Salicylate induces anxiety-like behaviour and slow theta oscillation and abolishes the relationship between running speed and fast theta oscillation frequency.
Jessica Winne1*, Rafael Franzon1*, Aron Miranda1, Thawann Malfatti1, João Patriota2, Sanja Mikulovic3, Katarina E. Leão1 & Richardson N. Leão1,3
1. Neurodynamics Lab, Brain Institute, Federal University of Rio Grande do Norte, Natal-RN, Brazil
2. Brain Institute, Federal University of Rio Grande do Norte, Natal-RN, Brazil 3. Department of Neuroscience, Uppsala University, Uppsala, Sweden
*. Equal contribution
Running title: Salicylate and theta
Correspondence:
Richardson N. Leão
Brain Institute, Federal University of Rio Grande do Norte, Av. Nascimento de Castro, 2155,
Natal-RN, Brazil
Grant Sponsors:
American Tinnitus Association The Brazilian Research Council
Key words
Salicylate, tinnitus, theta, ventral hippocampus, anxiety
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Article
Thi s article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which
Abstract
Salicylate intoxication is a cause of tinnitus in humans and it is often used to produce tinnitus-like perception in animal models. Here we assess whether salicylate induces anxiety-like electrophysiological and behavioural signs. Using microwire electrode arrays, we recorded local field potential in the ventral and, in some experiments dorsal hippocampus, in an open field arena 1 hour after salicylate (300mg/kg) injection. We found that animals treated with salicylate moved dramatically less than saline treated animals. Salicylate-treated animals showed a strong 4-6Hz (type 2) oscillation in the ventral hippocampus (with smaller peaks in dorsal hippocampus electrodes).
Coherence in the 4-6Hz-theta band was low in the ventral and dorsal hippocampus when compared to movement-related theta coherence (7-10Hz). Moreover, movement related theta oscillation frequency decreased and its dependency on running speed was abolished. Our results suggest that salicylate-induced theta is mostly restricted to the ventral hippocampus. Slow theta has been classically associated to anxiety-like behaviours. Here we show that salicylate application can consistently generate low frequency theta in the ventral hippocampus. Tinnitus and anxiety show strong comorbidity and the increase in ventral hippocampus low frequency theta could be part of this association.
Introduction
Acute salicylate toxicity is a common cause of tinnitus and is often used to model tinnitus in rodents (Chen et al., 2013). Peripherally, salicylate decreases the sensitivity of the cochlear sensory epithelium and also directly affects the auditory nerve (Puel and Guitton, 2007). Salicylate is also known to produce acute
electrophysiological changes in the auditory central nervous system and associated areas (e.g. the hippocampus and amygdala) (Bauer et al., 2000; Su et al., 2009; Chen et al., 2013; Gholami et al., 2015). For example, chronic exposure to salicylate alters plasticity in the hippocampus CA3-CA1 synapses decreasing long term potentiation (Gholami et al., 2015). Expression of immediate-early genes in CA1 is also affected by salicylate exposure (Wu et al., 2015).
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The limbic system is closely associated to anxiety and this condition has intimate relation with tinnitus (Pattyn et al., 2016) and a direct effect of salicylate in the limbic system may further increase tinnitus perception (Guitton et al., 2005). Interestingly, anxiety-inducing drugs associated with salicylate increase tinnitus perception in rodents (Guitton et al., 2005). Besides, tinnitus alters the connectivity between the auditory cortex and the hippocampus (Kraus and Canlon, 2012; Chen et al., 2017). Hence, anxiety related to salicylate poisoning and/or tinnitus could be caused both by a changes in auditory cortex/limbic system connectivity and via direct effect of salicylate in hippocampal circuits.
Theta oscillations of the hippocampus vary from 4 to 12 Hz and two types of theta have been described: the type 1 theta, atropine resistant, has a higher frequency (7 to 10HZ); and type 2 theta, atropine sensitive, has a lower frequency (4 to 7Hz) (Kramis et al., 1975; O’Keefe, 1993). Animals exposed to anxiogenic stimuli show low frequency theta oscillation in the ventral hippocampus (vHipp) (Sainsbury and Montoya, 1984; Adhikari et al., 2010) that is not associated with locomotion
(Montoya et al., 1989). On the other hand, fast (type 1) theta oscillation has a strong relationship with locomotion, especially in the dorsal hippocampus (dHipp) (Patel et al., 2012; Fuhrmann et al., 2015). Both theta frequency and power increase with running speed (Bender et al., 2015). A recent study has also shown that the linear relationship between movement-related theta frequency and speed is also modulated by anxiolytic drugs and novelty (Wells et al., 2013).
