Odor- and state-dependent olfactory tubercle local field potential dynamics in awake rats.

J Neurophysiol 111: 2109 –2123, 2014. First published March 5, 2014; doi:10.1152/jn.00829.2013.

Odor- and state-dependent olfactory tubercle local field potential dynamics in awake rats Kaitlin S. Carlson,1 Maggie R. Dillione,1 and Daniel W. Wesson1,2 1 Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio; and 2Department of Biology, Case Western Reserve University, Cleveland, Ohio

Submitted 22 November 2013; accepted in final form 14 February 2014

Carlson KS, Dillione MR, Wesson DW. Odor- and state-dependent olfactory tubercle local field potential dynamics in awake rats. J Neurophysiol 111: 2109 –2123, 2014. First published March 5, 2014; doi:10.1152/jn.00829.2013.—The olfactory tubercle (OT), a trilaminar structure located in the basal forebrain of mammals, is thought to play an important role in olfaction. While evidence has accumulated regarding the contributions of the OT to odor information processing, studies exploring the role of the OT in olfaction in awake animals remain unavailable. In the present study, we begin to address this void through multiday recordings of local field potential (LFP) activity within the OT of awake, freely exploring Long-Evans rats. We observed spontaneous OT LFP activity consisting of theta- (2–12 Hz), beta- (15–35 Hz) and gamma- (40 – 80 Hz) band activity, characteristic of previous reports of LFPs in other principle olfactory structures. Beta- and gamma-band powers were enhanced upon odor presentation. Simultaneous recordings of OT and upstream olfactory bulb (OB) LFPs revealed odor-evoked LFP power at statistically similar levels in both structures. Strong spectral coherence was observed between the OT and OB during both spontaneous and odor-evoked states. Furthermore, the OB theta rhythm more strongly cohered with the respiratory rhythm, and respiratory-coupled theta cycles in the OT occurred following theta cycles in the OB. Finally, we found that the animal’s internal state modulated LFP activity in the OT. Together, these data provide initial insights into the network activity of the OT in the awake rat, including spontaneous rhythmicity, odor-evoked modulation, connectivity with upstream sensory input, and statedependent modulation. olfaction; behavior; olfactory cortex; olfactory bulb

(OT) is a trilaminar structure located in the basal forebrain, where it is a part of the olfactory cortex (Cleland and Linster 2003; Wesson and Wilson 2011). The OT receives dense monosynaptic input from mitral and tufted cells in the olfactory bulb (OB) (Haberly and Price 1977; Nagayama et al. 2010; Schwob and Price 1984b; Scott et al. 1980; White 1965), as well as bisynaptic input from piriform cortex association fibers (Carriero et al. 2009; Johnson et al. 2000; Luskin and Price 1983). Precisely, mitral and tufted cells project into layer I of the OT (Nagayama et al. 2010; Schwob and Price 1978, 1984a; Scott et al. 1980), while the input to layers II and III originates from association fibers in the piriform cortex, dorsal peduncular cortex, and the ventral tenia tecta (Luskin and Price 1983; Schwob and Price 1984b). The role of the OT in olfactory processing is presently unknown. However, it is becoming increasingly clear that the OT shares similarities in odor information processing with

THE OLFACTORY TUBERCLE

Address for reprint requests and other correspondence: D. Wesson, Dept. of Neurosciences, Case Western Reserve Univ. School of Medicine, 2109 Adelbert Ave. Cleveland, OH 44106 (e-mail: [email protected]). www.jn.org

other principle olfactory structures. Of these, recent studies from our group have demonstrated that single units within the OT of anesthetized mice display odor-evoked activity in a similar manner as those within the piriform cortex, showing short latencies to odor-evoked spiking, respiratory coupling, and sometimes even narrow breadths of tuning (Payton et al. 2012; Wesson and Wilson 2010). Major questions still exist, however, including how odors are represented in the OTs of awake animals, and whether the OT is tightly coupled with odor output from the OB. To investigate the function of this highly understudied structure in odor processing in the awake animal, we sought to examine spontaneous and odor-evoked local field potentials (LFPs). LFPs reflect neural network activity at the population level and are commonly exploited to investigate sensory network function and the temporal relationships between interconnected neuronal groups (Fontanini and Bower 2006; Freeman 1975; Gervais et al. 2007; Kay et al. 2009; Salinas and Sejnowski 2001). The functional roles of LFPs have been of great interest for decades (e.g., Adrian 1942; Chapuis et al. 2009; Fletcher et al. 2005; Freeman and Viana Di Prisco 1986; Gelperin and Tank 1990; Kay et al. 2009; Ravel et al. 2003). Olfactory system LFPs have historically been divided into three spectral bands (with subtle variations between studies), including theta- (2–12 Hz), beta- (15–35 Hz), and gammabands (40 – 80 Hz) (for review of olfactory system LFPs, see Kay et al. 2009). These LFP bands are each hypothesized to play unique roles within the olfactory system. Theta oscillations are driven largely by intranasal sensory input and are often termed respiratory oscillations (Kay and Stopfer 2006; Komisaruk 1970; for review see Buonviso et al. 2006). Beta oscillations are robustly evoked by odors (Vanderwolf and Zibrowski 2001) and can last for two to four inhalation cycles (Kay and Beshel 2010). Beta oscillations are believed to reflect cooperative activity between interconnected structures (Kay and Beshel 2010; Kopell et al. 2000), and, possibly related to this, they may play a role in odor learning (Martin et al. 2004; Ravel et al. 2003). The highest frequency band, gamma, is smaller in power than beta and especially theta oscillations (Bressler and Freeman 1980) and originates near the end of an inhalation cycle (Rojas-Líbano and Kay 2008). The functional role of gamma oscillations has been investigated in the OB and olfactory cortex since the first studies conducted by Adrian (1942). These oscillations are believed to stem from the reciprocal dendrodendritic synapse between granule and mitral and tufted cells in the OB (Neville and Haberly 2003; Shepherd 1972). While growing literature is available regarding LFPs in both the OB and piriform cortex, much less is understood in

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other olfactory structures. In the present study, we sought to test specific questions regarding the nature of LFPs in the OT, including whether odor information shapes the structure of LFP activity in the OT, and if the OT LFP reflects similar aspects of information as do LFPs in the OB. Odor information processing in the OT is also likely modulated by the internal state of the animal (Narikiyo et al. 2014; Wesson and Wilson 2011). This may occur by means of noradrenergic locus coeruleus input (Inokuchi et al. 1988; Solano-Flores et al. 1980) and/or serotonergic input from the raphe nucleus (Hadley and Halliwell 2010). Moreover, sleeplike states modulate activity in the olfactory cortex (Barnes et al. 2011; Murakami et al. 2005; Wilson and Yan 2010), and sleep states shape spontaneous OT activity (Narikiyo et al. 2014). In addition, anesthesia impacts OB and piriform cortex network activity and functional connectivity (Fontanini and Bower 2005; Fontanini et al. 2003; Murakami et al. 2005; Wilson and Yan 2010), suggesting that odor information processing within the OT is likely dependent upon the internal state of the animal (i.e., sleep, anesthesia, arousal), although this has yet to be investigated. In the present study, we sought to characterize the odorevoked LFP activity within the OTs of awake, freely exploring rats. We hypothesized that odor information in awake rats is represented by structural and spectral changes in LFP activity. Our results reveal robust theta-, beta-, and gamma-band activities within the OT, which become enhanced during odor inhalation. We further found that LFP activity in the OT coheres with that in the OB. Finally, internal state significantly shaped spontaneous OT LFPs, but not odor-evoked LFPs. Our data provide the first characterization of odor-evoked LFP activity within the OT of awake rats and illustrate the likely importance of network oscillations and internal state to odor information processing in the OT. MATERIALS AND METHODS

