Temporal coordination of olfactory cortex sharp-wave activity with up- and downstates in the orbitofrontal cortex during slow-wave sleep

Simultaneous recordings of local field potentials and spike activities in the anterior piriform cortex (APC) and orbitofrontal cortex (OFC) during slow-wave sleep showed that APC sharp waves tended to occur during two distinct phases of OFC upstate: early phase, shortly after the down-to-up transition, and late phase, shortly before the up-to-down transition, suggesting that during slow-wave sleep, olfactory cortex and OFC areas communicate preferentially in the specific time windows.

Abstract

During slow-wave sleep, interareal communications via coordinated, slow oscillatory activities occur in the large-scale networks of the mammalian neocortex. Because olfactory cortex (OC) areas, which belong to paleocortex, show characteristic sharp-wave (SPW) activity during slow-wave sleep, we examined whether OC SPWs in freely behaving rats occur in temporal coordination with up- and downstates of the orbitofrontal cortex (OFC) slow oscillation. Simultaneous recordings of local field potentials and spike activities in the OC and OFC showed that during the downstate in the OFC, the OC also exhibited downstate with greatly reduced neuronal activity and suppression of SPW generation. OC SPWs occurred during two distinct phases of the upstate of the OFC: early-phase SPWs occurred at the start of upstate shortly after the down-to-up transition in the OFC, whereas late-phase SPWs were generated at the end of upstate shortly before the up-to-down transition. Such temporal coordination between neocortical up- and downstates and olfactory system SPWs was observed between the prefrontal cortex areas (OFC and medial prefrontal cortex) and the OC areas (anterior piriform cortex and posterior piriform cortex). These results suggest that during slow-wave sleep, OC and OFC areas communicate preferentially in specific time windows shortly after the down-to-up transition and shortly before the up-to-down transition.

NEW & NOTEWORTHY Simultaneous recordings of local field potentials and spike activities in the anterior piriform cortex (APC) and orbitofrontal cortex (OFC) during slow-wave sleep showed that APC sharp waves tended to occur during two distinct phases of OFC upstate: early phase, shortly after the down-to-up transition, and late phase, shortly before the up-to-down transition, suggesting that during slow-wave sleep, olfactory cortex and OFC areas communicate preferentially in the specific time windows.

slow-wave sleep provides a specific time window for offline communication among wide areas of the mammalian cerebral neocortex and subcortical structures in the forebrain without disturbance by sensory inputs from the external world (Contreras and Steriade 1995; Steriade and Contreras 1995; Steriade et al. 2001; Timofeev et al. 2001). During slow-wave sleep, the neocortex is isolated from external sensory inputs and shows intrinsically generated, coordinated population activity with large-amplitude, slow oscillations of local field potentials (LFPs) (Steriade et al. 1990, 1993b). This slow oscillatory activity propagates occasionally across whole areas of the neocortex, suggesting the coordination of activity across large-scale cortical networks (Massimini et al. 2004; Murphy et al. 2009; Vyazovskiy et al. 2011).

The neocortex slow oscillations consist of an upstate of enhanced neuronal activity and interareal communication and downstate of neuronal silence (Mölle and Born 2011; Steriade et al. 1993b). The upstate is characterized by depolarized membrane potential levels of neocortical neurons and is associated with depth-negative, surface-positive LFPs. During the downstate, in contrast, neocortex neurons are hyperpolarized, and each neocortex area shows depth-positive, surface-negative LFPs (Contreras and Steriade 1995; Steriade et al. 1993b). Because the neocortical activity associated with the slow oscillations propagates to the hippocampus and thalamus, activities of the hippocampus and thalamus are finely coordinated with the up- and downstates of the neocortex. Only during the neocortical upstate does hippocampus generate sharp-wave (SPW)/ripple events, and thalamus gives rise to spindles (Mölle and Born 2011). Thus during slow-wave sleep, the upstate of the neocortex provides a temporal framework in which reciprocal communication occurs between the neocortex and hippocampus and between the neocortex and thalamus.

The anterior piriform cortex (APC), a major area of the olfactory cortex (OC), generates characteristic SPW activity during slow-wave sleep (Manabe et al. 2011). The APC has direct axonal projections to the orbitofrontal cortex (OFC; lateral orbital cortex and ventrolateral orbital cortex) and agranular insular cortex, and these neocortical areas send top-down axonal projections back to the APC (Illig 2005; Ray and Price 1992). In addition, the APC indirectly interacts with these neocortical areas via the endopiriform nucleus (En) and thalamus. For example, pyramidal cells in the APC activate neurons in the En and claustrum, which then send signals to the agranular insular cortex and OFC (Fu et al. 2004; Lipowska et al. 2000). Furthermore, neurons in the deep part of the APC project axons to the mediodorsal nucleus (MD) and submedial nucleus of the thalamus, and these thalamic nuclei have reciprocal projections with the OFC and agranular insular cortex (Krettek and Price 1977; Neville and Haberly 2004; Ray and Price 1992).

These direct and indirect axonal connections between the APC and OFC raise the possibility that SPW events of the APC are coordinated with the up- or downstates of the OFC during slow-wave sleep. It has been shown in urethane-anesthetized rats that SPW activity of the piriform cortex occurs in synchrony with the slow-wave activity in the MD of the thalamus (Courtiol and Wilson 2014). However, it is not understood whether and how interactions between the APC and OFC occur during natural slow-wave sleep.

To address these questions, we recorded LFPs and spike activities simultaneously in the APC and OFC of freely behaving rats during the whole period of a natural sleep. Here, we demonstrate the presence of clear temporal coordination between the generation of SPWs in the APC and up- and downstates of the OFC. Results from multiunit recordings of the APC and OFC support the idea of enhanced interaction between the APC and OFC during the OFC upstate and reduced interaction during the downstate. These results suggest that the upstate of the slow oscillations in the OFC provides a common temporal framework for the large-scale interactions between the OFC and OC during slow-wave sleep.