In this work, we explore the effect of acute salicylate exposure on hippocampal oscillations during an open field behavioural test. It has been shown that low frequency theta activity in the ventral hippocampus is associated to anxiety-like behaviours (Adhikari et al., 2010). Hence, we targeted the ventral hippocampus CA1 with chronically implanted microelectrodes to assess whether salicylate induces anxiety-related theta oscillations. In some experiments, we also placed electrodes in the dorsal hippocampal CA1 region to investigate if dorsal hippocampal oscillations and the coherence between dorsal and ventral hippocampus local field potential are affected by acute salicylate exposure. We found that salicylate application can generate tinnitus perception in mice, decrease exploration in an open field test and drastically increase the power of low frequency theta oscillation in the ventral
hippocampus. Low-frequency theta oscillation power was not related to animal speed. We also found that there was a much smaller increase in low-frequency theta
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oscillation in the dorsal hippocampus and little low-frequency theta coherence between dorsal and ventral hippocampi. Moreover, salicylate application abolished the linear relationship between frequency of movement-related theta and running speed.
Material and Methods
Animals
Three to five weeks old C57BL/6 mice were housed on a 12h/12h day/night cycle to maintain their normal biorhythms and had free access to food and water. All
procedures were approved by the Animal Ethics Committee (CEUA) of the Federal University of Rio Grande do Norte (Protocol number 052/2015). Effort was made to minimize suffering and discomfort of animals and to reduce the number of the animals used.
Surgery
Electrode arrays were fabricated from insulated tungsten wires (50µm, impedance between 80 and 300KΩ or 35µm, impedance 200 to 1000KΩ, California Wires). Two different configurations of arrays were used: Type 1 array; 15 electrodes (3x5,
electrode spacing 200μm) targeting the ventral hippocampus or Type 2 array; 16-wire array with 10 electrodes (2x5, electrode spacing 200μm) also targeting the ventral hippocampus, and 6 electrodes (bundle) for targeting the dorsal hippocampus.
Animals were anesthetised with ketamine/xylazine (150/7mg/kg) diluted in saline and placed on a heat pad maintained at 37º to 38º by a temperature controller (Supertec). The head was then fixed to a stereotaxic frame and an incision was made to expose the skull and one or two holes were drilled to implant the electrodes. Coordinates for type 1 array were 3.2 mm AP, 3mm ML, 3.5mm DV. The electrode bundle for the dorsal hippocampus was inserted at the following stereotaxic coordinates: 2,2mm AP, 1.8mm ML and 2mm DV. A screw placed over the cerebellum served as reference electrode and three additional screws anchored the implant (held with dental cement). Animals were then housed individually and allowed to recover for at least 10 days.
Data acquisition and open field test
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Local field potentials (LFPs) were acquired by a 16-channel amplifier (Intantech) and custom software modified from the Intan RHA evaluation package (Intantech) or the OpenEphys (Siegle et al., 2017) software with an Intan RHD headstage. The Intan RHA headstage has a fixed 1Hz high pass filter while the RHD headstage was set to high pass filter from 0.1Hz. This difference in filtering did not affect low frequency theta power (we used synthetized signals to check whether the power from 2 to 10Hz was affected by the headstage filtering). Video was simultaneously recorded using a Basler camera (model acA1300-30um). Each camera frame was triggered by an Arduino board (precisely at 30 frames per second). The data acquisition software also recorded the trigger pulses. A maximum of three mice were recorded per day and the recordings were done during the night. Each mouse received an injection of 300mg/kg sodium salicylate (Sigma) in saline or saline intra peritoneal injection. After 1 hr each mouse was placed in a rectangular open field arena (40cm X 32cm X 15cm) made of opaque white plastic for electrophysiological and video recordings. Two sessions of ten minutes each were recorded. Mice were returned to the animal facility for seven days. Next, mice were brought to the experimental room and acclimatized for two hours. Animals i.p. injected with sodium salicylate in the first test round now received injection of saline and animals that received saline in the first test were injected with sodium salicylate in the second test (300mg/kg of sodium salicylate, diluted in saline at 500mg/mL). In other words, one week after the first set of experiments, animals that were initially injected with salicylate were injected with saline and vice-versa. We found no difference in behavioural or electrophysiological results between the two groups and we divided the sessions in control and salicylate sessions (Figure 1A). To pharmacologically separate the type of salicylate-induced theta, animals (10 mice) were injected i.p. with salicylate (300 mg / kg) and atropine sulphate (40 mg / kg). The same open field protocol as the first test was repeated. In another group (5 mice), animals were treated with lithium chloride (LiCl) (0.15 M, 12 ml / kg) or saline. We used LiCl as a nausea-inducing drug because it is a conventional emetic compound in rodent taste aversion studies and; its neurophysiological mechanisms are partially known (Spencer et al., 2012; Jahng and Lee, 2015). In a third group (4 mice), we performed the open field test in animals treated with 300mg/k g sodium salicylate in saline or salicylate + taurine (100mg/kg, Sigma). Finally, in forth group (6 mice), we performed the open field test in animals treated with taurine alone (100mg/kg, Sigma) (compared to saline control). In rodents, it has been demonstrated that taurine is an
