outside the categorization task — “The absence of neuronal responses to sound stimuli outside the categorization task (Itskov et al., 2012 ) seems at the first glance inconsistent with the results of our current experiment, where we have found that hippocampal neurons responded to the sound stimuli presented during passive wakefulness.” | claim_relations[0] |
| builds-on | Brankack and Buzsaki (1986) on short-latency hippocampal responses to sounds — “Short latency responses to sounds in hippocampus have previously described in the literature (Brankack and Buzsaki, 1986 ).” | claim_relations[1] |
| extends | Rudoy et al. (2009) sustained EEG response to auditory cues presented to sleeping humans — “The latter finding might be related to the sustained EEG response found in Rudoy et al. ( 2009 ) after presentation of auditory stimuli to humans during sleep.” | claim_relations[2] |
| qualifies | the same group's earlier finding that hippocampal neurons do not respond to sounds outside a discrimination task — “However, in those experiments hippocampal neurons did not respond to sounds when played to the animal not engaged in a discrimination task. These recordings were performed in highly over trained animals and it is possible that some of the features of neuronal responses were absent due to repetition of the same stimuli over many thousands of trials.” | claim_relations[3] |
| supports | reports that hippocampal neurons represent non-spatial variables including sounds, textures, odours and taste — “To investigate how hippocampal neurons code behaviorally salient stimuli, we recorded from neurons in the CA1 region of hippocampus in rats while they learned to associate the presence of sound with water reward.” | claim_relations[4] |
| supports | the established spatial selectivity of hippocampal neurons, reproduced here as location coding at the two ports — “Forty-one percent of tested neurons (24 out of 58) discriminated between right and left nosepokes (see Figure 3A ; p < 0.05, corrected, rank sum test on firing rates 400 ms before the nose pokes, see Materials and Methods).” | claim_relations[5] |
| sense | two distinct senses of hippocampal sound response are proposed: a short-latency onset response, present even in sleep and not sound-specific, versus a longer sound-specific response that needs task engagement — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | senses[0] |
| sense | sound-responsive is defined by ANY of four a-priori bin sizes surviving FDR correction - a disjunctive criterion, so the label mixes fast transient and slow sustained responses — “A neuron was considered a “sound or reward-encoding” neuron if any of the time bins showed a corrected p -value smaller than 0.05.” | senses[1] |
| sense | passive here means a different room, different floor, different light and different smell - the novelty of the context is part of the manipulation, not a nuisance — “Outside the context of the behavioral task, passive exposures to the awake rat were done in an unfamiliar dark plastic bin (33 × 22 × 35 cm) which differed from the training environment by geometry, texture of the floor and the walls, the lighting, and the smell.” | senses[2] |
| sense | the earlier null from this group's own work is reinterpreted as an artefact of overtraining rather than as an absence of the phenomenon — “However, in those experiments hippocampal neurons did not respond to sounds when played to the animal not engaged in a discrimination task. These recordings were performed in highly over trained animals and it is possible that some of the features of neuronal responses were absent due to repetition of the same stimuli over many thousands of trials.” | senses[3] |
| control | animals allowed to continue testing to satiation and given ad libitum water afterwards — held — rules out dehydration from the water restriction as a driver of behaviour and of the hippocampal responses - a thirsty animal is differently motivated throughout, not only at the reward — “To ensure that the animals did not suffer dehydration as a consequence of water restriction, they were allowed to continue the behavioral testing to satiation” | controls[0] |
| note | onset response —distinct-from→ sound-specific — the paper's central proposal: two response types separated by latency, duration and whether task engagement is required — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | concept_edges[0] |
| weight | onset response —distinct-from→ sound-specific — 0.9 — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | concept_edges[0] |
| outcome (stated by the tagger, no quotation) | control — held | controls[0] |
| for which use | limitation — every neuronal claim in the paper: NEURONAL DATA SUITABLE FOR ANALYSIS WAS COLLECTED FROM TWO ANIMALS — “Neuronal data suitable for analysis was collected from two animals.” | limitations[0] |
| for which use | limitation — any single-neuron property across sessions: THE NUMBER OF RECOR[ded neurons] is too small, even though the same neurons were tracked — “Although we were able to keep the same neurons across the recording sessions, as evidenced by the neuronal waveforms, autocorrelations and firing rates, we feel that the number of recorded neurons does not allow us to make any conclusive statements regarding a single neuron's properties across time and context.” | limitations[1] |
| for which use | limitation — the interneuron question specifically: LIMITED SIZE OF OUR DATASET DOES NOT ALLOW US TO MAKE ANY FURTHER CONCLUSIONS ON THIS ISSUE — “Unfortunately, limited size of our dataset does not allow us to make any further conclusions on this issue.” | limitations[2] |
| for which use | limitation — the population the result speaks for, with the whole funnel visible: OUT OF THE SIX ANIMALS TRAINED ... FOUR WERE IMPLANTED ... DATA SUITABLE FOR ANALYSIS WAS COLLECTED FROM TWO — “Out of the six animals trained to perform the behavioral task, four were implanted using microdrives for chronic recordings. Neuronal data suitable for analysis was collected from two animals.” | limitations[3] |
| duplicate of | limitation — limitations[1] — “Although we were able to keep the same neurons across the recording sessions, as evidenced by the neuronal waveforms, autocorrelations and firing rates, we feel that the number of recorded neurons does not allow us to make any conclusive statements regarding a single neuron's properties across time and context.” | limitations[4] |
| for which use | limitation — the same scope as limitations[1] - single neurons are not tracked across contexts despite the waveforms being available, because the sample is too small — “Although we were able to keep the same neurons across the recording sessions, as evidenced by the neuronal waveforms, autocorrelations and firing rates, we feel that the number of recorded neurons does not allow us to make any conclusive statements regarding a single neuron's properties across time and context.” | limitations[4] |
| for which use | limitation — the interneuron identification of the long-response cells: they are LIKELY TO BE PUTATIVE INTERNEURONS BECAUSE OF THE NARROW SPIKE WIDTH AND THE ABSENCE OF THE 3-4 ms PEAK IN THE AUTOCORRELATION PLOT (DATA NOT SHOWN) — “Both of these neurons are likely to be putative interneurons because of the narrow spike width and the absence of the 3–4 ms peak in the autocorrelation plot (data not shown). Unfortunately, limited size of our dataset does not allow us to make any further conclusions on this issue.” | limitations[5] |
| for which use | limitation — any claim that the passive responses carry sound identity: NONE OF THE THREE SOUND-RESPONDING NEURONS DISCRIMINATED BETWEEN DIFFERENT SOUNDS — “None of the three sound-responding neurons discriminated between different sounds i.e., their responses to different sounds were identical.” | limitations[6] |
| for which use | limitation — the population every result generalises to: SIX MALE RATS WEIGHING ABOUT 350 g — “Six male rats weighing about 350 g were housed individually and maintained on a 14 h/10 h light/dark cycle.” | limitations[7] |
| replication | contested | |
| limitation | Usable neuronal data came from only two of the six trained animals. — “Neuronal data suitable for analysis was collected from two animals.” | limitations[0] |
| limitation | Too few neurons were recorded to conclude anything about a single neuron's properties across sessions and contexts. — “Although we were able to keep the same neurons across the recording sessions, as evidenced by the neuronal waveforms, autocorrelations and firing rates, we feel that the number of recorded neurons does not allow us to make any conclusive statements regarding a single neuron's properties across time and context.” | limitations[1] |