MATERIALS AND METHODS

Animals.

All experiments were conducted in accordance with the guidelines of the Physiological Society of Japan and were approved by the Experimental Animal Research Committee of the University of Tokyo. Male Long-Evans rats, purchased from Japan SLC (Shizuoka Prefecture, Japan), were housed individually in plastic (345 × 403 × 177 mm) or metallic (350 × 400 × 300 mm) cages at 25°C under a 12-h light/dark cycle with light on at 5 AM.

Surgery for electrode implantation.

Details of the surgical preparation and chronic electrophysiological recording methods used in the present study have been reported previously (Manabe et al. 2011). Adult, male Long-Evans rats (294–475 g at the time of surgery) were anesthetized with ketamine (67.5 mg/kg ip) and medetomidine (0.5 mg/kg ip) for surgery to implant stimulation and recording electrodes at appropriate places in the brain.

For olfactory bulb (OB) stimulation, a twisted, stainless-steel electrode (75 μm diameter) was implanted in the OB (8.0 mm anterior to the bregma, 1.3 mm lateral to the midline, 2.5 mm deep from the skull surface). For recordings of single- and multiunit activities in the APC and OFC, a multisite microdrive array that allows targeting of 2 brain regions and independent adjustment of 16 individual tetrodes was implanted in the APC (0.0 mm anterior to the bregma, 4.2 mm lateral to the midline) and OFC (4.0 mm anterior to the bregma, 2.5 mm lateral to the midline). Individual tetrodes consisted of four twisted, polyimide-coated tungsten wires (12.5 μm diameter; California Fine Wire, Grover Beach, CA). The microdrive array was fixed to the skull with dental acrylic and anchor screws.

To record LFPs, twisted, polyimide-coated tungsten wires (50 μm diameter; California Fine Wire) were implanted into the OB (8.0 mm anterior to the bregma, 1.25 mm lateral to the midline, 2.3 mm deep from the skull surface), APC (2.0 mm anterior to the bregma, 2.8 mm lateral to the midline), posterior piriform cortex (PPC; 3.1 mm posterior to the bregma, 4.0 mm lateral to the midline), OFC (3.7 mm anterior to the bregma, 2.5 mm lateral to the midline, 4.0 mm from the skull surface), and medial prefrontal cortex (mPFC; 4.2 mm anterior to the bregma, 0.5 mm lateral to the midline, 2.0 mm from the skull surface). The positions of the electrode tips in layer III of the APC and PPC were determined by monitoring the configuration of OB-evoked LFPs. For neocortical EEG recording, a stainless-steel screw was threaded into the bone above the occipital cortex (6.3 mm posterior to the bregma, 3.0 mm lateral to the midline). Two further screws were threaded into the bone above the cerebellum for reference. For electromyogram recordings, Teflon-insulated, stainless-steel electrodes were implanted in the neck muscle. To monitor respiration pattern, a thermocouple (0.23 mm diameter, Teflon coated; World Precision Instruments, Sarasota, FL) was implanted in a nasal cavity through a hole made in the dorsal skull and secured to the skull with dental acrylic.

Electrophysiological recordings in freely behaving animals.

Animals were allowed to recover from surgery for at least 1 wk before recording. For recordings during sleep, rats were allowed to behave freely in a well-habituated, bedded cage. The recording electrodes were connected with an electrode interface board (NeuraLynx, Bozeman, MT) on the microdrive. Electrical signals were obtained using a Cheetah recording system (NeuraLynx). For unit recordings, the signals were sampled at 32 kHz and band-pass filtered at 600–6,000 Hz. For LFP recordings, the signals were sampled at 16 kHz and band-pass filtered at 0.1–6,000 Hz. Unit recordings were obtained with tetrode depth adjusted on each recording day to acquire activity from new neurons. The positions of the tetrode tips in the layer of the piriform cortex were determined by monitoring the configuration of the LFP evoked by electrical stimulation of the OB. After the experiments, small lesions were made by current injection for later histological examination of the recording sites.

Data analysis.

We used the KlustaKwik (by K. D. Harris) and MClust software (by A. D. Redish) for offline spike sorting (Harris et al. 2000) and Spike2 software (Cambridge Electronic Design, Cambridge, UK) for analysis. Slow-wave sleep was determined by the high slow-wave (0.5–1.5 Hz) and delta-wave (1.5–4 Hz) power of the neocortical EEG, the absence of movement signal in the neck muscle electromyogram, and stable, slow respiration. The high slow-wave and delta-wave activities were combined and defined as slow-wave activity in this report. SPWs were defined as large, sharp, depth-negative LFPs with a duration <200 ms (Manabe et al. 2011). To detect SPWs, the LFPs were downsampled to 100 Hz and band-pass filtered at 2–20 Hz. Mean and SD of the LFP amplitude were calculated during slow-wave sleep periods in each recording session (877–2,887 s). The threshold amplitude for SPW detection was set to 4 SDs above the mean amplitude. Tentative downstates in the OFC during slow-wave sleep were characterized as large, positive peaks of LFPs recorded in the deep layer (layers V/VI) of the OFC. To quantify the tentative downstate, LFP in the OFC was downsampled to 100 Hz and low-pass filtered at 20 Hz. Mean and SD of LFP amplitude were calculated. The threshold for tentative downstate detection was set to 2 SDs positive to the mean.

Recordings were performed for 1–3 h. The whole period of natural sleep that occurred during the recording period was used for analysis. The average duration of sleep was 2,507 s/rat. Mean numbers of events were 1,902 OFC downstate events, 1,850 mPFC downstate events, 1,939 APC-SPW events, 2,222 PPC-SPW events, and 2,370 OB-SPW events/rat. We made visual inspection of LFPs and confirmed that a clear artefactual event was not included in the events. For analysis, we used all events that were recorded during slow-wave sleep.