Surgical procedures and animal care. Long Evans female rats (n ⫽ 11, 2– 4 mo of age, 210 –250 g) were obtained from Charles River Laboratories (Wilmington, MA) and maintained within the Case Western Reserve University School of Medicine animal facility. Rats were housed on a 12:12-h (light-dark) cycle with food and water available ad libitum. All experiments were performed during the light cycle and conducted in accordance with the guidelines of the National Institutes of Health and approved by Case Western Reserve University Institutional Animal Care and Use Committee. Rats were anesthetized with isoflurane anesthesia (3.5–1% in 1.5 l/min O2) and mounted in a stereotaxic frame with a water-filled heating pad (38°C) positioned beneath to maintain body temperature. Anesthesia depth was verified by the absence of the toe-pinch reflex at which time rats were injected with Carprofen (Rimadyl, Pfizer Animal Care, 5 mg/kg sc). The scalp was shaved, cleaned with betadine and 70% ethanol, and an injection of lidocaine (0.1 ml of 1% in dH2O sc) was given in the surrounding scalp area prior to incision. A midline incision was then made from ⬃4 mm posterior to the nose, until ⬃2 mm posterior to lambda. The connective tissue was cleared away to expose the skull, which was subsequently cleaned with 3% H2O2, allowed to dry, and covered with Vetbond (3M, St. Paul, MN). Following focal craniotomies, each animal (n ⫽ 11) was implanted with a s/s bipolar electrode (0.005-in. outer diameter, Teflon coated to 0.007 in. outer diameter, A-M Systems, Carlsborg, WA) into the OT (2.0 mm anterior bregma, 1.5 mm lateral, 8.5 mm ventral). A subset of these animals (n ⫽ 7) also received a bipolar electrode implant (same material as above) into the ipsilateral OB (center of OB, 1.4

mm ventral) and/or a thermocouple (n ⫽ 5) into the contralateral dorsal nasal recess to access respiratory transients (catalog no. 5TCTT-K-36-36, Omega, Stanford, CT) (Uchida and Mainen 2003; Wesson 2013). Thermocouples (0.9 mm lateral of the midline, on nasal fissure) were lowered to extend ⬃0.3 mm into the nasal cavity and secured in place along with the electrodes using dental cement (Teets Cold Cure, Diamond Springs, CA). All rats were implanted with four 0 – 80 s/s screws into the skull for anchoring of the cement. A bare s/s wire (0.008 in., A.M. Systems) was connected to a contralateral skull screw to serve as a reference electrode. Finally, the entire assembly, which consisted of all leads presurgically soldered onto gold pins (A-M Systems) and inserted into a plastic female screw-plug adaptor (Emka Technologies, Falls Church, VA), was cemented onto the skull. After surgery, rats were returned to their home cages and allowed to recover on a heating blanket overnight. All rats received daily injections of Carprofen for 4 –5 days postsurgery and were singly housed for the remainder of the study. Data acquisition. Recordings of OT and OB LFP activity, intranasal respiration, and movement were collected via a screw-on radiofrequency transmitter (13 g weight, Emka RodentPACK, Falls Church, VA) secured to the head plug. Movement from an accelerometer, intrinsic to the transmitter, was also collected throughout all recordings. The movement signal was composed of integrated head movement along the x-, y-, and z-axes. LFP electrode activity was amplified and filtered (200-Hz cutoff) with respiration (20-Hz cutoff), and digitized/stored at 3 kHz, along with odor presentation events using a Tucker Davis Technologies amplifier and software (Alachua, FL). In the awake state, respiration was measured via the intranasal thermocouple. In the anesthetized state, the thermocouple was not sensitive enough to differentiate respiratory transients that were only weakly present among the shallow respiration; instead, a piezo foil (Parallax, Rocklin, CA) was placed under the rat’s chest to measure respiration (100⫻ gain, 20-Hz cutoff). Video was used to supplement behavioral state recordings and was digitized via a high-resolution USB camera positioned above the behavioral arena (to be defined below). Recordings. A summary timeline of the recording procedures is displayed in Fig. 1. Animals were allotted at least 1 wk of surgical recovery time prior to experiments. Following this, recordings were gathered over several days to allow assessment of spontaneous and odor-evoked LFP activity throughout changes in behavioral (awake, sleep-like) and anesthesia-induced states (urethane) within the same animal (Fig. 1). All behavioral procedures were performed in a dimly-lit, well-ventilated room maintained at 20 –22°C, except when the animals were left to fall asleep, in which case, the lights were kept on to aid in video-based indication of sleep-like states. Following surgical recovery, the rats were acclimated to the behavioral arena (Fig. 1A). The radio-frequency transmitter was screwed onto the head plug, and the animals were gently lowered into the glass testing arena (25 deep ⫻ 30 high ⫻ 30 wide; in cm) to freely explore for 20 –30 min and to acclimate to the experimental conditions. For all further recording sessions, the rats were similarly acclimated for 5 min prior to the start of the data acquisition. To acquire sleep-like state data, rats were left undisturbed and allowed to freely explore the behavioral arena for 3– 4 h, providing sufficient time to reach a sleep-like state. No odors were presented to animals in this session (Fig. 1A). Awake spontaneous data was acquired on the same day odors were presented. Rats were allowed to explore the behavioral arena prior to odor presentation, with exploration consisting of both external (rearing, sniffing) and self-directed (grooming) exploratory behaviors, as well as locomotion. Following 5 min of spontaneous data acquisition, odors were presented in recording sessions that typically lasted 15–20 min (see Stimulus delivery). Within 10 min following the awake spontaneous and odor-evoked recording, all rats were immediately anesthetized with urethane (1.5 g/kg ip) and positioned on a water-filled heating pad (38°C) to

J Neurophysiol • doi:10.1152/jn.00829.2013 • www.jn.org


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Fig. 1. Experimental timeline and electrode tip locations for olfactory tubercle (OT) and olfactory bulb (OB) recordings. A: rats were allowed 1 wk to recover following surgical implantation of the electrodes. Following this, all rats were acclimated to the behavioral arena and the head-mounted wireless transmitter for physiological recordings (outlined in order following “data” subheading). Recordings of spontaneous and odor-evoked local field potential (LFP) data occurred during awake, sleep-like, and anesthetized (urethane) states. Rat coronal stereotaxic panels displaying the location of recording electrode tips in the OB (B) and OT (C), obtained after histological staining and verification (n ⫽ 11 in OT, and n ⫽ 7 in OB). [Adapted from Paxinos and Watson 1997.]

maintain body temperature for recordings of spontaneous and odorevoked LFPs under urethane anesthesia (Fig. 1A). Five minutes of spontaneous activity was recorded before odor presentations. The odors were delivered in the same manner as in the awake paradigm (see Stimulus delivery). Following anesthetized recordings, rats were overdosed with an additional injection of urethane (1.5 g/kg ip) and transcardially perfused with 4°C 0.9% NaCl, followed by 10% formalin (Fisher Scientific, Waltham, MA). After perfusion, brains were removed and stored in 30% sucrose formalin at 4°C until sectioning. Stimulus delivery. Odors were presented through a custom airdilution olfactometer with independent stimulus lines up to the point of entry into a Teflon odor port, to eliminate any potential crosscontamination. In addition to a blank stimulus (clean air, N2), novel odor stimuli included isopentyl acetate, 2-heptanone, 1,7-octadiene,