| limitation | The dataset is too small to draw further conclusions about whether the long-responding cells were putative interneurons. — “Unfortunately, limited size of our dataset does not allow us to make any further conclusions on this issue.” | limitations[2] |
| limitation | usable neuronal data came from two animals, out of six trained and four implanted — “Out of the six animals trained to perform the behavioral task, four were implanted using microdrives for chronic recordings. Neuronal data suitable for analysis was collected from two animals.” | limitations[3] |
| limitation | the authors decline to track single neurons across contexts despite having the waveforms, because the sample is too small — “Although we were able to keep the same neurons across the recording sessions, as evidenced by the neuronal waveforms, autocorrelations and firing rates, we feel that the number of recorded neurons does not allow us to make any conclusive statements regarding a single neuron's properties across time and context.” | limitations[4] |
| limitation | the putative-interneuron identification of the long-response cells is explicitly not pursued for the same reason — “Both of these neurons are likely to be putative interneurons because of the narrow spike width and the absence of the 3–4 ms peak in the autocorrelation plot (data not shown). Unfortunately, limited size of our dataset does not allow us to make any further conclusions on this issue.” | limitations[5] |
| limitation | none of the passively responding neurons distinguished between the sounds — “None of the three sound-responding neurons discriminated between different sounds i.e., their responses to different sounds were identical.” | limitations[6] |
| limitation | six male rats only — “Six male rats weighing about 350 g were housed individually and maintained on a 14 h/10 h light/dark cycle.” | limitations[7] |
finding · quantity · variable · definition · key-term — grounded in the full text| finding | Forty-one percent of the neurons recorded during the task (24 of 58) discriminated between right and left nosepokes. — “Forty-one percent of tested neurons (24 out of 58) discriminated between right and left nosepokes” | content_tags.findings[0] |
| finding | Eleven of 58 task-recorded cells responded within the first 300 ms after sound onset. — “Out of 58 cells recorded in the task, 11 cells responded in the first 300 ms upon sound presentation” | content_tags.findings[1] |
| finding | Sound-responsive and reward-responsive populations were completely non-overlapping: no single cell responded to both events. — “There were no cells with responses to both sound and reward.” | content_tags.findings[2] |
| finding | During awake passive conditions, 3 of 25 recorded hippocampal neurons were classified as sound-sensitive. — “Three out of 25 neurons recorded during the awake passive conditions were classified as sound-sensitive” | content_tags.findings[3] |
| finding | During sleep, 8 of 33 recorded neurons (24%) showed significant sound-evoked responses. — “Twenty-four percent of recorded neurons (8 out of 33) exhibited significant sound-evoked responses.” | content_tags.findings[4] |
| finding | Response latencies during sleep fell between 50 and 150 ms after sound onset. — “Across neurons, latency ranged from 50 to 150 ms.” | content_tags.findings[5] |
| finding | Long-lasting responses to sounds occurred only during sleep. — “Long lasting responses to sounds were unique to responses recorded during sleep (Figure 6 ).” | content_tags.findings[6] |
| finding | Almost no neuron distinguished which sound was played: only one cell showed sound-identity-dependent responses across the two vowels and the novel noise. — “We found only one neuron with significant sound identity-dependent responses ( p = 0.0101, Kruskal–Wallis test) comparing responses to three sounds, two artificial vowels and the novel noise-like sound.” | content_tags.findings[7] |
| finding | Waiting time on unrewarded trials decreased over the session, suggesting the sound had become associated with reward and its absence a cue to switch ports. — “Importantly, in the “unlucky” trials the animals decreased waiting time over the course of the session, suggesting that the occurrence of the sound became associated with the water reward and absence of sound became a cue to go to the alternate nose poke (see Results).” | content_tags.findings[8] |
| finding | A significant subset of hippocampal neurons responds to passively presented sounds in both quiet wakefulness and sleep. — “In this study we show that a significant subset of hippocampal neurons responds to sounds presented passively to a rat during both quiet wakefulness and during sleep.” | content_tags.findings[9] |
| finding | 41% of neurons, 24 of 58, discriminated between the two nose pokes — “Forty-one percent of tested neurons (24 out of 58) discriminated between right and left nosepokes (see Figure 3A ; p < 0.05, corrected, rank sum test on firing rates 400 ms before the nose pokes, see Materials and Methods).” | content_tags.findings[10] |
| finding | 11 of 58 cells responded within 300 ms of sound onset and 14 responded at reward, with no overlap between the two sets — “Out of 58 cells recorded in the task, 11 cells responded in the first 300 ms upon sound presentation (19%, p < 0.05, rank sum tests done in 50 ms windows, corrected for testing six time points and also four different bin sizes, see Materials and Methods). Fourteen (24%) responded upon the reward delivery (Figure 3B ). There were no cells with responses to both sound and reward.” | content_tags.findings[11] |
| finding | three of 25 neurons were classified as sound-sensitive during passive wakefulness — “Three out of 25 neurons recorded during the awake passive conditions were classified as sound-sensitive (see Materials and Methods, p -values were 0.0029, 0.0000016, and 0.01, corrected).” | content_tags.findings[12] |
| finding | eight of 33 neurons responded during sleep, five decreasing and three increasing — “Twenty-four percent of recorded neurons (8 out of 33) exhibited significant sound-evoked responses. Across neurons, latency ranged from 50 to 150 ms. In five cases firing rate decreased after sound presentation, in three cases the firing rate increased.” | content_tags.findings[13] |
| finding | long-lasting responses were unique to sleep, where responses were almost twice as long and began slightly earlier — “Long lasting responses to sounds were unique to responses recorded during sleep (Figure 6 ). The responses to sounds during sleep were on average almost twice as long as the response to sounds presented during passive wakefulness (Figure 6F ). Responses to sounds during sleep also appeared slightly earlier than the responses during passive wakefulness (Figure 6E ).” | content_tags.findings[14] |
| finding | none of the passively responsive neurons distinguished between the sounds — “None of the three sound-responding neurons discriminated between different sounds i.e., their responses to different sounds were identical.” | content_tags.findings[15] |
| finding | waiting time on unrewarded trials fell across the session, showing the animals learned the sound-reward contingency — “The average time of waiting in no-sound-no-reward trials was 5.09 ± 0.19 s (SEM). Waiting time decreased over the course of the session ( p < 0.001, paired-sample t -test).” | content_tags.findings[16] |
| quantity | 24 out of 58 — neurons that discriminated between the right and left nosepoke (41% of tested neurons) — “Forty-one percent of tested neurons (24 out of 58) discriminated between right and left nosepokes” | content_tags.quantities[0] |
| quantity | 24% — proportion of neurons recorded during sleep (8 of 33) with significant sound-evoked responses — “Twenty-four percent of recorded neurons (8 out of 33) exhibited significant sound-evoked responses.” | content_tags.quantities[1] |
| quantity | 14 (24%) — task-recorded cells that responded at the moment of water reward delivery — “Fourteen (24%) responded upon the reward delivery (Figure 3B ).” | content_tags.quantities[2] |
| quantity | 5.09 ± 0.19 s — mean (± SEM) time the rats waited in the nosepoke on trials with no sound and no reward — “The average time of waiting in no-sound-no-reward trials was 5.09 ± 0.19 s (SEM). Waiting time decreased over the course of the session ( p < 0.001, paired-sample t -test).” | content_tags.quantities[3] |
| quantity | 50 to 150 ms — range of sound-response latencies across neurons recorded during sleep — “Across neurons, latency ranged from 50 to 150 ms.” | content_tags.quantities[4] |
| quantity | 1–2 s — duration of the long-lasting sound responses observed during sleep — “During sleep the short-latency responses in hippocampus are intermingled with long lasting responses which in the current experiment could last for 1–2 s.” | content_tags.quantities[5] |