Peri-downstate time histograms and peri-SPW time histograms were calculated in 10 ms bins. The tendency of spike discharges to become silent with the downstate was considered significant when the firing frequency of at least three bins among those within −100 to 0 ms from the peak time of the downstate fell to a mean − 2 SDs of background firing frequency (during −500 to −200 ms and during +200 to +500 ms; see Fig. 1, B and E). The tendency of spike discharges to synchronize with SPW events was considered significant when the firing frequency of at least one bin among those within −100 to +100 ms from the time of the trough of SPW exceeded a mean + 3 SDs of background firing frequency (during −300 to −200 ms and +200 to +300 ms; see Figs. 6 and 7).

Fig. 1.

Coordinated occurrence of orbitofrontal cortex (OFC) downstate and anterior piriform cortex (APC) downstate during slow-wave sleep. A: simultaneous recordings of local field potential (LFP) with multiunit spike activity (MUA) from the OFC and APC, together with the animal's respiration (Resp). Upward swing in the respiration monitor indicates inhalation. Black rectangles on the OFC-LFP indicate the peak of depth-positive potential (tentative downstate) in the OFC. Arrows show APC-sharp waves (SPWs). B: averaged time course of OFC-LFP aligned in reference to the peak of the OFC downstate (top). Bottom: histograms show the peri-downstate time histogram of the single-cell spike activity of 3 simultaneously recorded OFC neurons. The horizontal, dashed lines indicate the statistical threshold level (2 SDs below the background firing frequency) for detecting reduced activity. *, OFC unit activity decreases significantly during OFC downstates. C: filtered traces of OFC-LFP and APC-LFP in A. Each LFP recording was downsampled to 100 Hz and low-pass filtered at 4 Hz. D: solid line indicates mean coherence (n = 8 experiments from 3 rats, 2 or 3 experiments/rat) between OFC-LFP and APC-LFP. Dashed lines indicate ±SD. E: averaged time course of OFC-LFP aligned in reference to the peak of OFC downstate (top). Bottom: histograms show peri-downstate time histogram of single-cell firings of 3 simultaneously recorded APC neurons. The horizontal, dashed lines indicate the statistical threshold level (2 SDs below the background firing frequency) for detecting the reduced activity. *, APC unit activity decreases significantly during OFC downstates.

Fig. 6.

OFC neurons that showed enhanced spike discharges in synchrony with the APC-SPWs. Averaged time course of APC-SPW (top) and peri-SPW time histogram of 3 simultaneously recorded OFC neurons (bottom). Horizontal, dashed lines indicate the statistical threshold level (3 SDs above background firing frequency) for the detection of APC-SPW-associated, enhanced discharges. *, OFC unit activity increased significantly during the APC-SPW.

Fig. 7.

Analysis of the 2 types of APC-SPW. Peri-down-to-up APC-SPW time histograms (left) and peri-up-to-down APC-SPW time histograms (right) of spike discharges of 3 simultaneously recorded OFC neurons. Horizontal, dashed lines indicate the statistical threshold level (3 SDs above background firing frequency) for detecting the enhanced discharges. *, OFC unit activity increased significantly during APC-SPWs.

To compare the probability of APC-SPW events during the up- and downstate of the OFC, the following three periods were defined: 1) the upstate period shortly before the downstate onset (upstate 1); 2) the downstate period; and 3) the upstate period shortly after the down-to-up transition (upstate 2). The upstate 1 period was defined as the time window from −250 to −100 ms before the peak time of the downstate. The downstate period was defined as the time window within −100 to 0 ms from the peak time of the downstate. The upstate 2 period was the time window between 0 and +250 ms from the peak time of the downstate. Differences in APC-SPW generation among these three periods were statistically analyzed using one-way ANOVA with post hoc Tukey test (significance level, P < 0.05; see Figs. 3C and 9B).

Fig. 3.

Occurrence of APC-SPWs shortly after the down-to-up transition and shortly before the up-to-down transition of OFC slow-wave activity. A: superimposition of 10 traces of OFC downstates (top) aligned in reference to the peak of OFC downstates (dashed, vertical line). Gray trace is the averaged time course of OFC-LFP during downstates. Bottom: APC-LFPs with APC-SPWs superimposed in reference to the peak of OFC downstate. Arrows indicate the trough of APC-SPW. B: averaged time course of OFC-LFP during downstates (top) and peri-downstate time histogram of the troughs of APC-SPWs (bottom). C: frequency of APC-SPW occurrence among 3 periods (upstate 1, downstate, and upstate 2) around the OFC downstate. Upstate 1 period, −250 to −100 ms from the peak of the downstate; downstate period, −100 to 0 ms from the peak time; upstate 2 period, 0 to +250 ms from the peak time. Data represent means ± SD (n = 8 experiments from 3 rats, 2 or 3 experiments/rat). ***P < 0.001; n.s., not significant (one-way ANOVA with post hoc Tukey test).

Fig. 9.

Occurrence of APC-SPWs, PPC-SPWs, and OB-SPWs in synchrony with upstates of both OFC and mPFC. A: averaged time course of OFC downstates (left, top) and mPFC downstates (right, top) and peri-downstate time histogram of APC-SPWs, PPC-SPWs, and OB-SPWs (bottom). B: comparison of frequency of APC-SPW, PPC-SPW, and OB-SPW occurrence among the 3 periods (upstate 1, downstate, and upstate 2) around the OFC downstate (left) and the mPFC downstate (right). Data represent means ± SD (APC, OB, n = 4 experiments from 4 rats; PPC, n = 4 experiments from 3 rats, 1 or 2 experiments/rat). *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant (one-way ANOVA with post hoc Tukey test). C: phase histogram of APC-SPW, PPC-SPW, and OB-SPWs with the cycle of OFC slow-wave activity (left) and mPFC slow-wave activity (right). Error bars show SD. Zero degrees shows the peak of downstate, and 360° shows the next downstate. Phases I, II, and III indicate the initial one-third (0–120°), the middle one-third (120–240°), and the latter one-third (240–360°) of each slow-wave activity, respectively. Horizontal, dashed lines indicate means + 2 SDs of the APC-SPW, PPC-SPW, and OB-SPW ratio of phase II. SPW ratio in phase I or III that exceeded this line was considered a significant increase in SPW generation. D: peri-OFC downstate time histogram of averaged ratio of mPFC downstate events. The averaged ratio in each bin was calculated for all of the data from different data and from different animals (n = 4 rats).