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effective anxiolytic drug in several anxiety models (Kong et al., 2006). After sixty minutes open field electrophysiological and video recordings (2 x 10 min) mice were returned to the animal facility or sacrificed for histological procedures.
Data analysis
Power spectral densities (PSD) for all channels were computed using the Welch method (pwelch Matlab command). To allow merging of data from different animals, PSD values were normalised (PSDs were divided by the total power. A custom Matlab program (imaging processing toolbox) was used for tracking the animal in the field by thresholding the animal compared with the background. In some experiments, we correlate theta power with animal speed by down sampling total theta power vs. time obtained from a spectrogram (Matlab spectrogram command) of a recording channel placed at the stratum radiatum (SR) of both ventral and dorsal hippocampus. In the experiments involving dorsal and ventral hippocampus recordings, we
calculated the coherence between dorsal and ventral SR channels using the Matlab command mscohere. Data is presented by mean ±standard error of the mean (SEM). When possible, we tested for normality of the data using the Matlab command
vartest2 to calculate statistical significance with paired t test. For non-normal
distributed data we used the non-parametrical Friedman’s test to calculate statistical significance (Friedman, 1940). All custom software can be found at
http://github.com/cineguerrilha/Neurodynamics.
Results
To investigate anxiety-related behaviour mice were placed in an open field arena 1 hr after either saline or salicylate i.p. injection. When previously injected with salicylate, animals spent significantly less time in the arena’s centre than after saline injections (mean time in the centre in the saline group was equal to 110.81±13.15 s and 59.68±6.43 s for the salicylate group in a 10 min session, p=0.002, n=20, (Figure 1B-D). In addition, mice walked significantly less after salicylate injection (mean travelled distance equal to 21.53±1.30 m for the saline group and 12.33±1.19 m for the salicylate group, p=0.00003, n=20, (Figure 1D). Reduced time spent in the centre of the open field and reduced exploration of the arena indicates anxiety-like
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behaviour. Hence, these results suggest that salicylate injection produced anxiety-like behaviour in mice.
To test whether the effect of salicylate treatment generated an anxiety-like response (instead of pain, nausea or other effect that could decrease locomotor activity), we treated a separate group of mice (n=5) with salicylate or
salicylate+taurine (2 mice first injected with salicylate alone and 3 with
salicylate+taurine and a week after treatments were swapped – see above). We found that the association of salicylate with taurine completed reverted the anxiogenic effect of salicylate (Figure 2A and B). Animals treated with salicylate+taurine significantly showed greater locomotion (total travelled distance equal to 13.06±1.82m and 28.15±4.29m for salicylate and salicylate+taurine, respectively, p=0.005 n=10 mice, Figure 2B). Association of taurine with salicylate have also caused animals to spend more time in the centre of the arena (76.06±4.03s for salicylate and 166.01±21.10s salicylate+taurine, respectively, p=0.001, n=10 mice – Figure 2B). Animals treated with taurine alone showed no significant change in locomotion (26.42±5.8m for saline and 29.81±7.4 m for taurine, not significant, n=7 mice). However, animals treated with taurine spent more time in the arena centre (111.31±10.12s for saline and 179±11.42s for taurine, p=0.01, n=7 mice). Animals treated with the nausea-induced drug, LiCl, showed no changes in locomotion when compared to saline treated
animals (total travelled distance equal to 21.41±2.02m for saline and 23.73±2.33m for LiCl, not significant, n=10 mice). Conversely, lithium did not significantly influence the time the animals spent in the centre or border of the arena (125.86±12.98s and 105.92±13.58s for saline and LiCl, respectively, not significant, n=10 mice). We also compared locomotion and time in the centre in salicylate and lithium treated animals. Mice walked more after LiCl treatment when compared to control (13.06±1.82m vs. 23.73±2.33m for salicylate and LiCl, respectively, p=0.04, Friedman’s test, n=10 mice, Figure 2C). Time spent in the center was also higher for LiCl treatment (76.06±4.03s vs. 105.92±13.59s for salicylate and LiCl, respectively, p=0.01,
Friedman’s test, n=10 mice, Figure 2C). These results demonstrate that the anxiolytic drug taurine prevent the anxiety-like effects produced by salicylate and that the anxiogenic effect of salicylate cannot be explained by nausea-like symptoms.