| quantity | 0.2 ms — spike width (measured at 25% of spike amplitude) of the two example sleep-responsive neurons, suggesting putative interneurons — “Both neurons depicted on this figure had narrow spikes (Figures 5E and J ), 0.2 ms when measured on 25% of spike amplitude.” | content_tags.quantities[6] |
| quantity | two animals — number of implanted rats (out of six trained, four implanted) that yielded neuronal data suitable for analysis — “Neuronal data suitable for analysis was collected from two animals.” | content_tags.quantities[7] |
| quantity | 8% during passive wakefulness and 25% during sleep, as stated in the ABSTRACT — the abstract's summary percentages - see the results rows below, which do not reconstruct the 8% without assuming a narrower denominator — “When the sounds were played during passive wakefulness, 8% of neurons responded with short latency onset responses; 25% of neurons responded to sounds when they were played during sleep.” | content_tags.quantities[8] |
| quantity | three out of 25 neurons, which is 12% — the passive-wakefulness result as given in the RESULTS section — “Three out of 25 neurons recorded during the awake passive conditions were classified as sound-sensitive (see Materials and Methods, p -values were 0.0029, 0.0000016, and 0.01, corrected).” | content_tags.quantities[9] |
| quantity | two increased and one decreased — the direction split among those three - counting only the increases would give 2 of 25, which IS 8% — “Two of the neurons showed a significantly increased firing rate in response to the sound, and one a decreased the firing rate.” | content_tags.quantities[10] |
| quantity | 24%, eight out of 33 — the sleep result in the RESULTS section, against 25% in the abstract — “Twenty-four percent of recorded neurons (8 out of 33) exhibited significant sound-evoked responses. Across neurons, latency ranged from 50 to 150 ms. In five cases firing rate decreased after sound presentation, in three cases the firing rate increased.” | content_tags.quantities[11] |
| quantity | six trained, four implanted, two yielding usable data — the animal attrition — “Out of the six animals trained to perform the behavioral task, four were implanted using microdrives for chronic recordings. Neuronal data suitable for analysis was collected from two animals.” | content_tags.quantities[12] |
| quantity | 5.09 plus or minus 0.19 s — average waiting time on no-sound trials — “The average time of waiting in no-sound-no-reward trials was 5.09 ± 0.19 s (SEM). Waiting time decreased over the course of the session ( p < 0.001, paired-sample t -test).” | content_tags.quantities[13] |
| variable | types of sound stimuli (independent) — “Three types of sound stimuli (familiar vowel associated with the right well, familiar vowel associated with a left well, and a novel noise-like sound) were presented pseudo-randomly, so that no sound was repeated more than two times in a row.” | content_tags.variables[2] |
| variable | firing rate (dependent) — “Wilcoxon rank-sum test for two independent samples was used to compare the firing rates, bin by bin, in “lucky” trials versus not reward trials.” | content_tags.variables[3] |
| variable | waiting time (dependent) — “The average time of waiting in no-sound-no-reward trials was 5.09 ± 0.19 s (SEM). Waiting time decreased over the course of the session ( p < 0.001, paired-sample t -test).” | content_tags.variables[4] |
| variable | motivational state (control) — “Trials with and without sound presentation were compared, assuring that the animal was in exactly the same position and motivational state in both types of trials.” | content_tags.variables[5] |
| variable | inter-stimulus interval (control) — “The long inter-stimulus interval was intended to reduce the animal's adaptation to stimulus repetitions (Ulanovsky et al., 2004 ).” | content_tags.variables[6] |
| variable | sound presentation — “Trials with and without sound presentation were compared, assuring that the animal was in exactly the same position and motivational state in both types of trials.” | content_tags.variables[7] |
| definition | "location-encoding" neuron — “A neuron was considered to distinguish between the two nose pokes and therefore to be a “location-encoding” neuron if any of the time bins showed a corrected p -value smaller than 0.05.” | content_tags.definitions[0] |
| definition | sound-responsive neuron — “A neuron was considered sound-responsive if any of the time bins showed a corrected p -value smaller than 0.05.” | content_tags.definitions[1] |
| definition | latency of the response — “Latency of the responses was calculated as time of the first bin after sound onset with activity significantly different from baseline.” | content_tags.definitions[2] |
| definition | "lottery" task — “During the recording sessions (referred to as “lottery,” see below) the sounds were presented with 50% probability for a given nose poke entry.” | content_tags.definitions[3] |
| definition | two types of hippocampal neuronal responses to sounds — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | content_tags.definitions[4] |
| definition | sound-onset responses — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | content_tags.definitions[7] |
| definition | sound-specific responses — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | content_tags.definitions[8] |
| key-terms | hippocampus, CA1, hippocampal neurons, artificial vowels, nose poke, reward, passive listening, sleep, onset response, latency, tetrode, false discovery rate, adaptation, interneurons, spatial selectivity, lottery, water reward, novel, sound-evoked, peri-stimulus |
| concept | artificial vowels — not defined in this paper's own words; the stimuli, chosen for naturalness — “We chose artificial vowels as a more “naturalistic” class of stimuli than pure tones.” | concepts[2] |
| concept | nose poke — not defined in this paper's own words; the trial-initiating action and the location variable — “The animal initiated each trial by poking its snout into a nose poke.” | concepts[3] |
| concept | false discovery rate — not defined in this paper's own words; the multiple-comparison correction — “Benjamini–Hochberg false discovery rate corrections were used to correct for multiple comparisons (FDR, Benjamini and Hochberg, 1995 ).” | concepts[8] |
| concept | adaptation — not defined in this paper's own words; what the long inter-stimulus interval guards against — “The long inter-stimulus interval was intended to reduce the animal's adaptation to stimulus repetitions (Ulanovsky et al., 2004 ).” | concepts[10] |
| Wilcoxon rank-sum test | as written: Wilcoxon rank-sum test for two independent samples — “Firing rates in each time bin were compared using Wilcoxon rank-sum test for two independent samples (Siegel and Castellan, 1988 ).” | content_tags.statistics[0] |
| false discovery rate correction for multiple comparisons FDR | as written: Benjamini–Hochberg false discovery rate corrections — “Benjamini–Hochberg false discovery rate corrections were used to correct for multiple comparisons (FDR, Benjamini and Hochberg, 1995 ).” | content_tags.statistics[1] |
| sign test | as written: sign test for dependent measurements — “Firing rate values in each of them were compared to firing rate values in 50 ms baseline (sign test for dependent measurements, Siegel and Castellan, 1988 ).” | content_tags.statistics[2] |
| paired t-test p | as written: paired-sample t -test — “The average time of waiting in no-sound-no-reward trials was 5.09 ± 0.19 s (SEM). Waiting time decreased over the course of the session ( p < 0.001, paired-sample t -test).” | content_tags.statistics[3] |
| Kruskal-Wallis test p | — “We found only one neuron with significant sound identity-dependent responses ( p = 0.0101, Kruskal–Wallis test) comparing responses to three sounds, two artificial vowels and the novel noise-like sound.” | content_tags.statistics[4] |
| standard error of the mean ± | as written: (SEM) — “The average time of waiting in no-sound-no-reward trials was 5.09 ± 0.19 s (SEM). Waiting time decreased over the course of the session ( p < 0.001, paired-sample t -test).” | content_tags.statistics[5] |
| median and interquartile range (box plot) | as written: the central mark is the median, the edges of the box are the 25th and 75th percentiles — “On each box, the central mark is the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points not considering outliers; outliers are plotted individually as dots.” | content_tags.statistics[6] |