An OFC downstate that occurred in synchrony with an APC downstate (<50 ms difference in peak time of OFC and APC downstates) was defined as a global OFC-APC downstate. A local OFC downstate was defined when the OFC downstate did not accompany an APC downstate (within 50 ms). The difference in the firing frequency of OFC neurons between the global and local downstates (see Fig. 5C) was examined by calculating and comparing the averaged firing frequency of OFC neurons during global OFC-APC downstate and that during local downstate. The difference in the firing frequency of APC neurons between the global and local downstates was examined in the same way. To compare the frequency of APC-SPW occurrence shortly before and after the global downstate and that shortly before and after the local downstate, the averaged frequency of APC-SPW occurrence during −200 to +200 ms from the peak time of downstates was calculated. The group difference in the frequency of APC-SPW occurrence was verified by the paired t-test (significance level, P < 0.05; see Fig. 5E).

Fig. 5.

Tendency of APC-SPWs to occur more frequently around the global OFC-APC downstate than around the local OFC downstate. A: simultaneous recordings of LFPs and MUA in the OFC and APC during the global OFC-APC downstate (left) and local OFC downstate (right). B, left: peri-global OFC-APC downstate time histograms of OFC multiunit MUA (top) and APC MUA (bottom). Right: peri-local OFC downstate time histograms of OFC MUA (top) and APC MUA (bottom). Horizontal, dashed lines indicate average MUA frequency. C: averaged MUA frequency of OFC neurons (left) and that of APC neurons (right) during whole slow-wave sleep period (Ave.), during the global OFC-APC downstate (Global), and during local OFC downstates (Local). Data represent means ± SD (n = 8 experiments from 3 rats, 2 or 3 experiments/rat). ***P < 0.001, **P < 0.01, *P < 0.05; n.s., not significant (one-way repeated-measures ANOVA with post hoc Tukey test). D: peri-global OFC-APC downstate time histogram of APC-SPWs (left) and peri-local OFC downstate time histogram of APC-SPWs (right). Horizontal, dashed lines indicate the averaged APC-SPW frequency. E: frequency of APC-SPW occurrence around the global (left) and local (right) OFC downstates (n = 8 experiments from 3 rats, 2 or 3 experiments/rat). ***P < 0.001 (paired t-test).

To calculate the peri-OFC downstate time histogram (see Fig. 9D), the number of mPFC downstate events in each bin was divided by the total number of mPFC downstate events within −250 to +250 ms from the peak time of the OFC downstate. The ratio of mPFC downstate events in each bin was then calculated for each animal. The averaged ratio of mPFC downstate events in each bin was then calculated using all of the data from different experiments and from different animals.

To obtain peri-downstate-phase histograms of APC-SPW, PPC-SPW, and OB-SPW events, one cycle of slow-wave activity in the OFC and mPFC (i.e., a down-up-down transition) was divided into 360° (1 cycle = 30 bins). The number of SPW events in each bin was divided by the total number of SPW events in one cycle. The ratio of SPW events in each bin was then calculated for each animal. The averaged ratio of SPW events in each bin for all of the data from different experiments and from different animals was then calculated (see Figs. 4D and 9C).

Fig. 4.

APC-SPWs occurring at the 2 distinct phases of the OFC upstate. A: timing of APC-SPW occurrence in reference to each cycle (down-up-down) of the 15 traces of OFC slow-wave activity with different duration. Each trace was aligned with the peak of the OFC downstate (vertical, dashed line, 0 s), and short black lines indicate the peak of the next downstate. Gray lines show the time of APC-SPW occurrence. B: an enlarged example of trace #7 from A. The peak of the OFC downstate was set to 0 s (dashed line), and the short, black line with dashed line shows the peak of the next downstate. Gray line shows APC-SPW. C: phase histogram of APC-SPW events that occurred in the 15 traces of A. One cycle of slow-wave activity is divided into 360° (with 12° bins). The peak of the OFC downstate is shown by 0°, whereas the peak of the next downstate is indicated by 360°. D: phase histogram of APC-SPWs with reference to the cycle of OFC slow-wave activity (n = 8 experiments from 3 rats, 2 or 3 experiments/rat, error bars show SD). Zero degrees shows the OFC downstate, and 360° shows the next downstate. Phases I, II, and III indicate the initial one-third (0–120°), middle one-third (120–240°), and the latter one-third (240–360°) of the slow-wave activity, respectively. Horizontal, gray, dashed line indicates mean +2 SDs of the APC-SPW ratio of phase II. APC-SPW ratio of phases I and III exceeded this line, and the probability of APC-SPW occurrence in both phases I and III was considered significantly higher than that in phase II.

Histology.

After the experiments, the animals were deeply anesthetized with urethane and perfused first with PBS, followed by 4% paraformaldehyde in phosphate buffer, pH 7.4. Brains were postfixed in 4% paraformaldehyde in phosphate buffer at 4°C. Coronal sections of the brain (40 μm thick for brains with unit recordings, 50 μm thick for brains with LFP recordings) were cut on a vibratome, mounted on glass slides, and stained with cresyl violet.