In order to assess changes in ventral/intermediate hippocampal theta oscillations after salicylate injection, we first electrophysiologically localised the electrode targeting the CA1 stratum radiatum (SR). It has been described that, in CA1
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SR, movement-related theta oscillation phase modulate high gamma oscillation (Figure 3A and B and (Scheffer-Teixeira et al., 2012)). Electrode localization was further confirmed by post-hoc histological analysis (Figure 3A). Local field potential (LFP) was recorded during the open field test under control conditions (60 min after saline injection) and 60 min after salicylate injection. Theta was separated in 2 bands, 4-6Hz and 7-10Hz, similar to type 2 and type 1 theta bands described elsewhere (Kramis et al., 1975; Sainsbury and Montoya, 1984; Montoya et al., 1989). After saline injections, power spectrum analysis reviewed a large peak between 7 and 10Hz (Figure 3C-F). Salicylate injections instead produced a lower frequency peak between 4 and 6Hz (Figure 3C-F). Mean 4-6Hz power was equal to 2060.21±379.05 µV2/Hz for the saline group and 3410.43±509.58 µV2/Hz for the salicylate group (p=0.0132,
n=10, Figure 3E). Furthermore, salicylate injection caused significant difference in
7-10Hz mean power (2274.16±495.90 µV2/Hz for saline and 1723.16±441.58 µV2/Hz for the salicylate, p=0.038, n=10, Figure 3F). No change in 4-6Hz power was
observed for salicylate+taurine or LiCl injection (Figure 4). These data indicates that salicylate induces low frequency theta oscillation in the intermediate/ventral
hippocampus.
Classical studies on theta oscillations have separated type 1 and type 2 theta using the cholinergic blocker atropine (Sainsbury and Montoya, 1984; Bland, 1986; Montoya et al., 1989). Hence, we tested whether salicylate-induced theta in the intermediate/ventral hippocampus was sensitive to atropine (Figure 5). The mean 4-6Hz theta power was significantly reduced by injection of atropine sulphate
(3322.43±509.58 µV2/Hz for the salicylate group and 1607.01±286.74 µV2/Hz for the atropine+salicylate group, p=0.026, n=10, Figure 5A-C). We found a small but not significant decrease in 7-10Hz theta power after atropine application (1861.23±786.56 µV2/Hz for the salicylate group and 1160.67±345.26 µV2/Hz for the
atropine+salicylate group, p=0.325, n=10, Figure 5C). These results suggest that salicylate modulates circuits associated with type 2 theta.
Movement-related theta oscillation (7-10Hz) power is positively affected by walking speed. Thus, we next asked whether the 4-6Hz salicylate-induced theta oscillations relate to the animal’s speed in the open field. We have extracted the instantaneous amplitude of the 4-6Hz and 7-10Hz LFP components by averaging the mean signal at these frequency bands (by applying short-time Fourier transform to the LFP signal). We found that there was no correlation between speed and 4-6Hz power
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(Figure 6). However, as expected, there was a positive correlation between speed and 7-10Hz power (Figure 6). Mean correlation (amplitude vs. animal speed) coefficient (r) for the 7-10Hz-signal component was equal to 0.2619±0.0551 (Figure 6C). However, the 4-6Hz component show either little or negative amplitude vs. animal speed correlation (Figure 6). Mean r for the 46Hzsignal component was equal to -0.1630±0.0708 (p=0.00016 when compared to the mean r from the 7-10Hz
component, n=10, Figure 6C). No significant correlation was found between both theta frequency bands and acceleration. These results show that, differently from movement-related theta, salicylate-induced theta oscillation power is not dependent on locomotion speed.