| cluster analysis (automatic spike sorting) | as written: presorted automatically using KlustaKwik — “Spikes were presorted automatically using KlustaKwik (Harris et al., 2000 ), using waveform energy on each of the four channels of the tetrode as coordinates in a 4 dimensional feature space.” | content_tags.statistics[7] |
| Wilcoxon rank-sum test in 50 ms bins | — “Forty-one percent of tested neurons (24 out of 58) discriminated between right and left nosepokes (see Figure 3A ; p < 0.05, corrected, rank sum test on firing rates 400 ms before the nose pokes, see Materials and Methods).” | content_tags.statistics[8] |
| Benjamini-Hochberg false discovery rate | — “Benjamini–Hochberg false discovery rate corrections were used to correct for multiple comparisons” | content_tags.statistics[9] |
| four a-priori bin sizes with a disjunctive criterion | — “To take into account the variability of the responses (e.g., fast transient or sustained responses), we used four a-priori defined bin sizes (50 ms, 500 ms, 1 and 2 s).” | content_tags.statistics[10] |
| claim type | existence — a second reading reaching the same value; recorded: existence — “Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to sounds: sound-onset responses with very short latency and longer-lasting sound-specific responses that are likely to be present when the animal is actively engaged in the task.” | alternatives.claim_type |
| method | tetrode recording from CA1 during a 50% reward lottery task with sound cues, then passive listening in a novel box and during sleep — a second reading DIFFERING from the recorded value; recorded: electrophysiology — “Rats learned to alternate between two reward ports at which, in 50% of the trials, sound stimuli were presented followed by water reward after a 3-s delay. Sound at the water port predicted subsequent reward delivery in 100% of the trials and the absence of sound predicted reward omission.” | alternatives.method |
| phenomenon | non-spatial sensory coding in hippocampus — a second reading DIFFERING from the recorded value; recorded: hippocampal neuronal responses to sounds during sleep — “To investigate how hippocampal neurons code behaviorally salient stimuli, we recorded from neurons in the CA1 region of hippocampus in rats while they learned to associate the presence of sound with water reward.” | alternatives.phenomenon |
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Abstract. To investigate how hippocampal neuronsterm·key code behaviorally salient stimuli, we recorded from neurons in the CA1term·key region of hippocampusterm in rats while they learned to associate the presence of sound with water rewardterm·key. Rats learned to alternate between two rewardterm·key ports at which, in 50% of the trials, sound stimuli were presented followed by water rewardterm·key after a 3-s delay. Sound at the water port predicted subsequent rewardterm·key delivery in 100% of the trials and the absence of sound predicted rewardterm·key omission. During this task, 40% of recorded neurons fired differently according to which of the two rewardterm·key ports the rat was visiting. A smaller fraction of neurons demonstrated onset responseterm to sound/nosepoke (19%) and rewardterm·key delivery (24%). When the sounds were played during passive wakefulness, 8% of neurons responded with short latencyterm·key onset responsesterm; 25% of neurons responded to sounds when they were played during sleepterm. During sleepterm the short-latency responses in hippocampusterm are intermingled with long lasting responses which in the current experiment could last for 1–2 s. Based on the current findings and the results of previous experiments we described the existence of two types of hippocampal neuronal responses to soundsterm·def: sound-onset responsesterm·def with very short latencyterm·key and longer-lasting sound-specific responsesterm·def that are likely to be present when the animal is actively engaged in the task. Introduction Hippocampusterm is located on top of the cortical hierarchy (Felleman and van Essen, 1991 ; Burwell et al., 1995 ; Burwell and Amaral, 1998 ; Vertes, 2006 ; van Strien et al., 2009 ) and is necessary for the formation of new episodic memories (Scoville and Milner, 1957 ; Aggleton and Brown, 1999 ; Eacott and Norman, 2004 ; Cipolotti and Bird, 2006 ). It is also necessary for the formation of spatial memory and navigation (Morris et al., 1982 ). During the last 40 years of study of the firing properties of hippocampal neuronsterm·key, knowledge about the spatial variables that drive hippocampal firing became extensive in both phenomenology and details (O'Keefe, 1976 ; O'Keefe and Nadel, 1978 ; Muller and Kubie, 1987 ; Kubie et al., 1990 ; O'Keefe and Recce, 1993 ; Wilson and McNaughton, 1993 ; O'Keefe and Burgess, 1996 ; Wood et al., 1999 ; Jensen and Lisman, 2000 ; Anderson and Jeffery, 2003 ; Wills et al., 2005 ; Leutgeb et al., 2005a , b ; Colgin et al., 2008 ). Non-spatial variables are also represented by hippocampal neuronsterm·key and studies using various sensory modalities demonstrate that hippocampal neuronsterm·key respond to sounds (Berger et al., 1976 ; Christian and Deadwyler, 1986 ; Sakurai, 2002 ; Moita et al., 2003 ; Takahashi and Sakurai, 2009 ; ItskovpersName et al., 2012 ), textures (ItskovpersName et al., 2011 ), odors (Wood et al., 1999 ; Wiebe and Staubli, 1999 , 2001 ; Komorowski et al., 2009 ), and gustatory cues (Ho et al., 2011 ). The majority of placeterm unrelated sensory responses in hippocampusterm have been demonstrated in animals actively engaged in a behavioral task. We recently developed a categorization task (ItskovpersName et al., 2011 , 2012 ) which allowed us to demonstrate that hippocampal neuronsterm·key discriminate between sounds. However, in those experiments hippocampal neuronsterm·key did not respond to sounds when played to the animal not engaged in a discrimination task. These recordings were performed in highly over trained animals and it is possible that some of the features of neuronal responses were absent due to repetition of the same stimuli over many thousands of trials. In the current experiment we characterized responses of hippocampal neuronsterm·key to behaviorally relevant sounds which were played during sleepterm and to awake passively listening rats. In the behavioral task, the sounds cued the upcoming release of a rewardterm·key. Outside the task, the sounds were unrelated to the animal's behavior. Materials and methods Ethics statement All experiments were conducted in accordance with standards for the care and use of animals in research outlined in European Directive 2010/63/EU, and were supervised by a consulting veterinarian. Subjects Six male rats weighing about 350 g were housed individually and maintained on a 14 h/10 h light/dark cycle. Animals were placed on water-restricted diet 1 day prior to the beginning of the experiments. To ensure that the animals did not suffer dehydration as a consequence of water restriction, they were allowed to continue the behavioral testing to satiation and were given access to ad lib drinking water for 1 h after the end of each recording session. The animals' body weight and general state of health was monitored throughout the experiments. Out of the six animals trained to perform the behavioral task, four were implanted using microdrives for chronic recordings. Neuronal data suitable for analysis was collected from two animals. Stimuli The stimuli were chosen so that they were short sounds, easily perceived by rats. We chose artificial vowelsterm as a more “naturalistic” class of stimuli than pure tones. Artificial vowelsterm are a simplified version of vocalization sounds used by many species of mammals, and have been used in studies of the ascending auditory pathway from the auditory nerve (Cariani and Delgutte, 1996 ; Holmberg and Hemmert, 2004 ) to the auditory cortex (Bizley et al., 2009 , 2010 ). The spectra of natural vowels are characterized by “formant” peaks which result from resonances in the vocal tract of the vocalizing animal (Schnupp et al., 2011 ). Formant peaks therefore carry information about both the size and the configuration of the vocal tract, and human listeners readily categorize vowels according to vowel type (e.g., /a/ vs /o/, Peterson and Barney, 1952 ) as well as according to speaker type (e.g., male vs. female voice) or speaker identity (Gelfer and Mikos, 2005 ). Many species of animals, including rats (Eriksson and Villa, 2006 ), chinchillas (Burdick and Miller, 1975 ), cats (Dewson, 1964 ), monkeys (Kuhl, 1991 ), and many bird species (Kluender et al., 1987 ; Dooling and Brown, 1990 ) readily learn to discriminate synthetic vowels. The stimuli were generated using binary click trains at the fundamental frequency of 330 Hz and 100 ms duration which were bandpass filtered with a bandwidth equal to 1/50th of the formant frequency