RESULTS

To examine the possibility that the OFC interacts directly or indirectly with the APC at specific time windows during slow-wave sleep, we recorded LFPs and multiunit spike activities simultaneously in the deep layer (layers V and VI) of the OFC and the deep layer (layer III) of the APC of freely behaving rats. During slow-wave sleep periods, the LFP in the OFC showed slow-wave activity (0.5–4 Hz), with each oscillatory cycle consisting of a large, slow, positive potential, followed by a longer, slow, negative potential (Fig. 1A; OFC-LFP). Multiunit spike activities in the OFC indicated that OFC neurons showed enhanced spike activities during the slow, negative potential, indicating that the occurrence of the slow, negative potential corresponds in time with the upstate of the OFC (Fig. 1, A and B) (Contreras and Steriade 1995; Steriade et al. 1993b). In striking contrast, the slow, positive potential was associated with silencing of the spike activities of OFC neurons (Fig. 1, A and B). This shows that the slow, positive potential corresponds in time with the downstate of the OFC.

To estimate roughly the time window of the OFC downstate, based on the configuration of the slow oscillatory potentials, we first assumed four different levels of tentative threshold potential that differentiate the depth-negative (upstate) and depth-positive (downstate) potential (Fig. 2A). With the comparison of the timing of the downstate defined by this method and that of the diminished spike activities of OFC neurons, we found that the downstate, determined by the threshold of mean + 2 SDs, provided the best fit with the temporal framework of the downstate determined by the diminished spike discharges (Fig. 2, A and B). Accordingly, we set the threshold to detect the downstate as a mean + 2 SDs of the filtered OFC LFP in this study. Figure 2C shows examples of the spike activities of individual OFC neurons during the downstate, detected by the above method. All of the OFC neurons recorded in Fig. 2C showed reduced spike discharges during the OFC downstate. Furthermore, a majority of the OFC neurons (73%, 134/183 cells) showed a significant silence (see materials and methods) during the downstate (Fig. 1B).

Fig. 2.

Estimation of OFC downstate based on configuration of the OFC LFP. A: LFPs in the OFC, filtered traces of the OFC-LFP, and spike histogram of simultaneously recorded OFC neurons. Horizontal, black, dashed line shows the mean of the filtered LFP. Horizontal, colored, dashed lines show the means + various SDs of the filtered OFC-LFP (blue, 1 SD; green, 1.5 SDs; red, 2 SDs; yellow, 2.5 SDs). B: peri-downstate time histogram of OFC multiunits (all 22 cells in C). Tentative peak of the downstate is shown at 0 s. Downstates were detected in 4 different ways with distinct threshold levels (blue, means +1 SD; green, means +1.5 SDs; red, means +2 SDs; yellow, means +2.5 SDs). C: peri-downstate time histogram of 22 simultaneously recorded, single neurons in the OFC. The threshold for detecting tentative downstates was set as the mean +2 SDs (2 SD of A and B). Vertical, red, dashed lines show the tentative peak of the OFC downstate.

LFPs in the APC also showed slow-wave activity during slow-wave sleep (Fig. 1A). Although the APC shows slow oscillatory potentials in phase with the respiration cycle during wakefulness (Mori et al. 2013), the slow-wave activity during slow-wave sleep occurred relatively independently of the respiration cycle (Fig. 1A). Each cycle of APC slow-wave activity consists of a slow depth-positive potential and a slow depth-negative potential.

Simultaneous recordings of LFPs in the OFC and APC indicated that slow depth-positive and -negative potentials in the APC tended to synchronize with those in the OFC. LFPs in the APC showed high coherence with those of the OFC in the range between 1 and 4 Hz (Fig. 1, C and D). These results suggest that slow-wave activity in the APC tends to occur in synchrony with slow-wave activity in the OFC during slow-wave sleep. In accordance with this idea, approximately one-fifth of APC neurons (22%, 27/121 cells) showed significantly reduced spike activities during the OFC downstates (Fig. 1E).

It should be noted that the downstate in the OFC did not always accompany the downstate in the APC and that some APC neurons were occasionally active during the OFC downstate (Fig. 1A). In other words, downstates in the OFC can be classified into two types: 1) OFC downstates with an accompanying APC downstate (global OFC-APC downstate) and 2) OFC downstates without an accompanying APC downstate (local OFC downstate), as will be described in detail later.

We previously reported that APC shows characteristic SPWs that were superimposed on slow, negative potentials (APC upstate) during slow-wave sleep (see Fig. 1A) and that each SPW was associated with highly synchronized spike discharges of APC neurons (Manabe et al. 2011). Given that numerous APC neurons show synchronized spike discharges during APC-SPW events (Manabe et al. 2011) and that APC-SPWs occurred mainly during the slow, negative potential in the APC (Fig. 1A), we further examined the temporal relationship between SPW events in the APC and slow-wave activity in the OFC during slow-wave sleep. As shown in Fig. 3, A–C, the occurrence of SPW events in the APC was greatly reduced during the downstate of the OFC. A majority of APC-SPW events temporally coincided with the upstate of the OFC.

To examine more closely the timing of APC-SPW occurrence in relation to the time course of the OFC upstate, a convenience sample of 15 traces of OFC slow-wave cycles with various durations was arbitrarily selected and aligned in reference to the peak of OFC downstate (Fig. 4, A and B). Figure 4A clearly shows that APC-SPWs tend to occur shortly before and shortly after the OFC downstate. We tentatively divided one cycle of OFC slow-wave activity into 360° [from the peak of a downstate (0°) to the peak of next downstate (360°); 1 bin = 12°] and calculated histograms of APC-SPWs in reference to a cycle of OFC slow-wave activity (Fig. 4C). As shown in Fig. 4C, APC-SPWs tended to occur shortly before and shortly after the OFC downstate. The same analysis of the phase histogram of APC-SPWs was performed for all recordings during sleep (n = 8 experiments from 3 rats; Fig. 4D). We tentatively defined three phases for each slow-wave activity: 1) an initial one-third (0–120°, phase I) that starts from the peak of the downstate (0°) and includes the down-to-up transition, 2) a middle one-third (120–240°, phase II), and 3) a final one-third (240–360°, phase III) that includes the up-to-down transition and ends at the peak of the next downstate (360°; Fig. 4D). APC-SPW events occurred during the upstate of the OFC at all three phases of the slow-wave cycle. However, the timing of APC-SPW occurrence indicated that the APC-SPWs tended to occur more frequently during time windows shortly after the transition from the downstate to the upstate of the OFC (during phase I) and shortly before the transition from the upstate to the downstate of the OFC (during phase III) compared with the time window during phase II (Fig. 4D). These results indicate that these transitions in OFC and APC-SPWs tend to be coherent.