We next examined the effect of salicylate on oscillations in the dorsal hippocampus by implanting 5 mice with the type 2 wire array (see Methods). We again identified an electrode targeting the SR of the dorsal hippocampus using post-hoc histological analysis (Figure 7A) and theta-gamma modulation profile (Figure 7B). In the dorsal hippocampus, the faster theta oscillation was predominant in both saline and salicylate conditions (Figures 7C and D). However, salicylate lowered the peak theta frequency by approximately 1Hz without affecting normalised mean power of the 7-10Hz oscillation (Figure 7C and D). However, there was a significant
increase in the mean 4-6Hz oscillation power (Figure 7D and E). Mean 4-6Hz power was equal to 1361.76 ±308.41 µV2/Hz for the saline group and 2474.50±456.98 µV2/Hz for the salicylate group (p=0.04, n=5, Figure 7E). We have also found that the fast (7-10Hz) theta oscillation peak frequency decreased after salicylate
application. Peak 7-10Hz theta frequency was equal to 7.6±0.2Hz in saline and
8.8±0.1Hz in salicylate (p=0.001, n=5, Figure 7D). Taken together, these results show that salicylate induces a smaller increase in low frequency theta oscillation in the dorsal hippocampus when compared to the ventral hippocampus.
We asked if the dorsal and ventral hippocampus show coherent salicylate induced theta oscillation. We measure coherence in theta oscillations from pairs of electrodes implanted in the SR of dorsal and ventral hippocampus (type 2 arrays). Dorsal and ventral hippocampus fast theta coherence was high in both saline and salicylate conditions (Figure 8). However, mean coherence between 4-6Hz was slightly higher (but not significantly different) in the salicylate condition (mean coherence for the saline group was equal to 0.0638±0.0142 and 0.1796±0.0518 for the salicylate group, p=0.0633, n=5, Figure 8B). Thus, coherence analysis indicates that
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faster theta oscillation generated in the dorsal hippocampus (Patel et al., 2012) is efficiently transmitted to the ventral hippocampus while salicylate-induced theta oscillation is mostly restricted to the ventral hippocampus.
Lastly, we checked whether the decrease in fast (7-10Hz) theta peak frequency in salicylate-treated animals was caused by changes in locomotion speed, as
locomotion-related theta oscillation frequency varies with running speed (Bender et al., 2015). Saline and salicylate-treated animals were placed on a treadmill with controlled speed (0 to 11m/s) (Figure 9A). The effect of running speed in fast theta frequency in the dorsal hippocampus is illustrated in Figure 8A. Salicylate not only reduced peak fast theta frequency but also eliminate the effect of running speed in theta frequency (Figure 9B). Mean correlation coefficient (r) for saline-treated animals was equal to 0.17±0.03 and -0.06±0.05 for salicylate-treated animals
(p=0.02, n=5, Figure 9C). No differences were found in the correlation between theta power and speed between saline- and salicylate-treated animals (data not shown). It has been suggested that theta-frequency vs. velocity intercept alters with anxiety (Wells et al., 2013). However, we could not test this hypothesis given the non-significant correlations between theta frequency and speed after salicylate injection. These results show that salicylate practically abolishes the effect of running speed in type 1 theta frequency.
Discussion
We have recorded LFP in the ventral and dorsal hippocampus during an open field experiment to assess whether an excessive dose of salicylate produce anxiety-like electrophysiological changes. Several other studies have demonstrated that acute administration of salicylate (at the dose used in this study) induces tinnitus-like perception in rodents (for review, see Chen et al., 2013). We show that salicylate can induce tinnitus-like behaviour in a mouse gap-detection test. Salicylates furthermore cause animals to explore less an open field arena and salicylates induce an increase in power of low frequency theta oscillation in the ventral (and dorsal, to some extent) hippocampus. This oscillation power was not dependent on locomotion speed. Besides, we found that there is a slight increase of low frequency theta power in the dorsal hippocampus after salicylate injection. Type 2 (4-6Hz) theta coherence was low when compared to high frequency movement-related theta. The peak frequency
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of locomotion-related fast theta oscillation also decreased following salicylate application. Moreover, the linear relationship between running speed and fast theta was abolished after salicylate treatment.