using Malcolm Slaney's Auditory Toolbox. Formant center frequencies were 6.912 and 15.008 kHz, respectively. The spectra of the stimuli are shown in Figure 1B . The formant frequencies chosen here lie within rat and human sensitivity range (Heffner et al., 1994 ). As demonstrated in our previous experiments (ItskovpersName et al., 2012 ) the rats can perceive and discriminate similar artificial vowelsterm. A third, control sound used in passive recordings as a novelterm·key stimulus was a naturalistic noise-like sound (see the spectrum in Figure 1B ). The stimuli were ramped on and off (supplementary audio files 1–2). They were presented at a sound level of 70 dB SPL, from a speaker located above the experimental apparatus. Figure 1 Apparatus and the stimuli. (A) Experimental apparatus, view from the top. Nose pokesterm are marked with arrows. A water well was positioned behind each nose poketerm. (B) Frequency spectrum of the two artificial sounds used in the behavioral sound (blue and red) and a control noise-like sound also used in passive listeningterm experiments. Sounds were presented through Visaton FRS 8 speaker, which has a flat frequency response (<±2 dB) between 200 Hz and 10 kHz. The speaker was driven through a standard PC sound card controlled by LabView (National Instruments, Austin, TX, USA). Apparatus The arena for the behavioral task consisted of a dim-lit sound-attenuated box (35 × 30 × 40 cm). One of the walls had two round holes (called “nose pokesterm” throughout the text, see Figure 1A ). A water well was placed behind each of the nose pokesterm. Infrared sensors at the edge of the nose pokesterm monitored the rat's entry and exit from the holes. Outside the context of the behavioral task, passive exposures to the awake rat were done in an unfamiliar dark plastic bin (33 × 22 × 35 cm) which differed from the training environment by geometry, texture of the floor and the walls, the lighting, and the smell. For recordings during the sleepterm we used a tall box (26 × 20 × 60 cm) lined with thick cotton tissue. Light sensors, sound, and water delivery were controlled by a custom-written LABview script, operating a National Instruments card (National Instruments Corporation, Austin, TX, USA). Surgery and electrophysiological recordings The rats were implanted with chronic recording electrodes. They were anesthetized with a mixture of Zoletil (30 mg/kg) and Xylazine (5 mg/kg) delivered i.p. A craniotomy was made above left dorsal hippocampusterm, centered 3.0 mm posterior to bregma and 2.5 mm lateral to the midline. A Neuralynx “Harlan 12” microdrive loaded with 12 tetrodesterm·key (25 μm diameter Pl/Ir wire, California Fine Wire) was mounted over the craniotomy. Tetrodesterm·key were loaded perpendicular to the brain surface and individually advanced in small steps of 40–80 μm, until they reached the CA1term·key area, indicated by the amplitude and the shape of the sharp wave/ripples. After surgery, animals were given antibiotic (Baytril; 5 mg/kg delivered through the water bottle) and the analgesic caprofen (Rimadyl; 2.5 mg/kg, subcutaneous injection) for postoperative analgesia and prophylaxis against infection, and were allowed to recover for one week to 10 days after surgery before training started. After recovery from electrode implantation the animals were trained, during which neuronal responses were recorded from the tetrodeterm·key array using TDT data acquisition equipment (RZ2, Tucker and Davis Technologies, Alachua, FL, USA). We usually recorded from 1 to 4 single units per tetrodeterm·key. Spikes were presorted automatically using KlustaKwikterm·stat (Harris et al., 2000 ), using waveform energy on each of the four channels of the tetrodeterm·key as coordinates in a 4 dimensional feature space. The result of the automatic clustering was inspected visually after importing the data into MClust (kindly provided by A. D. Redish). Each single unit was recorded only in one session. Behavioral task The task was designed in a way that allowed us to examine neuronal responses to behaviorally relevant auditory stimuli. We took into account the richness of location-related neuronal signals in hippocampusterm that are known to influence sensory responses recorded in different spatial locations (ItskovpersName et al., 2012 ). The animal initiated each trial by poking its snout into a nose poketerm. During the recording sessions (referred to as “lotteryterm·key,” see below) the sounds were presented with 50% probability for a given nose poketerm entry. Trials with and without sound presentationterm·var were compared, assuring that the animal was in exactly the same position and motivational stateterm·var in both types of trials. Pre-training The day prior to behavioral testing, animals were placed on a water restricted dry food diet. To accustom the animals to the set-up and the task they were given four pre-training sessions with 100% rewardterm·key probability (Figure 2A ). During these four sessions the rats learned to alternate between two water spouts, collecting a rewardterm·key at each site. After receiving water in one of the spouts, the rat had to go to the second spout to receive rewardterm·key, and then back to the first spout. Attempts to collect water from the same spout twice were neither rewarded nor punished. Initially water rewardterm·key was delivered immediately upon the nose poketerm. During the following sessions, rewardterm·key was delayed by 1 s on each successive session. Thus, by the end of fourth pre-training session, delay was 3 s. At this point, the rat was introduced to the main “lottery” taskterm·def (see below). The rats typically started to perform the alternation task at the end of the first or beginning of the second session. By the end of the fourth session, rats reached stable performance of ~300 trials per session (206–510, mean 309). No sounds were played during the pre-training. Figure 2 Behavioral task. (A) Pre-training. The animal had to collect water rewardterm·key by continuously alternating between the two nose pokesterm (right and left). During the first pre-training session water was delivered instantly upon nose poketerm entry. The delay between the nose poketerm and water delivery increases during subsequent sessions (see Materials and Methods). (B) “Lottery” taskterm·def: now water was delivered only in 50% of the trials. This sequence of pictures outlines the case when the animal had a “lucky” trial in one of the nose poketerm. Other “unlucky” trials have neither sound nor rewardterm·key, the animal proceeded to the opposite nose poketerm (see Materials and Methods). “Lotteryterm·key” On the fifth day of the training, the reinforcement schedule was changed to increase the salience of the rewardterm·key and, especially, to make the rat attend to novelterm·key sounds associated with rewardterm·key. Now the water was delivered only on 50% (not 100%, as before) of nose pokesterm (Figure 2B ). The rat still had to alternate between right and left nose pokesterm and wait for at least 3 s for the water rewardterm·key delivery. Whether the current trial would turn out to be “lucky” or “unlucky” was determined randomly by software. On “lucky” trials, a sound was played immediately upon entry into the nose poketerm, predicting the upcoming rewardterm·key. There were two sounds used in the behavioral task (two artificial vowelsterm, Figure 1B ), each of them associated with just one of the rewardterm·key spouts. This task was designed so that we could examine neuronal responses to sound presentationterm·var in awake behaving animals. Importantly, in the “unlucky” trials the animals decreased waiting timeterm·var over the course of the session, suggesting that the occurrence of the sound became associated with the water rewardterm·key and absence of sound became a cue to go to the alternate nose poketerm (see Results). Passive listeningterm To record hippocampal responses to sounds in a context unrelated to the behavioral task we placed the rat in a novelterm·key environment 2–3 h after completion of the behavioral session. Three types of sound stimuliterm·var (familiar vowel associated with the right well, familiar vowel associated with a left well, and a novelterm·key noise-like sound) were presented pseudo-randomly, so that no sound was repeated more than two times in a row. Inter-stimulus intervalterm·var varied randomly from 25 to 30 s. The sounds were presented irrespectively of the animal's action and location. Each stimulus type was presented 43 times on average. The long inter-stimulus intervalterm·var was intended to reduce the animal's adaptationterm to stimulus repetitions (Ulanovsky et al., 2004 ). Passive sound presentationterm·var during sleepterm started 5 min after the animal had fallen