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and ethyl butyrate (Sigma Aldrich, St. Louis, MO). These molecularly diverse odors were diluted in their liquid state to 133.32 Pa (1 Torr) in mineral oil and were then further diluted to ⬃10% (vol/vol) in the air-dilution olfactometer by mixing 206 ml odor vaporized N2 with 1,694 ml clean N2 (Medical grade; Airgas, Independence, OH). Thus stimuli were delivered at a total flow rate of 1.9 l/min. The same olfactometer and odor port were utilized throughout all experiments. During awake recordings, the odor port was positioned at the top corner of the arena to provide optimal odor delivery, while still allowing space for rapid clearance. During recordings under anesthesia, the odor port was positioned ⬃2 cm in front of the nose. In both states, 5 min of spontaneous activity were recorded, followed by novel stimulus presentation, which consisted of 5-s presentations of all four odors, presented pseudorandomly and counterbalanced, a total of four times each (n ⫽ 16 total trials) with an intertrial interval of 30 s. A mass flow exhaust vent was positioned ⬃1 m over the behavioral arena to provide clearance of the odor prior to the next trial. The odor delivery paradigm in the awake state allowed odor presentation independent of training and/or learning and therefore was well-suited for an initial characterization of odor-evoked LFP activity in the OT. Electrode placement verification. Electrode tip locations were verified with postmortem histological examinations, referencing a rat brain atlas (Paxinos and Watson 1997). Coronal brain sections (40 ␮m) were mounted on gelatin-subbed slides and stained with 0.1% cresyl violet. OB recordings sites (n ⫽ 7, total of 7 rats) were found primarily in the granule cell layer (Fig. 1B), and OT recording sites (n ⫽ 11, total of 11 rats) were located mostly within layers I or II of the anterior aspect of the OT (Fig. 1C). Data analysis. Extraction and analysis of LFP data was performed in Spike2 (Cambridge Electronic Design), similarly across all brain structures. LFP (0.1–100 Hz) and respiration (0.1–50 Hz) were filtered using a second-order low-pass Butterworth filter. Using these filtered data, we performed several specific analyses as outlined below. Throughout all of these analyses, LFP spectral bands were defined as theta (2–12 Hz) (Kay 2003a), beta (15–35 Hz) (Kay and Beshel 2010), and gamma (40 – 80 Hz) (Cenier et al. 2009; Kay et al. 2009). We additionally analyzed for differences in both low (40 – 65 Hz) and high (65– 80 Hz) gamma-band activity (Kay 2003b). For analysis of spontaneous activity (awake, sleep-like, anesthetized), two ⬃200-s time epochs of fast Fourier transform (FFT) power spectra (1.34-s Hanning window, 0.75-Hz resolution) were taken from each rat in each state and were averaged to obtain spontaneous state spectra. The epochs were taken from the freely exploring activity in the awake state (n ⫽ 11 for OT, n ⫽ 7 for OB), the sleep-like state (n ⫽ 6 for OT, n ⫽ 6 for OB), and before odor presentation when the animal was anesthetized (n ⫽ 11 for OT, n ⫽ 7 for OB). Sleep-like states were defined by minimal accelerometer movement, which was confirmed by video analysis, in addition to prominent changes in LFP amplitude, including the occurrence of sharp waves which are present in slow-wave sleep in the OT (Narikiyo et al. 2014) (see Fig. 2). To quantify sharp waves, full-band LFP was filtered to 2–20 Hz and events greater than 4 SDs below the mean were trough detected (Narikiyo et al. 2014). The average of the power spectra for each rat in each state was normalized to the minimum/maximum and finally averaged across all rats to obtain a single mean power spectrum for spontaneous awake, sleep, and anesthetized states in both the OB and OT. For analysis of odor-evoked spectral activity (1.34-s Hanning window, 0.75-Hz resolution), prestimulus epochs were compared with during stimulus epochs. In the awake state, the animals moved freely within the arena as odors were presented, and thus the odors reached the rats at variable times, depending upon rat position and snout orientation. Manual inspection of each rat’s sniffing behavior indicated that novel odor orienting responses did not occur with a fixed latency following odor onset. Therefore, when investigating odorevoked LFP modulation, upon visual inspection of traces, we selected 2-s epochs prior to odor-evoked activity to compare to 2-s epochs


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Fig. 2. Physiological classification of sleep state for state-dependent analysis. Example full-band OT LFP (0 –100 Hz), sharp wave quantification, and movement trace from a single rat during free-exploration of the behavioral arena, including epochs identified as sleep. Movement from the head-mounted accelerometer displays an integrated head movement on the x-, y-, and z-axes. Data taken for analyses from this rat include two epochs of 200 s each (“sleep”). During these times, OT LFP amplitude and the rate of sharp waves increased (Narikiyo et al. 2014), while accelerometer movement ceased. Sleep states were further confirmed by video.

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during odor-evoked activity. Time epochs usually included the last 2 s of odor presentation. For comparison of odor-evoked LFP modulation to non-odor-evoked modulations in spontaneous LFP, we selected consecutive 2-s epochs during spontaneous activity (prior to odor presentation), which we refer to as the “shuffle.” Any trial confounded by an artifact (grooming, head bumping into the arena wall or floor, etc.) was excluded from analysis. In the urethane-anesthetized rats, the stimulus epochs were selected based directly on the odor presentation and duration windows. The intervals chosen for comparison were preodor (⫺5 to 0 s) and during odor (0 to 5 s) epochs. From these prestimulus and during stimulus epochs, odor-evoked magnitudes were calculated within each spectral band (theta, beta, and gamma) using established methods (Kay and Beshel 2010). For this, we normalized the power spectra during odor (2 s in the awake state, 5 s in the anesthetized state) to the power spectra before odor (2 or 5 s, respectively). These values were used to calculate the band powers (theta, beta, gamma), which were then converted to decibel power ratios using the formula 10 ⫻ log10 (band power during odor/band power preodor) (Kay and Beshel 2010). A value close to zero would indicate little change in odor-evoked band power, while a negative or positive value would indicate a decrease or increase in odor-evoked band power, respectively (Kay and Beshel 2010). LFP coherence calculations (Chabaud et al. 1999) were created using the COHER open-ware script freely available at (http://www. ced.co.uk), as described previously (Wesson et al. 2011; Wilson and Yan 2010). For the COHER performance, the OB (for OT-OB analysis) or respiration (for respiration-OB/OT analysis) was set as the reference signal. Data from all rats with appropriate signals (i.e., confirmed electrode tracks, no artifacts present, consistent signal) contributed to our data analyses. Furthermore, data that contained artifacts within epochs of interest (odors) were omitted from analyses. In no other instances were any animals excluded from analysis. For all statistical comparisons, individual spectral band activity means or power ratio means across trials for each rat were averaged to obtain the final mean spectral band activity or final mean power ratio. Each individual rat thus contributed a single mean value in its respective band or power ratio for use in statistical comparisons. Residual degrees of freedom may vary considerably based on the number of animals being compared. All statistical analyses were performed in Microsoft Excel, StatVIEW (SAS Institute, Cary, NC) or MATLAB (Mathworks, Waltham, MA), and all data are reported as means ⫾ SE unless noted otherwise. RESULTS

In the present study, we recorded the spontaneous and odor-evoked LFP activity in the OTs of awake rats using