There is a strong association between tinnitus and mood disorders. A study in Sweden showed that the comorbidity between tinnitus and depression is above 60% (Zöger et al., 2001). While the salicylate dose used in this study has been shown to generate tinnitus-like signs in rodents (Chen et al., 2013), we cannot directly associate the anxiety-like behaviour following salicylate injection with tinnitus. However, to our knowledge, no study has demonstrated a direct anxiogenic effect of salicylate. Importantly, mice showed normal open-field exploration when salicylate was associated with taurine (at a dose known to produce anxiolytic effects in rodents) (Kong et al., 2006). Of note, our results and previous studies have shown that taurine exert similar anxiolytic effects to diazepam (El Idrissi et al., 2009; Hazim et al., 2014). Side effects of salicylate in humans may include headache and nausea (Pearlman and Gambhir, 2009). We used LiCl to produce nausea (Nakajima, 2018) during the open field to further support our claim that salicylate induces anxiety-like behaviour. Lithium-treated animals explored the arena’s centre and border in a similar manner, suggesting no anxiety-like effects in the exploratory pattern (Seibenhener and Wooten, 2015). Hence, it is plausible that the lower mobility and centre crossing displayed by our subjects after salicylate administration indicates an anxiety-like behaviour rather than pain or nausea.
Salicylate administration led to an increase in low frequency theta oscillation in the ventral hippocampus. The little correlation between salicylate-induced theta and movement suggest that this oscillation is mechanistically different from movement-induced theta. It has been demonstrated that theta oscillations in the ventral
hippocampus appear when animals display anxiety-like behaviours (Adhikari et al., 2010). Moreover, several classical studies showed that type 2 (low frequency,
atropine-dependent) theta oscillation appears when the animal is exposed to predators and anxiogenic environment (Kramis et al., 1975, p 2; Montoya et al., 1989, p 2), during anticipatory anxiety (McNaughton and Gray, 2000) and conditioned freezing behavior, showing in this case theta synchronization in amygdalohippocampal pathways (Seidenbecher et al., 2003). The lower frequency and the little correlation with movement of salicylate-induced theta suggest that this oscillation resembles type 2 theta oscillation (Bland, 1986). Atropine-sensitivity further evince that salicylate
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increases type 2 theta (Kramis et al., 1975; Sainsbury and Montoya, 1984; Montoya et al., 1989).
The association of slow type 2 theta oscillation with anxiety was demonstrated more than 30 years ago (Sainsbury and Montoya, 1984). Recently, Tendler and
Wagner have shown that the limbic system synchronises in low frequency theta during fearful stimuli while a fast theta appears during non-fearful social stimuli (Tendler and Wagner, 2015). However, that work did not directly investigated the ventral hippocampus. Hence, slow theta from the ventral hippocampus may synchronise other limbic regions during anxiety-like behaviour after salicylate
injections. Given the strong association of anxiety with tinnitus and the association of the latter with slow theta oscillation generation, it will be interesting to show whether anxiolytic drugs (that also affects theta oscillation) (Yeung et al., 2016) could
improve the performance of mice in the GPIAS test.
We found that salicylate induces a small increase in low-frequency theta in the dorsal hippocampus and cause a small change in movement-related theta, possibly due to the lower locomotion speed after salicylate injection. Interestingly, after salicylate injections, there was still high movement-related theta coherence in the dorsal and ventral hippocampus while the salicylate-induced theta coherence was small. This finding could suggest that salicylate-induced theta may be confined to the ventral hippocampus. As mentioned above, movement-related theta is initially
generated in the dorsal hippocampus and, through lamellar connections, spreads to ventral regions (Lubenov and Siapas, 2009; Patel et al., 2012). However, salicylate-induced theta does not seem to significantly invade the dorsal hippocampus. Previous studies have shown that, during decision making, a low frequency theta oscillation appear in the almost exclusively ventral hippocampus while the fast theta component is only prominent in the dorsal hippocampus (Schmidt et al., 2013). Moreover, Schmidt and others (2013) also demonstrated high fast-theta coherence in the dorsal and ventral hippocampus during decision making despite the decrease in power of fast theta in the ventral hippocampus. Hence, it seems plausible that the circuits involved in the generation of salicylate-induced theta differs from those responsible to fast theta rhythmogenesis.