asleep, as determined by total immobility. The animal was monitored continuously via remote video camera. As was the case during the session in which the rat remained awake in a novelterm·key environment, three types of sound stimuliterm·var (familiar vowel associated with the right spout, familiar vowel associated with a left spout, and a novelterm·key noise-like sound) were presented pseudo-randomly, so that no sound repeated more than two times in a raw. Inter-stimulus intervalterm·var varied randomly from 25 to 30 s. Each stimulus type was presented 49 times on average. To ensure the proper timing of the responses, we recorded a copy of the signal sent to the loudspeaker along with the neuronal signals. Statistical procedures Firing ratesterm·var of hippocampal neuronsterm·key typically vary over the course of a single trial, since they tend to respond to different aspects of the task. The timing and the latency of the responseterm·def can vary across individual cells. Taking this into account, to quantify the responses to sounds we used a method intended to (1) take into account multiple time points, (2) provide a single measure of statistical significance of the effect, (3) take into account variability in the timescale of the responses, fast in some cases (on the order of tens of milliseconds, Brankack and Buzsaki, 1986 ) and slow (hundreds of milliseconds) in others. Statistical significance of neuronal responses in the behavioral task To quantify firing rateterm·var changes related to animal's location in space we measured firing rateterm·var in non-overlapping 50 ms bins during the approach to the nose pokesterm (see Figure 3A ). Firing ratesterm·var in each time bin were compared using Wilcoxon rank-sum test for two independent samplesterm·stat (Siegel and Castellan, 1988 ). Benjamini–Hochberg false discovery rate correctionsterm·stat were used to correct for multiple comparisons (FDR, Benjamini and Hochberg, 1995 ). A neuron was considered to distinguish between the two nose pokesterm and therefore to be a “location-encoding” neuronterm·def if any of the time bins showed a corrected p -value smaller than 0.05. Figure 3 Encoding of spatial and sensory features of the task. Each panel with a peri-stimulusterm·key histogram and a raster plot shows responses of an individual neuron. On each plot zero time corresponds to nose poketerm, upon which in “lucky” trials a sound was triggered. On plots B – D line at 3 s corresponds to rewardterm·key delivery in “lucky” trials. In unlucky trials the animal always stayed in the nose poketerm for at least 3 s (see Results). (A) Location-responsive neuron. (B) Responses of reward-responsive neuron during nosepokes to the left nosepoke. This neuron responded upon water delivery (at 3 s). (C) Responses at the right nosepoke of the sound-responsive neuronterm·def. This neuron responded to the sound presentationterm·var. (D) Responses at the right nosepoke of sound-responsive neuronterm·def. This neuron responded to the nosepoke, but fired at higher frequency when the sound was presented. All of the demonstrated neurons showed very similar robust responses at the opposite nosepokes, but fired at smaller peak rates (data not shown). Bin size for peri-stimulusterm·key histograms is 200 ms with 190 ms overlap. To quantify a neuron's responses to sound presentationterm·var and water rewardterm·key delivery during the behavioral task, we compared firing ratesterm·var in the “lucky” trials to firing ratesterm·var in trials in which no sound was played and no water delivered. To take into account the variability of the responses (e.g., fast transient or sustained responses), we used four a-priori defined bin sizes (50 ms, 500 ms, 1 and 2 s). In case of 50 ms bins, we measured firing rateterm·var in six non-overapping bins (0–300 ms). Wilcoxon rank-sum test for two independent samplesterm·stat was used to compare the firing ratesterm·var, bin by bin, in “lucky” trials versus not rewardterm·key trials. Benjamini–Hochberg FDR correction were used to correct for testing multiple time points ( n = 6 in 50 ms bins) and bin sizes (50 ms, 500 ms, 1 and 2 s, FDR, Benjamini and Hochberg, 1995 ). A neuron was considered a “sound or reward-encoding” neuron if any of the time bins showed a corrected p -value smaller than 0.05. Statistical significance of sound-evokedterm·key responses in passive listeningterm and during sleepterm To quantify sound-evokedterm·key responses during passive presentation, we compared post-stimulus firing rateterm·var with the baseline. To take into account the variability of responses we again used four a-priori defined bin sizes (50 ms, 500 ms, 1 and 2 s). For each analysis, baseline firing ratesterm·var were defined as the firing rateterm·var in the time immediately preceding the presentation of the sounds. We used baseline bin size equivalent to the time bin size used to measure evoked activity (50 ms, 500 ms, 1 or 2 s). In the case of the smallest bin size, firing rateterm·var was calculated in 50 ms non-overlapping bins in first 300 ms after sound onset, yielding six individual time points. Firing rateterm·var values in each of them were compared to firing rateterm·var values in 50 ms baseline (sign test for dependent measurementsterm·stat, Siegel and Castellan, 1988 ). This yielded six tests for each neuron, which were corrected for using FDR. Latency of the responsesterm·def was calculated as time of the first bin after sound onset with activity significantly different from baseline. In the case of 500 ms bins, we took a mean firing rateterm·var in 500 ms baseline and compared it with mean firing rateterm·var in the 500 ms post-stimulus interval. In the case of 1 s bins we took a mean firing rateterm·var in 1 s baseline and compared it with mean firing rateterm·var in 1 s post-stimulus interval. In the case of 2 s bins we took a mean firing rateterm·var in 2 s baseline and compared it with mean firing rateterm·var in 2 s post-stimulus. This yielded four measurements for each neuron: p -value for 50 ms bins (already corrected for comparing multiple bins), and p -values for 500 ms, 1 and 2 s bins. These p -values were corrected for multiple testing using FDR. A neuron was considered sound-responsive if any of the time bins showed a corrected p -value smaller than 0.05. For the purposes of illustration, the firing rateterm·var was calculated in a 50 ms wide window sliding in steps of 25 ms along the whole duration of the trial (see Figures 3 – 4 ). Figure 4 Short lasting neuronal responses to passive sound presentationterm·var during quiet wakefulness and sleepterm. Each panel with a peri-stimulusterm·key histogram and a raster plot shows responses of an individual neuron. Zero time corresponds to the time of sound presentationterm·var. Trials with three different sounds are pooled together (vowel 1, vowel 2 and a novelterm·key noise-like sound). (A) Neuron responsive to sound presentationterm·var during passive listeningterm. (B – D) Neurons responsive to sound presentationterm·var during sleepterm. Bin size for panels peri-stimulusterm·key histograms is 50 ms with 40 ms overlap. Histology After conclusion of the recording experiments, the animals were overdosed with intraperitoneal injection of the anesthetic urethane and transcardially perfused with 4% paraformaldehyde solution. Brains were sectioned in the coronal plane and stained with cresyl-violet. Electrode tracks were localized on the serial sections in CA1term·key field. Results Behavior This study set out to test how neurons in rat hippocampusterm represent auditory stimuli in the context of a behavioral task, during passive exposure and during sleepterm. Familiar sounds that during the training preceded water rewardterm·key were played in the novelterm·key context (new enclosure) or during sleepterm. Rats were given four pre-training sessions, during which they learned to alternate between two water spouts to get water rewardterm·key (see Figure 2A and Materials and Methods). In the main experimental session (“lotteryterm·key”, Figure 2B ) the water rewardterm·key contingencies were changed: now water was given only in 50% of the entries to the nosepokes. On these “lucky” trials one of the sounds was played immediately upon the nosepoke entry. Distinct sounds were used for each of the two nose pokesterm (see Figure 1B and Materials and Methods). Neuronal representation of the behavioral task In awake rats, hippocampal neuronsterm·key exhibit strong spatial selectivityterm·key: they encode the animal's position, head direction, and other spatial variables (O'Keefe and Dostrovsky, 1971 , reviewed in Moser et al., 2008 ). Consistent with the existing literature, the neurons recorded in this task exhibited significant sensitivity to animal's