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bipolar stainless steel electrodes and radio telemetry. In this design, the majority of rats contributed to within subjects’ measures of both spontaneous and odor-evoked activity. Spontaneous LFP activity in the OT of awake rats. For an initial description of LFP activity in the OT of awake rats, we recorded spontaneous activity during free exploration of the behavioral arena following a period of acclimation. We found OT LFP activity to consist of high-frequency (⬎15 Hz) activity riding on top of phasic slow-wave oscillations (⬍6 Hz) (Fig. 3A). Comparison of OT LFP with intranasal respiration reflects that the slow oscillations often coincide phasically with respiratory transients, as seen in other olfactory structures, including the OB (Cenier et al. 2009; Courtiol et al. 2011; Kay and Laurent 1999) and piriform cortex (Buonviso et al. 2006; Litaudon et al. 2008; Poo and Isaacson 2009). Furthermore, occasional bursts of both beta- and gamma-band oscillations appeared to correspond with respiration (Fig. 3A). For an initial quantification of this LFP activity, we employed an FFT analysis across data from 11 rats with quality OT LFP signals (see MATERIALS AND METHODS) to quantify the ongoing spontaneous spectral power within the OT (Fig. 3B). Theta-band activity was significantly greater within the OT than either beta[F(1,20) ⫽ 15.278, P ⫽ 0.0009] or gamma-band activity [F(1,20) ⫽ 17.718, P ⫽ 0.0004] (Fig. 3B), with beta-band power largely dominating over that of gamma-band [F(1,20) ⫽ 13.254, P ⫽ 0.0016]. Furthermore, low gamma-band was significantly greater than high gamma-band activity [F(1,20) ⫽ 11.118, P ⫽ 0.0033] (data not shown). Odor-evoked LFP activity in the awake OT. A hallmark of the olfactory system is the prominent increase in theta-, beta-, and gamma-band LFP activity upon odor inhalation (Adrian 1950; Buonviso et al. 2003; Freeman 1975; Kay et al. 2009; Laurent and Davidowitz 1994). Therefore, we next addressed how LFP activity within the OT is shaped through odor stimulation by presenting odors to five rats freely exploring the behavioral arena (same day as spontaneous recording in Fig. 3). We found that odor stimulation evoked increases in OT LFP activity in both the beta- and gamma-bands. The example in Fig. 4A displays OT LFP activity and respiration from an awake rat during presentation with the odor 2-heptanone. Following odor onset (and diffusion of the odor through the arena), the rat initiated high-frequency sniffing, reflecting iden-



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Fig. 3. OT LFP activity in awake rats. A: example respiratory and OT LFP traces from a rat during a period of free exploration. Shown are band-pass filtered traces of beta (15–35 Hz) and gamma (40 – 80 Hz) activity, as well as full band LFP (0 –100 Hz). A magnified trace from this data is also shown (right) to illustrate high-frequency activity (⬎15 Hz) riding on top of phasic slow-wave oscillations (⬍6 Hz). Black bar indicates point of trace used in example. Respiration (50 Hz low-pass) was acquired from a nasal thermocouple; movement was acquired from head-mounted accelerometer. Vertical gray lines indicate respiration peaks (maximum inhalations). B: mean normalized fast Fourier transform (FFT) power spectrum showing the distribution of energy from 0 to 80 Hz across 11 rats, clipped at 0.6 power due to a large theta-band peak at ⬃4 Hz. Inset: histogram (left) displays quantification of normalized power in the theta-, beta-, and gamma-bands. A histogram of gamma power on a finer scale (right) is included to illustrate the inclusion of gammaband power in the OT LFP. Data were normalized to min/max with two ⬃200-s epochs from each rat. ***P ⬍ 0.001, **P ⬍ 0.01, ANOVA followed by Fisher’s paired least-significant difference (PLSD).

tification of the stimulus (Fig. 4A, arrow) (Wesson et al. 2008). Soon after this sniffing response, the rat was observed orienting to the stimulus, indicated by the prominent increases in the accelerometer signal acquired from the head transmitter (“movement”; Fig. 4A, bottom). Overall, the behavioral and LFP activity was relatively conserved across rats, with subtle variability in the latency to sniffing and LFP responses inherent in our presentation paradigm. We calculated odor-evoked power ratios between a 2-s during-odor epoch and a 2-s preodor epoch (Fig. 4B). This method accounts for minor variations in electrode placement and electrode impedance present in LFPs which can otherwise contribute variability to across-animal analyses (Kay and Beshel 2010). In addition to calculating odor-evoked power

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ratios, we also computed power ratios of spontaneous LFP activity by pseudorandomly selecting consecutive 2-s epochs during the spontaneous activity. These “shuffled” power ratios allow for direct statistical comparisons between odor-evoked and spontaneous (shuffled) LFP power. As predicted, the shuffled power ratios were typically values close to zero (Fig. 4B), reflecting little fluctuations in ongoing spontaneous LFP activity. Shuffled power ratios were most variable in the theta-band, displaying moderate fluctuations relative to zero (Fig. 4B), possibly resultant from the impact of free exploration (sniffing, whisking, movement) on theta-band activity (Kay 2005; Macrides et al. 1982; Vanderwolf 1992). As suggested by the traces in Fig. 4A, we found positive odor-evoked power ratios across all rats and all spectral bands (Fig. 4B). Compared with the shuffled power ratios, we found a significant increase in odor-evoked activity in the beta- [F(1,8) ⫽ 33.033, P ⫽ 0.0004] and gamma-bands [F(1,8) ⫽ 12.159, P ⫽ 0.0082], and an increase in the theta-band, although this was not significant [F(1,8) ⫽ 5.292, P ⫽ 0.0504] (Fig. 4B). Odor-evoked power ratios in the low and high gamma-bands were statistically similar [F(1,8) ⫽ 3.244, P ⫽ 0.1094]. These data demonstrate that OT LFP activity in awake rats, specifically that within the beta- and gamma-bands, is strongly modulated by odors. LFP activity within the OT compared with the OB. While a considerable amount of information is available on odor information processing in the OB and on the transmission into and coupling of this information with downstream structures (e.g., piriform cortex, entorhinal cortex, hippocampus) (Chapuis et al. 2009, 2013; Staubli et al. 1995), whether or not this occurs between the OB and the OT is unknown. We predicted that both the OT would share aspects of spontaneous activity with the OB (i.e., prominent theta rhythm), and spontaneous and odor-evoked LFP activity within the OT would mimic that within the OB. To test this, we implanted seven rats with bipolar electrodes in both the OB and OT, in addition to intranasal thermocouples to access respiratory transients (see MATERIALS AND METHODS). We first explored spontaneous LFP activity in both regions. Both the OT and OB displayed mostly similar timing of theta-band oscillations and bursts in beta- and gamma-band activity (Fig. 5A). The overall power of these bands, however, did differ in a qualitative manner, especially within the thetaband. Furthermore, in some rare instances, the OT LFP theta rhythm deviated from respiration, whereas the OB did not. For example, in Fig. 5A, the OB displays two large theta cycles (green arrows) coinciding with the timing of two discrete respiratory cycles. In contrast, the OT displays two additional theta cycles that do not correspond with obvious rhythmicity in either OB LFP or respiration (Fig. 5A, red arrows). FFT analysis of this spontaneous data revealed that both the OT and OB display quantitatively similar distributions of power across all three spectral bands, albeit with subtle variations (Fig. 5B). Although the OB displayed qualitatively greater power of high theta (⬃10 Hz) and low beta (⬃20 Hz) spectra than the OT in some cases (Fig. 5 power spectrum), theta- [F(1,16) ⫽ 1.676, P ⫽ 0.214], beta- [F(1,16) ⫽ 0.0004, P ⫽ 0.986] and gamma-band power [F(1,16) ⫽ 0.427, P ⫽ 0.523] (Fig. 5B, inset), both low [F(1,16) ⫽ 0.400, P ⫽ 0.5359] and high [F(1,16) ⫽ 0.501, P ⫽ 0.4891], were not statistically different between regions.