As described above, the OFC downstate did not always synchronize with the APC downstate: whereas the downstate of the OFC sometimes coincided with the downstate of the APC (global OFC-APC downstate in Fig. 5A), the OFC downstate occasionally occurred when the APC was at the upstate (local OFC downstate in Fig. 5A). Approximately 31% of OFC downstate events were global OFC-APC downstates, whereas the remaining 69% were local OFC downstates. OFC neurons were silent during both the global and local downstates. On the other hand, APC neurons showed reduced spike activity only during the global OFC-APC downstate and did not show reduced spike activity during the local OFC downstate (Fig. 5, B and C). Averaged frequency of multiunit activity of OFC neurons (33.2 ± 16.0 Hz, mean ± SD, averaged across all slow-wave sleep period) decreased significantly both during global OFC-APC downstate (9.5 ± 8.7 Hz) and local OFC downstate (14.7 ± 11.3 Hz; one-way repeated-measures ANOVA with post hoc Tukey test; Fig. 5C). Averaged frequency of multiunit activity of APC neurons (15.7 ± 6.8 Hz, mean ± SD) also decreased during the global OFC-APC downstate (7.9 ± 4.8 Hz) but did not decrease significantly (13.0 ± 7.0 Hz) during the local OFC downstate (one-way repeated-measures ANOVA with post hoc Tukey test; Fig. 5C). We compared the frequency of APC-SPW occurrence shortly before and after the global OFC-APC downstate (Fig. 5D) with that shortly before and after the local OFC downstate (Fig. 5D) and found that APC-SPWs occurred more frequently in the time windows before and after the global OFC-APC downstate than before and after the local OFC downstate (Fig. 5E). These results suggest that OFC and APC are coherent in the time windows shortly before and after the global OFC-APC downstates compared with those shortly before and after the local OFC downstates.

Because numerous APC neurons show synchronized spike discharges during APC-SPWs (Manabe et al. 2011), we examined whether individual OFC neurons show spike discharges in synchrony with the APC-SPWs using a simultaneous recording of APC-SPWs and single-unit spikes in the OFC. Figure 6 shows the peri-APC-SPW time histogram of spike discharges of three simultaneously recorded OFC neurons. Approximately 45% of recorded neurons in the OFC (82/183 OFC neurons) showed enhanced spike discharges during the APC-SPWs. These observations support the idea that APC spike activity and OFC spike activity tend to be coherent during the time windows shortly after the down-to-up transition and shortly before the up-to-down transition of OFC.

The enhanced OFC and APC coherence suggests the following three possibilities of the direction of signal transfer: 1) SPW-related, synchronized discharges of APC neurons directly or indirectly drive numerous OFC neurons; 2) synchronized discharges of OFC neurons during the OFC upstate directly or indirectly drive the synchronized discharge of APC neurons and generate APC-SPWs; and 3) the OFC-APC coherence is mediated by a common influence from a third brain region. As a first step to address the question of the direction of signal transfer, we compared the timing of spike generation between OFC and APC neurons with reference to APC-SPWs.

As exemplified in Fig. 6, the probability of spike discharges of OFC neurons peaked at various phases of the APC-SPW event. Approximately 60% (49/82) of OFC neurons showed a peak of spike discharge probability in the descending phase (before the trough) of the APC-SPW, whereas the remaining 40% (33/82) of OFC neurons showed a peak in the ascending phase of the APC-SPW. We previously reported that the probability of spike discharge for most APC neurons peaks in the middle of the descending phase of APC-SPW events (Manabe et al. 2011). Comparison of the present and these previous results suggests that although the peak of spike discharge probability of APC neurons tends to precede that of OFC neurons during the APC-SPW events, the timing of the discharge of OFC neurons largely overlaps that of APC neurons. These initial results thus did not provide the clue for answering the question of the signal flow direction.

As described above, a majority of APC-SPWs occurred shortly before and shortly after the OFC downstate. We therefore classified APC-SPWs into those that occurred shortly after the down-to-up transition (down-to-up APC-SPWs) and those that occurred shortly before the up-to-down transition (up-to-down APC-SPWs; Fig. 7). We then examined whether each of the 82 OFC neurons that fired in synchrony with APC-SPWs showed spike discharges in synchrony with down-to-up, up-to-down, or both types of APC-SPWs. Approximately 49% of the OFC neurons (40/82 neurons) showed significantly increased discharges during down-to-up APC-SPWs, 13% neurons (11/82 neurons) showed significantly enhanced discharges during up-to-down APC-SPWs, and 15% neurons (12/82 neurons) showed enhanced discharges during both up-to-down and down-to-up APC-SPWs. These results suggest that OFC-APC coherence can occur during both down-to-up and up-to-down APC-SPWs but tends to be more frequent during the down-to-up type.