location in space. Forty-one percent of tested neurons (24 out of 58) discriminated between right and left nosepokes (see Figure 3A ; p < 0.05, corrected, rank sum test on firing ratesterm·var 400 ms before the nose pokesterm, see Materials and Methods). On the “lucky” trials (50% of trials, selected randomly), upon entry to the nosepoke the rats heard a sound and received water rewardterm·key after a 3 s delay. Two different sounds were used in two different locations (nose pokesterm). In “unlucky” trials (50% of trials) the rat heard no sound and received no water rewardterm·key (see Figure 2B and Materials and Methods). The rats stayed still with their snouts in the nosepoke waiting for 3 s between the nosepoke entry/sound onset and the water rewardterm·key delivery. The average time of waiting in no-sound-no-reward trials was 5.09 ± 0.19 s (SEM). Waiting timeterm·var decreased over the course of the session ( p < 0.001, paired-sample t -testterm·stat). To examine responses to sound and rewardterm·key, we aligned neuronal activity on the entry to the nosepoke and compared firing rateterm·var observed in the two types of trials, separately for the two water wells. The responses are illustrated on Figures 3B–D ; the line at 3 s denotes rewardterm·key delivery. Out of 58 cells recorded in the task, 11 cells responded in the first 300 ms upon sound presentationterm·var (19%, p < 0.05, rank sum tests done in 50 ms windows, corrected for testing six time points and also four different bin sizes, see Materials and Methods). Fourteen (24%) responded upon the rewardterm·key delivery (Figure 3B ). There were no cells with responses to both sound and rewardterm·key. Passive sound exposure The aim of this experiment was to characterize the representation of sound identity during passive listeningterm. We played three different sounds to the animals outside the context of the behavioral task, in a novelterm·key box. Two of the sounds were artificial vowelterm stimuli associated with water in the two different nosepokes and familiar to the animals and one of the sounds was a dissimilar novelterm·key band pass filtered noise stimulus. Our intention was to compare the responses to familiar and relevant sounds with the responses to novelterm·key and behaviorally irrelevant ones. The animals were placed in a novelterm·key context 24 h after they performed the “lotteryterm·key” behavioral task. Sounds were triggered with no relation to the animal's posture or movement, in a random sequence lasting approximately 50 min with a random interstimulus intervalterm·var ranging from 25 to 30 s. The rats did not respond to the sounds with any overt behavior. Three out of 25 neurons recorded during the awake passive conditions were classified as sound-sensitive (see Materials and Methods, p -values were 0.0029, 0.0000016, and 0.01, corrected). The responses are illustrated in Figure 4A . The latencies of response to passive sound presentationterm·var were less than 150 ms in two cases and less than 50 ms in the third case (see Figure 4A ). Two of the neurons showed a significantly increased firing rateterm·var in response to the sound, and one a decreased the firing rateterm·var. It would be interesting to know whether the neurons that responded to sounds were also responsive to events occurring within the task, such as sounds, rewardterm·key, or locations. Although we were able to keep the same neurons across the recording sessions, as evidenced by the neuronal waveforms, autocorrelations and firing ratesterm·var, we feel that the number of recorded neurons does not allow us to make any conclusive statements regarding a single neuron's properties across time and context. None of the three sound-responding neurons discriminated between different sounds i.e., their responses to different sounds were identical. Auditory responses during sleepterm During sleepterm sessions, the rats were placed in a small familiar box in which they soon curled up and fell asleep. Animals' activity was monitored remotely via webcam, placed above the box. Five minutes after the animals curled up and fell asleep, the sound stimuli were turned on. The animal's sleepterm status was constantly monitored online by the experimenter and sound presentationterm·var did not cause the animals to wake up. The sounds were presented the entire time when the animals were asleep (117–180 times, 49 presentations per sound type on average) and were turned off when they awoke. Only the trials where sounds did not cause any visible behavioral response from the sleeping animal were analyzed further. The responses to sounds during sleepterm are illustrated in Figures 4B–D and Figure 5 . Twenty-four percent of recorded neurons (8 out of 33) exhibited significant sound-evokedterm·key responses. Across neurons, latencyterm·key ranged from 50 to 150 ms. In five cases firing rateterm·var decreased after sound presentationterm·var, in three cases the firing rateterm·var increased. Figure 5 Sound-evokedterm·key responses during sleepterm can last for over 1 s. Panels (A–E) and (F–J) depict responses and results of statistical analysis for two individual neurons recorded from two rats. Panels (A, F) show peri-stimulusterm·key time histograms, PSTH (0 ms corresponds to sound onset; trials with three sounds are pooled together); (B, G) show raster plots on the same time scale as the PSTH. Panels (C, H) show p -value (sign-test for dependent measures, 50 ms windows). Box plots in panels (D, I) present comparison of 2 s long baseline and post-stimulus periods. On each box, the central mark is the median, the edges of the box are the 25th and 75th percentiles, the whiskers extend to the most extreme data points not considering outliers; outliers are plotted individually as dots. p -values are indicated at the outset (paired-sample sign testterm·stat). Panels (E, J) depict average waveforms on each of the four channels of the tetrodeterm·key for the corresponding neuron. Spike width (at 25% max spike amplitude) and firing rateterm·var for neuron on panels (A–E) are 0.2 ms and 1.8 Hz, respectively; for the neuron in panels (F, J) , 0.2 ms and 1.8 Hz, respectively. Bin size for panels (A, C, F, H) is 50 ms with 40 ms overlap. Figure 5 illustrates neurons with significant differences between baseline and post-stimulus activity lasing more than 1 s, even when considered in short 50 ms long bins (e.g., black line in Figure 5C ). In some cases an initial time-locked response was followed by a long sustained activity response (Figures 5A,B,C,D ). In five cases we observed significant differences between mean firing rateterm·var in 2 s post-stimulus and the baseline ( p < 0.05, corrected). A neuron in Figure 5 (panels F-G-H-I-J) illustrates the case. A short-latency onset responseterm was followed by a sustained, long lasting response (Figure 5F ). Panel I plots average firing rateterm·var in 2 s pre-and post-stimulus windows. p -value is indicated at the outset ( p = 0.0035, sign testterm·stat for dependent measures). Both neurons depicted on this figure had narrow spikes (Figures 5E and J ), 0.2 ms when measured on 25% of spike amplitude. Both of these neurons are likely to be putative interneuronsterm because of the narrow spike width and the absence of the 3–4 ms peak in the autocorrelation plot (data not shown). Unfortunately, limited size of our dataset does not allow us to make any further conclusions on this issue. Long lasting responses to sounds were unique to responses recorded during sleepterm (Figure 6 ). The responses to sounds during sleepterm were on average almost twice as long as the response to sounds presented during passive wakefulness (Figure 6F ). Responses to sounds during sleepterm also appeared slightly earlier than the responses during passive wakefulness (Figure 6E ). Figure 6 Time course of sound-evokedterm·key responses: transient and sustained responses. (A) normalized PSTHs of neurons with significant responses on a fast time scale (window size 50 ms), recorded during sleepterm; (B) normalized PSTHs of neurons with significant responses on a slow time scale (window size 500 ms), recorded during sleepterm; (C) normalized PSTHs of neurons with significant responses on a fast time scale (window size 50 ms), recorded during awake passive listeningterm; (D) normalized PSTHs of neurons with significant responses on a slow time scale (window size 500 ms), recorded during passive listeningterm. Out of 25 neurons only three showed small, but significant responses; (E, F) Absolute values of normalized PSHT (pulled data from neurons that significantly increase and decrease the firing rateterm·var). Black trace represents the average of neurons recorded during sleepterm ( n = 8 out of 33), Gray trace is the mean activity of neurons