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Fig. 4. Odor-evoked modulation of OT LFP activity in awake rats. A: example respiratory and OT LFP traces from a rat just prior to and during presentation with the odor, 2-heptanone. Shown are band-pass filtered traces of beta (15–35 Hz) and gamma (40 – 80 Hz) activity, as well as full band LFP (0 –100 Hz) and respiration (50 Hz low-pass). Arrow indicates onset of odor-evoked sniffing. B: mean odor-evoked power ratios across four odors (dark gray bars) compared with “shuffle” spontaneous power ratios (light gray bars, see MATERIALS AND METHODS). Significant increases in beta- and gamma-band power were observed in response to odor compared with shuffled data. ***P ⬍ 0.001, **P ⬍ 0.01: ANOVA followed by Fisher’s PLSD. n.s., not significant.

Next, we examined odor-evoked LFP activity within both structures by presenting the rats with odors while they were freely exploring the behavioral arena. The example traces in Fig. 6A display evoked activity in the OT and OB upon presentation with the odorant, 2-heptanone. While there are subtle differences in spontaneous theta-band power, both structures displayed noticeable increases in not only theta-, but also beta- and gamma-band power upon odor presentation (Fig. 6A). Figure 6B shows the odor-evoked power ratios from the OB, along with the previously quantified OT odor-evoked power ratios (Fig. 4B) from the same rats for comparison. In both the OT and OB, odor presentation elicited increases in odor-evoked power ratios across all three spectral bands compared with the shuffled power ratios. This was significant in both the beta- and gamma-bands in the OT [theta: F(1,8) ⫽ 5.292, P ⫽ 0.0504; beta: F(1,8) ⫽ 33.033, P ⫽ 0.0004; gamma: F(1,8) ⫽ 12.159, P ⫽ 0.008] with no difference in odor-evoked activity between low and high gamma-band activity [F(1,8) ⫽ 3.244, P ⫽ 0.1094], and across all bands in the OB [theta: F(1,8) ⫽ 6.724, P ⫽ 0.032; beta: F(1,8) ⫽ 13.178, P ⫽ 0.007; gamma: F(1,8) ⫽ 22.754, P ⫽ 0.0014], with low gamma- dominating high gamma-band activity [F(1,8) ⫽ 19.439, P ⫽ 0.0023]. Odor-evoked modulation was not statistically different between the OT and OB within each spectral band [theta: F(1,8) ⫽ 2.598, P ⫽ 0.146; beta: F(1,8) ⫽ 0.838, P ⫽ 0.387; gamma: F(1,8) ⫽ 1.259, P ⫽ 0.294 (Fig. 6B); low gamma: F(1,8) ⫽ 1.019, P ⫽ 0.3423; high gamma: F(1,8) ⫽ 2.863, P ⫽ 0.1291]. These results suggest that odor-evoked activity in the OB is mostly well-maintained by the OT. Does the similarity of odor-evoked activity between these monosynaptically coupled structures reflect functional coherence? To test this, we calculated the spectral coherence between both structures in the awake rats (same sessions as in Fig. 6). Two second epochs were selected both before (“spon-

taneous”) and during odor [n ⫽ 5–9 epochs/rat, 7 ⫾ 1.6 (mean ⫾ SD)] and subjected to a spectral coherence analysis, where 1 indicates completely coherent waveforms and 0 indicates completely incoherent waveforms in their frequency content. We found that both spontaneous and odor-evoked epochs corresponded with functional coherence across all spectral bands (⬎0.6) (Fig. 7). No significant effects were observed within bands when comparing spontaneous to odor-evoked conditions, nor across bands within condition (odor vs spontaneous, P ⬎ 0.1, ANOVA followed by Fisher’s paired least-significant difference). Thus, in both spontaneous and odor-evoked conditions, the functional connectivity of the OT and OB aligns in a manner that would allow for the coupling of information during both spontaneous and odor processing modes. The strong coherence found within the theta-band (Fig. 7) suggests that OB and OT LFP couple on a cycle-to-cycle basis, perhaps relative to respiration. To test this, we analyzed theta cycle peaks in the OB and OT relative to the inhalation onset from five rats (⬎3K inhalation-triggered events/rat). As shown in Fig. 8A from a single rat, and across all rats in Fig. 8B, inhalation-triggered OT theta cycles lagged behind those observed in the OB. OT theta cycle onset was broadly distributed over time, possibly reflecting both dual arrival of bisynaptic input from the piriform cortex and/or OB mitral and tufted cells (Ishikawa et al. 2007) and potential centrifugal influences. The latency of inhalation-triggered OT theta cycles was significantly slower than those in the OB [F(1,8) ⫽ 7.385, P ⫽ 0.0264] (Fig. 8C). Given the strong relationship between the OT and OB theta peaks and respiration, we also performed spectral coherence analysis between respiration (0 –15 Hz) and OT and OB theta-band activity (Fig. 8D). This analysis revealed, as suggested by Figs. 5A and 8, that the OB theta rhythm differentially coheres with respiration compared with the OT theta



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Frequency (Hz) Fig. 5. Spontaneous LFP activity within the OT compared with the upstream OB. A: example respiratory and LFP traces of awake spontaneous activity in the OT and OB. Traces are band-pass filtered for beta (15–35 Hz) and gamma (40 – 80 Hz) activity. Full band LFP (0 –100 Hz) and respiration (50 Hz low-pass) are also displayed. Green arrows indicate moments of theta cycles that cohere between the OT and OB and discrete respiratory cycles. Red arrows indicate theta cycles in the OT without obvious relationships to those in the OB or respiratory cycles. Gray vertical lines indicate respiration peaks. B: mean FFT power spectrum and band power comparison (inset) for OT and OB. FFT averaged from two ⬃200-s spontaneous epochs/rat in OB and OT. The power spectrum is clipped at 0.6 power due to a large theta-band peak at ⬃4 Hz. ANOVA followed by Fisher’s PLSD.

rhythm (P ⫽ 0.0086, Kolmogorov-Smirnov test), with the OB rhythm being more coherent [F(1,16) ⫽ 12.615, P ⫽ 0.0027] (coherence across all frequency bins in OB vs. OT) (Fig. 8D). Impact of anesthetic- and behavioral-state on spontaneous OT LFP activity. Sensory processing, including that within the olfactory system, is highly state-dependent (Bouret and Sara