As reported previously, APC-SPWs propagate widely to numerous areas of the OC (anterior olfactory nucleus, PPC, and olfactory tubercle) and OB (Komano-Inoue et al. 2014; Manabe et al. 2011; Narikiyo et al. 2014). Furthermore, neocortical slow-wave activity generates synchronized up- and downstates across wide areas of the prefrontal cortex during slow-wave sleep (Massimini et al. 2004; Murphy et al. 2009; Vyazovskiy et al. 2011). These results suggest that coordination of the upstate in the prefrontal cortex and the SPW events in the OC occurs not only between the APC and OFC but also across many areas of both the OC and prefrontal cortex. To examine this possibility, we made simultaneous recordings of LFPs from the deep layers of the OFC, mPFC, APC, and PPC (Fig. 8). Furthermore, because APC-SPWs propagate to the OB, we also recorded LFPs from the deep layer (granule cell layer) of the OB. As shown in Fig. 8, A–C, up-to-down and down-to-up transitions of slow-wave activity tended to synchronize not only between neocortex areas (OFC and mPFC) but also between the neocortex and OC areas (APC and PPC), albeit that the slow-wave synchronization between the OFC and mPFC was more coherent than that between the OFC and APC. Weak synchronization of slow-wave activity was also noted between the neocortex areas (OFC and mPFC) and OB (Fig. 8, A–C). Figure 9, A and B, shows that the occurrence of APC-SPW, PPC-SPW, and OB-SPW events was greatly reduced during both the OFC and mPFC downstates. Furthermore, not only APC-SPWs but also PPC-SPWs and OB-SPWs tended to occur shortly after and shortly before the OFC downstates (Fig. 9C). The same results were observed in the mPFC downstate (Fig. 9C). This is in agreement with the observation that a majority of OFC downstate occurred in synchrony with mPFC downstate (Fig. 9D).

Fig. 8.

Coordinated slow-wave activities in wide areas of the prefrontal cortex and central olfactory system. A: simultaneous recordings of LFPs in the OFC, medial prefrontal cortex (mPFC), APC, posterior piriform cortex (PPC), and olfactory bulb (OB). Vertical bars on the OFC-LFP and mPFC-LFP indicate the peak of depth-positive potential (tentative downstate) in the OFC and mPFC. Arrows show APC-SPWs, PPC-SPWs, and OB-SPWs. B: filtered traces of OFC-LFP, mPFC-LFP, APC-LFP, PPC-LFP, and OB-LFP. Each LFP was downsampled to 100 Hz and low-pass filtered at 4 Hz. C: averaged coherence of slow-wave activity between OFC and mPFC, OFC and APC, OFC and PPC, OFC and OB, mPFC and APC, mPFC and PPC, and mPFC and OB. Data represent means ± SD (n = 3 rats). **P < 0.01, ***P < 0.001; n.s., not significant (one-way ANOVA with post hoc Tukey test).

These observations suggest that APC-SPWs, OFC upstate, and mPFC upstate tend to be coherent. Given that APC-SPWs are transmitted to the OB and olfactory tubercle (Komano-Inoue et al. 2014; Manabe et al. 2011; Narikiyo et al. 2014), these observations suggest that APC-SPW signals that occur in temporal coordination with upstate of both the OFC and mPFC may be transmitted to the OB and olfactory tubercle. In other words, SPW-associated, synchronized neuronal activity of the large-scale networks of the central olfactory system (APC, PPC, olfactory tubercle, and OB) may occur in temporal association with the enhanced neuronal activities of the large-scale networks of the OFC and mPFC during the time windows of shortly after the down-to-up transition and shortly before the up-to-down transition of the prefrontal cortex.

DISCUSSION

Temporal coordination of OC SPWs with upstate of OFC slow oscillations.

These results indicate that SPW-associated, synchronized discharges of APC neurons occur in phase with the upstate of the OFC and that these APC neurons tend to show reduced activity during the downstate of the OFC (Fig. 1). Given that OFC neurons show enhanced spike activity during the OFC upstate, these results suggest that the upstate of the OFC provides a time window for enhanced communication between OFC and APC neurons. Consistent with this notion, ∼45% of recorded OFC neurons showed enhanced spike discharges in synchrony with APC-SPWs, a period in which numerous APC neurons show synchronized discharges (Manabe et al. 2011). However, present experiments do not address the question of whether APC and OFC neurons are directly interacting during the slow-wave sleep.

It can be speculated that APC and OFC coherence is mediated by direct and indirect axonal connections between these areas. First, APC and OFC have mutual direct axonal connections (Illig 2005; Neville and Haberly 2004; Ray and Price 1992) (Fig. 10).　Pyramidal cells in the APC give rise to a direct, feedforward axon projection to the OFC, whereas pyramidal cells in the OFC send a direct top-down projection to the deep layer (layer III) of the APC (Illig 2005). Given that APC-SPWs are generated by excitatory synaptic inputs to the deep layer of the APC (Manabe et al. 2011), it is possible that the top-down signal transmission from the OFC to the APC may contribute to their generation. Second, APC and OFC may interact through indirect axonal connections via the MD of the thalamus. Pyramidal and multipolar cells in the deep layers of the APC project axons to the MD (Neville and Haberly 2004; Ray and Price 1992), and the thalamocortical neurons in the MD send axons to the OFC (Fig. 10). Because APC-SPWs are considered to be generated by excitatory synaptic inputs to the deep layer of APC, the synchronized activity of APC neurons during APC-SPWs might be transmitted to the OFC via these indirect transthalamic pathways. Finally, neurons in the En receive axonal inputs from APC pyramidal cells and project axons to the OFC (Behan and Haberly 1999), raising the possibility that En neurons might be involved in the interaction between the APC and OFC during APC-SPWs. In addition, it is possible that the APC and OFC coherence can be mediated by a common influence from a third brain region. Future experiments are necessary to elucidate the circuit mechanisms of the communication between APC and OFC during slow-wave sleep.

Fig. 10.

Schematic diagram illustrating possible pathways for communication between the APC and the OFC during slow-wave sleep. Direct axonal connections: pyramidal cells (Py) in the APC project axons directly to the OFC, whereas Py in the OFC send top-down axons to the deep layer of the APC. Indirect connections: APC Py and multipolar cells in layer III project axons to the mediodorsal nucleus (MD) of the thalamus, and thalamocortical neurons (TC) in the MD project axons to the OFC. APC neurons also project axons to the endopiriform nucleus (En), and multipolar cells (MPC) in the En send axons to the OFC. For simplicity, local inhibitory interneurons are omitted from this diagram. I, II, III, layers in the APC; I, II/III, V/VI, layers in the OFC.