responsive during awake conditions ( n = 3 out of 25). The majority of cells did not discriminate between different sounds. We found only one neuron with significant sound identity-dependent responses ( p = 0.0101, Kruskal–Wallis testterm·stat) comparing responses to three sounds, two artificial vowelsterm and the novelterm·key noise-like sound. Taken together, these results indicate that hippocampal neuronsterm·key showed sound-evokedterm·key responses during awake passive listeningterm. One quarter of recorded neurons showed reliable sound-evokedterm·key responses during sleepterm. Short latencies of the responses (less than 50 ms) were observed alongside with long-lasting sustained activity. Discussion In this study we show that a significant subset of hippocampal neuronsterm·key responds to sounds presented passively to a rat during both quiet wakefulness and during sleepterm. During sleepterm the responses were characterized by a sharp, short latencyterm·key onset, and in some cases by long-lasting sustained activity. These responses were as strong as the responses to the sounds are associated with water rewardterm·key and the novelterm·key sounds that had never been played to the animal before. The absence of neuronal responses to sound stimuli outside the categorization task (ItskovpersName et al., 2012 ) seems at the first glance inconsistent with the results of our current experiment, where we have found that hippocampal neuronsterm·key responded to the sound stimuli presented during passive wakefulness. We think that this discrepancy may be explained by the differences in the behavioral task and experimental conditions. In the former study highly familiar sounds (used during 4–5 weeks of training) were played in the familiar context after the animal had quenched its thirst and was quietly sitting in the apparatus. In the present study the animals were thirsty during passive presentation and were placed in a completely novelterm·key dim environment, which could have facilitated attention to the auditory stimuli. In this situation we observed strong sound evokedterm·key responses in passive listeningterm conditions. Thus, there are three possible differences that could have explained the difference between these two results: motivational stateterm·var of the animal (thirsty/satiated), familiarity of the stimuli and familiarity of the context. Short latencyterm·key responses to sounds in hippocampusterm have previously described in the literature (Brankack and Buzsaki, 1986 ). This study described responses of extremely short latencyterm·key evoked potentials with first component around 27 ms after the click onset and neuronal responses with latencyterm·key similar latencyterm·key (see their Figure 5 ). Several reports have demonstrated that stimulus-elicited responses appear in hippocampal neuronsterm·key with latencyterm·key of around 80 ms after the stimulus onset (Christian and Deadwyler, 1986 ; Moita et al., 2003 ). What is probably even more important is the similarity of the behavioral paradigms that we and the above mentioned authors used in their studies: the use of naive animals (Moita et al., 2003 ) or the lack of overtraining due to fast learning in a simple behavioral task (Christian and Deadwyler, 1986 ). Unfortunately it is not possible to compare response specificity to individual sound stimuli in these studies since the authors did not consider this feature in their experimental design. In addition to the abovementioned facts, one study that used tactile stimuli in anesthetized and awake naive passively stimulated rats (Pereira et al., 2007 ) have found similar short latencyterm·key responses to trigeminal nerve shock. In over-trained, behaving animals tactile responses have longer latencyterm·key (ItskovpersName et al., 2011 ). Furthermore, the stimulus-specific responses to behaviorally relevant sensory stimuli can persist for at least several seconds after the stimulus has disappeared, as demonstrated using tactile-guided task (ItskovpersName et al., 2011 ). Based on our observations we suggest that there might be two types of neuronal responses in the hippocampusterm to sensory stimuli: Short-latency responses (from ~27 ms latencyterm·key to tens of milliseconds). These responses are not present in the over-trained and/or satiated animals suggesting that they might disappear when the animal expects the stimuli. Long-latency responses. These responses are likely to be evoked by behaviorally relevant stimuli. This second type of hippocampal responses to sounds is likely to encode object identity e.g., are specific to particular behaviorally relevant sounds (ItskovpersName et al., 2012 ) and textures (ItskovpersName et al., 2011 ). We posit that these responses are associated with the behavioral “meaning” of the sound. The evoked responses in the CA1term·key region may be generated by perforant path terminals, originating in the entorhinal cortex or polisynaptically via the Schaffer collaterals. The origin of this short-latency signal in the entorhinal cortex is not known; previous studies suggest that it is unlikely to be of cortical origin, because bilateral ablation of the somatosensory cortex had no effect on the amplitude or the depth distribution of tooth pulp evoked responses in the hippocampusterm (Brankack and Buzsaki, 1986 ). The long-latency responses might be routed to hippocampusterm through the same entorhinal cortex pathway or perhaps even through medial prefrontal cortex and thalamus, contributions of which remain, to the best of our knowledge, unexplored. It is likely that long-latency responses originate from higher-level cortices with more elaborated “object-related” rather sensory feature processing. Stimulus-specific representations can persist in hippocampusterm for at least several seconds after the offset of the behaviorally relevant stimulus (ItskovpersName et al., 2011 ). This memory trace could be stored by sustained activity, previously described in entorhinal cortex (Egorov et al., 2002 ), or by some other cellular or network mechanisms in higher order associative cortices or hippocampusterm itself. Hippocampal responses during sleepterm It has been recently demonstrated that re-exposure to olfactory and auditory stimuli during sleepterm can enhance memory for specific events, associated with those stimuli (Rasch et al., 2007 ; Rudoy et al., 2009 ). Rasch and co-authors ( 2007 ) cued new memories in humans during sleepterm by an odor that had been presented as context during prior learning, and showed that this manipulation enhanced subjects' performance. Re-exposure to the odor during slow-wave sleepterm improved the retention of hippocampusterm dependent declarative memories but not of hippocampus-independent procedural memories. Re-exposure was ineffective during rapid eye movement sleepterm or wakefulness or when the odor had been omitted during prior learning. Concurring with these findings, functional magnetic resonance imaging revealed significant hippocampal activation in response to odor re-exposure during slow wave sleepterm. Rudoy and co-authors ( 2009 ) trained participants to associate each of 50 unique object images with a location on a computer screen before a nap. Each object was paired with its characteristic sound (e.g., cat with meow and kettle with whistle). Presentation of the sounds during sleepterm resulted in better memory performance. Interestingly, the memory enhancement was specific to the played sounds and did not generalize to the other stimuli; those corresponding sounds were not played during sleepterm. Average electroencephologram (EEG) amplitudes measured over the interval from 600 to 1000 ms after sound onset were larger when there was less forgetting. In the present study we investigated the hippocampal responses to behaviorally relevant sounds during re-exposure to the sounds during awake state and sleepterm. We found short-latency responses to sound presentationterm·var during sleepterm, as well as long-lasting (more than 1 s) sustained responses. The latter finding might be related to the sustained EEG response found in Rudoy et al. ( 2009 ) after presentation of auditory stimuli to humans during sleepterm. Rasch et al. ( 2007 ) reported an increased BOLD-signal in hippocampusterm after presentation of the odors. There results suggest that long-lasting changes in neuronal activity contribute to stimulus-evoked responses observed in previous studies, in addition to previously shown local field potential change and short-latency onset responsesterm. It is hoped that further studies will clarify how exactly the presentation of the relevant sensory cues during sleepterm facilitates the hippocampus-dependent memory. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.