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2002; Fontanini and Katz 2008; 2006; Lavin et al. 1958; Li et al. 2012; McDonald et al. 1964; Wachowiak et al. 2013). Recent work in the OT has demonstrated state-dependent changes in LFP activity, where occasional sharp waves occur during spontaneous sleep states (Narikiyo et al. 2014). To further explore whether spontaneous activity is shaped by internal state, and to test whether odor-evoked activity in the OT is also influenced by internal state, we monitored OT and OB LFP activity during transitions in and out of sleep and also, separately, under urethane anesthesia. This design allowed within-subjects comparisons of awake, sleep-like, and anesthetic-induced states (see MATERIALS AND METHODS). We hypothesized that the OT would display considerable state-dependent dynamics like those observed in the OB and piriform cortex (Li et al. 2012; Murakami et al. 2005; Narikiyo et al. 2014; Wilson et al. 2011). Particularly, we predicted that both urethane and sleep-like states would quantitatively impact the levels of spontaneous power across all LFP spectral bands and also odor-evoked power in the OT. We found striking state-dependent differences in spontaneous LFP activity in the OT and, as reported elsewhere, in the OB (Li et al. 2012) (Fig. 9A). Prominent differences in theta-band structure were observed, likely resultant from differential patterns of respiration throughout these states (Fig. 9A). Furthermore, in both sleep-like and urethane anesthesia states, we observed sharp-wave activity in the OT (Fig. 9A, arrows). Since the prominence and role of sharp-wave activity in the OT has been reported elsewhere (Narikiyo et al. 2014), we did not analyze its occurrence here. To quantify possible state-dependent changes in OT LFP activity, we averaged two 200-s epochs of spontaneous activity from each rat (n ⫽ 6 –11) in awake, sleep-like, and anesthetized states (Fig. 9B). OT beta-band activity was significantly greater in the awake [F(1,20) ⫽ 10.193, P ⫽ 0.005] and sleep-like states [F(1,15) ⫽ 14.913, P ⫽ 0.002] compared with the anesthetized state. OT beta-band activity in the awake state was similar to that in the sleep-like state [F(1,15) ⫽ 1.596, P ⫽ 0.226]. Similarly, OT gamma-band activity was significantly greater in the awake [F(1,20) ⫽ 11.892, P ⫽ 0.003] and sleep-like states [F(1,15) ⫽ 14.610, P ⫽ 0.002] compared with the anesthetized state. Similar to that seen in beta power, OT gamma-band activity in the awake state did not differ from that in the sleep-like state [F(1,15) ⫽ 3.433, P ⫽ 0.084] (Fig. 9B, left). OT theta-band activity was significantly greater in the sleep-like state [F(1,15) ⫽ 7.971, P ⫽ 0.013], but not in the awake state [F(1,20) ⫽ 4.278, P ⫽ 0.052] compared with the anesthetized state. Finally, awake and sleep-like spontaneous state theta-band activity were similar [F(1,15) ⫽ 0.053, P ⫽ 0.821] (Fig. 9B, left). In contrast to the OT, state-dependent effects in the OB were less frequently observed (Fig. 9B, right). OB beta-band activity did not significantly differ across states [awake vs. urethane: F(1,12) ⫽ 2.877, P ⫽ 0.116; sleep-like vs. urethane: F(1,11) ⫽ 3.137, P ⫽ 0.104; awake vs. sleep-like: F(1,11) ⫽ 1.263, P ⫽ 0.285] and, similarly, OB gamma-band activity did not differ across states [awake vs. urethane: F(1,12) ⫽ 3.926, P ⫽ 0.071; sleep-like vs. urethane: F(1,11) ⫽ 2.100, P ⫽ 0.175; awake vs. sleep-like: F(1,11) ⫽ 1.275, P ⫽ 0.283] (Fig. 9B, right). OB theta-band activity was, however, significantly greater than


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β Beta Fig. 6. Similar odor-evoked power in the OT and OB. A: example respiratory and LFP traces of awake odorevoked activity in the OT and OB. Shown are bandpass filtered traces of beta (15–35 Hz) and gamma (40 – 80 Hz) activity, as well as full band LFP (0 –100 Hz) and respiration (50 Hz low-pass). B: mean odorevoked power ratios across four odors (dark shaded bars) compared with “shuffle” spontaneous power ratios (light shaded bars, see MATERIALS AND METHODS). n ⫽ 12–24 shuffle or odor epochs/rat. Significant increases in beta- and gamma-band power were observed in response to odors compared with shuffled data. No significant differences were found between OT and OB in any bands. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001: ANOVA followed by Fisher’s PLSD.

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urethane in the awake state [F(1,12) ⫽ 5.140, P ⫽ 0.043], but, compared with the sleep-like state, it was not [F(1,11) ⫽ 0.762, P ⫽ 0.401]. Our findings confirm and extend previous observations of state-dependent influences on spontaneous OT LFP activity (Narikiyo et al. 2014) and suggest a greater impact of internal state on OT dynamics relative to OB dynamics. Impact of anesthesia on odor-evoked LFP activity. Do these observed state-dependent changes in spontaneous OT LFP activity also impact odor-evoked activity? To test this, we directly compared odor-evoked power ratios in the awake state to those acquired under urethane anesthesia within the same recording session. Given that odor-evoked responses in the OB are prominently impacted by anesthesia (Adrian 1950; Rinberg

**

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et al. 2006; Wachowiak et al. 2013), and in some cases can be heightened (Rinberg et al. 2006), we hypothesized that, in the anesthetized state, odor-evoked power ratios in the OT would be greater than when awake. The example traces in Fig. 10A are from a single rat both before and 20 min after injection with urethane. Most noticeably, urethane resulted in a slowing of respiratory frequency, which altered the temporal structure of odor-evoked activity in the OT, likely resultant from changed odor inhalation patterns. Nevertheless, between states, odor-evoked activity appeared qualitatively different (Fig. 10A). To quantify anesthesia-dependent changes in odor-evoked LFP activity, we calculated odor-evoked power ratios (5 s pre vs. 5 s during, see MATERIALS



mean spectral coherence +/- SEM

0.75

spontaneous

DISCUSSION

odor

In the present study, we examined odor information processing in the OT of awake rats by means of LFP recordings. As part of this objective, we also determined whether LFP responses in the OT coincide with those in the upstream OB and how these may be shaped by the internal state of the animal. Specifically, the present results provide novel evidence that the OT LFP in awake animals 1) represents odors with strikingly similar spectral powers compared with the OB, 2) is strongly coherent with the OB during both spontaneous and odorevoked states, and 3) is shaped by the internal state of the animal. Together, our results provide new insights into how spontaneous and odor-evoked LFP activity is represented in the OT of awake animals, supporting and, in some cases, raising new hypotheses regarding the role of the OT in mammalian olfaction. Odor processing in the OT of awake animals. We hypothesized that odor information in the OTs of awake rats is represented by changes in LFP spectral power. This straightforward hypothesis follows suit with known aspects of odorevoked LFP activity in the OB and piriform cortex, with each displaying increases in spectral power during odor inhalation (e.g., Buonviso et al. 2003; Cenier et al. 2008; Chapuis et al. 2009; Freeman and Baird 1987; Kay and Beshel 2010). In addition, this hypothesis agrees with the prominent recruitment of neurons within the OT upon odor presentation (Carlson et al. 2013; Payton et al. 2012; Rampin et al. 2012). Among the spectral bands that have received the most attention in terms of being shaped by odor and odor quality, are beta and gamma (Cenier et al. 2008; Lepousez and Lledo 2013; Lowry and Kay 2007; Martin et al. 2006, 2007). We found that the OT is also characterized by considerable beta-band power and detectable, yet minor, gamma-band power. Furthermore, we found that the power of these bands becomes significantly elevated during odor presentation, a finding that is in agreement with studies in other olfactory structures, including the OB, piriform cortex, and anterior olfactory nucleus (e.g., Adrian 1950; Beshel et al. 2007; Bressler and Freeman 1980; Manabe and Mori 2013; Neville and Haberly 2003). While our analysis did not allow for a direct test of how these high-frequency oscillations are coupled with respiration, the timing of the beta- and gammaband bursts suggests that these oscillations may on occasion coincide with respiration, as seen elsewhere in the olfactory system (Cenier et al. 2009; Manabe and Mori 2013; Roux et al. 2007). Similar experiments in the future using wavelet-based LFP analysis (Roux et al. 2007) would be useful in testing this prediction. An additional significance of our observations of both spontaneous and odor-evoked beta- and gamma-band oscillations resides in manners whereby these spectral bands may impact functional connectivity of information transfer. Based upon the hypothesized roles for beta-band oscillations in the olfactory network (Kay et al. 2009), our results confirm that the OT is involved in interregional processing of odor information in behaving animals, as suggested by previous anatomical and physiological studies (Carriero et al. 2009; Chiang and Strowbridge 2007; McNamara et al. 2004; Schwob and Price 1984a; Scott et al. 1980). Whether LFP oscillations aid in the coupling of odor-evoked and/or behaviorally relevant spiking activity into downstream structures remains to be tested.