Detailed examination of the timing of APC-SPW occurrence with reference to the time course of OFC upstate showed that APC-SPWs occur preferentially either at the beginning of upstate, shortly after the down-to-up transition of OFC slow-wave activity, or at the end of upstate, shortly before the up-to-down transition. This observation suggests that enhanced coherence between APC and OFC neurons occurs at specific time windows shortly after the down-to-up transition and shortly before the up-to-down transition of OFC slow-wave activity. The upstate of the neocortex slow oscillations provides the time window for the generation of not only APC-SPWs but also spindle activities during sleep. During slow-wave sleep, the neocortex shows two types of spindle that differ in frequency and generation area: 1) fast spindles with peak frequencies between 12 and 15 Hz and widespread distribution over the central and parietal areas and 2) slow spindles with peak frequencies between 9 and 12 Hz and focused distribution over the frontal cortical areas (Anderer et al. 2001; De Gennaro and Ferrara 2003; Mölle et al. 2011; Terrier and Gottesmann 1978). Interestingly, whereas fast spindles in the central and parietal areas are synchronized with the down-to-up transition of slow-wave activity, the slow spindles in the frontal cortical areas occur in association with the up-to-down transition (Mölle et al. 2011). These results suggest that during upstates, the time windows shortly after the down-to-up transition and shortly before the up-to-down transition play distinct, functional roles in thalamocortical and corticocortical communications. The emergence of down-to-up transition in the neocortex may drive thalamic spindle generation and mediate communications between the central and parietal cortices and thalamus at the early phase of upstate (Anderer et al. 2001; Mölle et al. 2011). In contrast, the generation of slow spindles during up-to-down transitions may originate in the frontal cortex areas at the late phase of upstate (Anderer et al. 2001; Mölle et al. 2011), raising the possibility that the slow spindles might be involved in the generation of downstate.

Consistent with the two types of spindle, our study identified two types of APC-SPW: 1) initial-phase APC-SPWs, which occur during the initial phase of upstate shortly after the down-to-up transition, and 2) late-phase APC-SPWs, which occur during the last part of the upstate shortly before the up-to-down transition. In analogy with the functional difference in the two types of spindles, we speculate that initial-phase APC-SPWs differ from late-phase APC-SPWs in the functional meaning of APC-OFC communication. At the initial phase of upstate, initial-phase APC-SPWs may drive OFC neurons, or synchronized activity of OFC neurons drives the initial-phase APC-SPWs to generate and maintain upstate in both the OFC and APC. At the late phase of upstate, on the other hand, late-phase APC-SPWs might mediate distinct interactions between the APC and OFC to generate a synchronized downstate in the OFC and APC. Consistent with this notion, recent experimental results revealed that the up-to-down transition (onset of downstate) is more synchronous between remote populations of neurons than the down-to-up transition (onset of upstate) (Chen et al. 2012), suggesting the presence of a neural circuit mechanism that actively generates the downstate synchronously across the OFC and APC. Further studies are necessary to elucidate the functional roles of APC-OFC communication at the initial and late phases of upstate.

Grouping influence of frontal cortex slow-wave activity on OC SPWs.

Hippocampal SPW/ripple activities and thalamic spindle activities occur in temporal coordination with neocortical upstates during slow-wave sleep (Isomura et al. 2006; Mölle and Born 2011; Siapas and Wilson 1998; Sirota et al. 2003; Steriade et al. 1993a, b, c). Neocortical fast oscillatory activity (20–60 Hz) also occurs in synchrony with the upstates (Steriade 2006). The present results indicate that OC SPW activities also occur in temporal coordination with the neocortical upstate. Our results thus support the idea that neocortical upstate provides key time windows for the sleep-time communications across large-scale neuronal networks that involve OC areas, hippocampal regions, thalamic nuclei, and neocortex areas, including the frontal cortex.

It has been shown that the firing pattern of hippocampal cornu ammonis 1(CA1) neurons that occurs during awake exploratory behavior appears to be replayed in association with hippocampal SPW/ripple events during the upstate of slow-wave activity in the neocortex (Ji and Wilson 2007). Coordinated activity between hippocampal SPW/ripple events and the neocortical upstate is hypothesized to play a role in the process of the memory consolidation (Girardeau et al. 2009; Ji and Wilson 2007; Siapas and Wilson 1998; Sirota et al. 2003). Our present results suggest the possibility that SPW-associated, synchronized activities of APC neurons provide the means to communicate widely with the OFC during the upstate. We speculate that coordinated activities of the APC and OFC during the OFC upstate of slow oscillation might represent the reactivation of neuronal firing patterns that occurred during wakefulness and that the coordination between APC-SPWs and OFC upstate may be associated with olfactory memory consolidation.

This study showed that not only APC-SPWs but also PPC-SPWs and OB-SPWs occurred during the early and late phases of the upstate of both the OFC and mPFC. In other words, SPW events in large-scale networks of olfactory areas are coordinated in precise time windows during the upstates of the large-scale networks of the frontal cortex. This suggests that many OC areas, neocortex areas, thalamic regions, and hippocampal regions communicate with each other during the early and late phases of the upstate of neocortical slow-wave activity. Further studies are necessary to elucidate the functional roles of this communication among the large-scale networks of the forebrain during slow-wave sleep.

GRANTS

Support for this work was provided by Grants-in-Aid for Scientific Research on Innovative Areas (25135708) and for Young Scientists (B; 25830003) and Takeda Science Foundation (to H. Manabe).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

N.O., H.M., and K.M. conceived and designed research; N.O. and H.M. performed experiments; N.O. and H.M. analyzed data; N.O., H.M., and K.M. interpreted results of experiments; N.O. prepared figures; N.O., H.M., and K.M. drafted manuscript; N.O., H.M., and K.M. edited and revised manuscript; N.O., H.M., and K.M. approved final version of manuscript.