0.65

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Fig. 7. Spectral coherence of OT and OB LFP activity. Mean spectral coherence of OT and OB waveforms, organized by LFP band, in both spontaneous and odor-evoked conditions (2-s epochs) is shown. 1 ⫽ most coherent, 0 ⫽ no coherence. n ⫽ 4 rats. AND METHODS)

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from both conditions. Odor-evoked power ratios were similar between OT and OB during both the awake state (same awake-state data and stats as in Fig. 6B) and the anesthetized state [theta: F(1,8) ⫽ 0.167, P ⫽ 0.694; beta: F(1,8) ⫽ 0.002, P ⫽ 0.964; and gamma: F(1,8) ⫽ 0.178, P ⫽ 0.684] (Fig. 10B). Across both structures and spectral bands, however, there was an overall effect of anesthesia on odorevoked power ratios [F(1,28) ⫽ 12.275, P ⫽ 0.0009], which was similarly observed within the OT itself [F(1,28) ⫽ 4.69, P ⫽ 0.039] and also in the OB [F(1,28) ⫽ 7.434, P ⫽ 0.011]. Within OT spectral bands specifically, though, LFP odorevoked power ratios were similar between awake and anesthesia conditions [theta: F(1,8) ⫽ 0.552, P ⫽ 0.479; beta F(1,8) ⫽ 4.413, P ⫽ 0.069; and gamma: F(1,8) ⫽ 4.037, P ⫽ 0.079] (Fig. 10B). The restricted effect of anesthesia on the power ratios was recapitulated in the OB, but, in this structure, a significant difference in the theta-band was observed [theta: F(1,8) ⫽ 8.767, P ⫽ 0.018; beta: F(1,8) ⫽ 1.664, P ⫽ 0.233; gamma: F(1,8) ⫽ 3.411, P ⫽ 0.102]. Odor-evoked low and high gamma-band activity were not statistically different in the OT [F(1,8) ⫽ 0.122, P ⫽ 0.7360] or the OB [F(1,8) ⫽ 0.146, P ⫽ 0.7125]. To overcome the prominent interanimal variations, and allow for a more clear assessment of the effect of anesthesia on odor-evoked activity, we also explored the change in each band’s power at the individual rat level. In both the OT and OB, across all spectral bands, most rats displayed increases in odor-evoked power ratios under anesthesia compared with the awake condition (Fig. 10C). In fact, only one rat (out of 5) displayed reduced odor-evoked activity while anesthetized. Finally, while odor-evoked power was qualitatively greater in the OB than the OT (Fig. 10C), both regions again displayed statistically similar levels of change (%change, awake vs. urethane) in the theta [F(1,8) ⫽ 1.486, P ⫽ 0.2576], beta [F(1,8) ⫽ 0.699, P ⫽ 0.4273], and gamma-bands [F(1,8) ⫽ 1.236, P ⫽ 0.2986]. These results suggest that, at least within the context of this odor presentation paradigm, the representation of odors by LFP activity in the OT is largely independent of internal state.


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Based on our present results, we hypothesize that odors may be represented by LFPs in the OT in terms of their identity, concentration, and timing (present or not). We also predict that the robust input of odor information into the OT, together with the OT’s connectivity with numerous structures necessary for

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decision-making and motivation (for review, see Wesson and Wilson 2011), may support the role of the OT as a critical node in a network linking odor information with higher-order emotional and cognitive aspects. It will be informative in future work to determine whether the patterning and power of these oscillations are modified based on cognitive and perceptual demands, as they are in the OB (Beshel et al. 2007). Data to support cognitive modulation of odor-evoked activity in the OT will be essential in assessing the behavioral-relevance of this structure and further may provide insights into possible roles of OT neural dysfunction in neurological disorders associated with impaired ventral striatum function. Similarities and differences of odor processing in the OT and the OB. How might OT LFP activity be reflecting upstream influences from initial odor information processing in the OB? To investigate this question, we recorded LFPs simultaneously in the OT and OB. While this method cannot account for, nor rule out, possible centrifugal influences on OT processing from those originating from within the OB, several findings are worth discussing. First, we found that odor presentation elicited statistically similar increases in beta- and gamma-band powers in both structures. While the level of significance in the beta-band was greater within the OT vs. in the OB (P ⬍ 0.0001 vs. P ⬍ 0.01) (Fig. 6), there were no differences when comparing between the regions. The strong spectral power similarities between the OT and OB raise the possibility that the OT LFP may indeed represent odors in a similar manner as the OB, at least at the network level. Second, the spectral coherence between the OB and OT was considerably strong during both spontaneous and odor-evoked epochs (Fig. 7). This level of coherence is comparable to that reported previously between other primary olfactory structures (Kay and Beshel 2010; Wesson et al. 2011; Wilson and Yan 2010). Importantly, however, some aspects of the LFP differed between the OT and OB in the awake animal. Specifically, our results demonstrate that the OB is more tightly coupled to the respiratory cycle than the OT. Whereas the bulk of the theta cycles in the OB corresponded with respiratory events immediately preceding them, the theta cycles in the OT were characterized by greater diffusion relative to respiration, and a longer latency. This could be interpreted as reflecting bisynaptic input into the OT from principle OB output neurons and/or input via piriform cortex association fibers which are known to functionally impact OT activity over distributed time Fig. 8. Temporal dynamics of theta cycles in the OT and OB relative to respiration. A: raster plot of theta cycles which were peak detected in the OB (red) and OT (blue) and plotted across and relative to the onsets of ⬃5,000 inhalation cycles from a single recording in 1 rat. Averaged respiration waveform for the data in A is displayed on top of the raster plot. Vertical dashed line ⫽ moment of inhalation onset. B: staircase plot and cumulative probability (dashed lined) of theta cycle peaks across 5 rats (⬎4,000 inhalation cycles/rat, 1 session each), including the rat used for A. These population-level data illustrate that theta cycle peaks appear in the OB prior to the OT relative to inhalation onset. Red and blue vertical arrows indicate moment of theta cycles which occurred prior to the aligned inhalation onset, likely driven by the preceding inhalation. C: mean peak theta cycle latency relative to inhalation onset across all 5 rats (1 peak value/rat). As expected by the data in B and C, theta cycle peaks occurred first in the OB followed by the OT. P value ⫽ ANOVA followed by Fisher’s PLSD. D: mean spectral coherence between respiration and OB (red) or OT (blue) theta rhythm across 5 rats (same data as in B and C) (data filtered between 0.1 and 15 Hz prior to coherence analysis). OB theta rhythm displayed differential coherence with respiration than that in the OT. P value ⫽ Kolmogorov-Smirnov test.



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Fig. 9. Behavioral and anesthetic state impact the spontaneous LFP activity in the OT. A: example OT and OB traces of spontaneous LFP activity in awake (left), sleep-like (middle), and urethane-anesthetized (right) states. Shown are band-pass filtered traces of beta (15–35 Hz) and gamma (40 – 80 Hz) activity, as well as full band LFP (0 –100 Hz) and respiration (50 Hz low-pass). Downward vertical arrows indicate occurrence of sharp-wave activity. Respiration in the sleep-like state possessed low-amplitude noisy transients from the nasal thermocouple due to minute air transients during sleep. Respiration during urethane was recorded with a chest piezo foil for this reason of difficulty detecting nasal respiration in nonactively respiring rats with the thermocouple. B: mean FFT power spectrum and band power comparison (inset) for OT (left) and OB (right) organized by state. FFT was averaged from two 200-s spontaneous epochs/rat in OB and OT. The power spectrum is clipped at 0.6 power due to a large theta-band peak at ⬃4 Hz. Different rat numbers contributed to each state, depending on signal quality. *P ⬍ 0.05, **P ⬍ 0.01: ANOVA followed by Fisher’s PLSD.

scales (Carriero et al. 2009). Alternatively, this could reflect differential centrifugal input to the OT (Hadley and Halliwell 2010; Inokuchi et al. 1988; Solano-Flores et al. 1980), which may modulate the structure’s intrinsic rhythmicity. Perhaps supporting this alternative, we observed occasional OT theta cycles which occurred independent of obviously corresponding theta cycles present in the OB and/or respiration (Fig. 5).

State-dependent changes in OT network activity. We further recorded OT activity in the rats while awake, in sleep-like states, and under urethane anesthesia. Sleep-like states have been shown to modulate several types of neural activity in the olfactory cortex (Barnes et al. 2011; Manabe and Mori 2013; Murakami et al. 2005; Wilson and Yan 2010), and, relevant to this particular study, spontaneous OT activity is altered during


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sleep (Narikiyo et al. 2014). The network activity in the OB and piriform cortex is also well known to be impacted by anesthesia, including urethane (Fontanini and Bower 2005; Fontanini et al. 2003; Murakami et al. 2005; Wilson and Yan 2010). Therefore, we allowed rats to fall asleep and used combinations of respiratory frequency, video-based measures (immobility), and accelerometer signal to assay time points which we defined as “sleep-like.” It is important to note that we cannot concretely validate that the animals were in fact asleep without additional measures (e.g., neocortical EEG). Neverthe-

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Fig. 10. Impact of anesthetic state on odorevoked LFP in the OT. A: example OT and OB traces of odor-evoked LFP activity in the awake (left) and urethane-anesthetized (right) states. Shown are band-pass filtered traces of beta (15–35 Hz) and gamma (40 – 80 Hz) activity, as well as full band LFP (0 –100 Hz) and respiration (50 Hz low-pass). Black bar indicates duration of odor presentation. B: histogram displaying overview of odor-evoked power ratio comparison for OT and OB in the awake and anesthetized states. In all cases, mean activity was greater in the anesthetized state than during the awake state, albeit not always significantly. *P ⬍ 0.05, ANOVA followed by Fisher’s PLSD. C: line graph comparison of changes in theta-, beta-, and gammaband activity in the OT (blue) and OB (red) from the awake state to the anesthetized state for individual rats (n ⫽ 5).

less, our data from this sleep-like state replicated specific previous findings of OT physiology in sleeping rats, including the presence of sharp waves (Narikiyo et al. 2014) (see Fig. 2). Indeed, contributing to this growing body of literature on state-dependent influences on olfactory system function, we found that spontaneous OT activity is strongly shaped by state. The most prominently affected spontaneous activity was observed in the beta- and gamma-bands, with each modulated by both sleep-like and urethane states (Fig. 9). Thus our present results are in agreement with the hypothesis that gamma



oscillations, modulated in their presence by the state of the animal, may be important for shaping odor processing (Manabe and Mori 2013; Mori et al. 2013). In contrast, no significant changes were observed in the OB among these spectra under urethane, although all spectra showed decreases in power. Mostly in agreement with this, decreased OB betaand gamma-band powers have been reported as a consequence of urethane (Li et al. 2012). Taken together, our results suggest that the OT might be more greatly affected by urethane anesthesia than the OB. While urethane significantly affected spontaneous OT LFP activity, it did not significantly alter odor-evoked activity (Fig. 10). Only odor-evoked OB theta rhythm was significantly affected by urethane; all other spectral bands showed only insignificant trends in increasing odor-evoked power during urethane anesthesia. It is interesting to consider how state can dramatically alter spontaneous OT activity while sparing major influences on odor-evoked activity. One reason that this may be the case is that odor-evoked changes in OT dynamics are proportionally small on top of the already dramatically impacted spontaneous LFP activity. Separately, as seen also during spontaneous activity (Fig. 9), OT sharp waves, which are believed to originate from the piriform cortex (Narikiyo et al. 2014), were observed during the course of odor presentation while animals were under urethane anesthesia (Fig. 10). Their quantitative investigation, however, was beyond the scope of this study. Whether these sharp waves more frequently occur during odor and/or impact the processing of odor information within the OT requires further investigation. Conclusions. Together, these data provide initial insights into the network activity of the OT in the awake rat, including spontaneous rhythmicity, odor-evoked modulation, connectivity with upstream sensory input, and state-dependent modulation. We should note that our results do not show that the OT itself is necessary in odor processing or if it is just simply reading-out information from the OB. To answer this question, experiments specifically targeting OT activity need to be performed, along with accompanying behavioral assays for changes in olfactory processing. Nevertheless, our results describing odor information processing in the OT of awake animals, together with the established connectivity of the OT with areas essential for motivation, emotion, and higher-order functions (Alheid and Heimer 1988; Ikemoto 2007), add to the hypothesis that the OT may play a unique role in shaping and relaying odor information into behaviorally relevant downstream structures (Wesson and Wilson 2011). We anticipate, based on these observations, that these features of odor-evoked activity in the OT will be essential for odor-guided behaviors. ACKNOWLEDGMENTS We thank Dr. Marie Gadziola for comments on an earlier version of this manuscript. GRANTS This work was supported by National Science Foundation Grant IOS1121471 to D. W. Wesson. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s).

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Anesthetic regimes modulate the temporal dynamics of local field potential in the mouse olfactory bulb.

Olfactory tubercle stimulation alters odor preference behavior and recruits forebrain reward and motivational centers.

Coding odor identity and odor value in awake rodents.

'Silent' mitral cells dominate odor responses in the olfactory bulb of awake mice.

Odor memory and odor learning in rats with lesions of the lateral olfactory tract and mediodorsal thalamic nucleus.

Theta activity in local field potential of the ventral tegmental area in sleeping and waking rats.

Development of the anterior olfactory nucleus in normal and unilaterally odor deprived rats.

Processing of Intraoral Olfactory and Gustatory Signals in the Gustatory Cortex of Awake Rats.

Anesthetic-dependent field potential interactions in the olfactory bulb.

Chromatic and Achromatic Spatial Resolution of Local Field Potentials in Awake Cortex.

Astrocytes Modulate Local Field Potential Rhythm.

Complementary codes for odor identity and intensity in olfactory cortex.

Differential entrainment and learning-related dynamics of spike and local field potential activity in the sensorimotor and associative striatum.

Unilateral lesions of the olfactory tubercle modifying general arousal effects in the rat olfactory bulb.

Millisecond Coupling of Local Field Potentials to Synaptic Currents in the Awake Visual Cortex.

Illustrated Review of the Ventral Striatum's Olfactory Tubercle.

Dopamine-sensitive adenylate cyclase within laminae of the olfactory tubercle.

Catecholamine innervation of the basal forebrain. III. Olfactory bulb, anterior olfactory nuclei, olfactory tubercle and piriform cortex.

Membrane Potential Dynamics of Spontaneous and Visually Evoked Gamma Activity in V1 of Awake Mice.

On the photovoltaic effect in local field potential recordings.

Odor memories regulate olfactory receptor expression in the sensory periphery.

Cortical Feedback Decorrelates Olfactory Bulb Output in Awake Mice.

Extracellular brain glucose levels reflect local neuronal activity: a microdialysis study in awake, freely moving rats.

Predicting the response of olfactory sensory neurons to odor mixtures from single odor response.