Interestingly, salicylate has also abolished the relationship between movement-related theta and running speed. Speed vs. fast theta frequency is also affected by anxiolytic drugs (Wells et al., 2013). It has been suggested that the
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increase in type 1 theta frequency following increase in speed is modulated by septal cholinergic inputs to the hippocampus and entorhinal cortex (Newman et al., 2013). It is important to note that type 2 theta has is independent of movement and entorhinal cortex input (Burgess, 2008). A recent study has shown that most of medial septum cells respond to broadband noise (Zhang et al., 2018). Septal neurons receive an important auditory input from the cochlear nucleus (relayed by the pontine reticular nucleus and the pontine central grey) (Zhang et al., 2018). There is a growing
evidence that a non-canonical auditory pathway to the medial septum that respond to noise is essential in noise-cued fear conditioning (Zhang et al., 2018). This pathway could possibly be involved in tinnitus and its comorbid anxiety.
In summary, we found that salicylate application generates anxiety-like behaviours and a slow type of theta oscillation and eliminate the effect of running speed in fast theta frequency. It will be interesting to investigate the response of medial septum neurons to salicylate. Future studies should also assess LFP and anxiety-like behaviour in other models of tinnitus (e.g. acoustic trauma) and its relationship to different type of theta oscillations and information processing along the dorso/ventral hippocampal axis (Brozoski and Bauer, 2016).
Acknowledgments
This work was supported by the American Tinnitus Association and the National Council for Scientific and Technological Development (Brazil), Grant no.
440793/2016-5.
Figure Legends
Figure 1. Salicylate decreases locomotion and increases anxiety-like behaviour. A. Example plots of locomotion in the arena for saline (top) and salicylate (bottom).
Black and grey circles highlight the movement at the centre and border area,
respectively. B. Example graph of speed versus time for the same animal shown in A. Time spent on centre is illustrated by green dashes. C. Example of animal’s speed in time for saline and salicylate injection. D. Boxplot showing mean total distance travelled for mice treated with saline and salicylate showing reduced locomotion activity for salicylate group. E. Boxplot of total time spent in centre for mice treated
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with saline and salicylate indicating increased anxiety-like behaviour. *p=0.002, **p<0.001 (paired t test).
Figure 2. Taurine prevents anxiety-like behaviour in salicylate treated animals. A. Example plots of locomotion in the open field for salicylate (left) and
salicylate+taurine (bottom) treated animals. Black and grey circles highlight the movement at the centre and border area, respectively. B. Boxplot showing the total distance travelled (left) and total time spent in centre (right) for mice treated with salicylate or salicylate+taurine. *p<0.01. C. Boxplot showing the total distance travelled (left) total time spent in centre (right) for mice treated with salicylate or LiCl. *p=0.04, **p=0.01 (Friedman’s test).
Figure 3. Salicylate increases type 2 theta in the ventral hippocampus. A. left:
Schematic drawing of the localization of electrodes in the ventral hippocampus (*).
Right: Bright field image showing positioning of electrode in stratum radiatum
(arrow) on the ventral hippocampus. B. Example of cross-frequency modulation analysis for one channel used together with histology to verify electrode position in the stratum radiatum. C. Representable example of a raw trace signal from one channel from the same animal for saline (black trace) and salicylate (red trace). D. Example spectrogram for the channel in 'C' for saline (top) and salicylate (bottom). E. Mean power spectrum density showing an increase in type 2 theta in salicylate
compared to saline (shaded areas represent SEM). F. Mean power was significantly different between saline and the salicylate group for 4-7Hz oscillations (type 2 theta) while no difference were found for 7-10Hz oscillations (type 1 theta). *p<0.05.
Figure 4. Lithium does not interfere with type 2 theta. A, B. Mean power spectrum
density for saline vs. lithium (top) and saline vs. taurine+salicylate (shaded areas represent SEM). C, D. Boxplots showing the mean power for the A and B groups for 4-7Hz oscillation (type 2 theta) and for 7-10Hz oscillation (type 1 theta).
Figure 5. Salicylate-induced theta is atropine-sensitive. A. Representable example
of LFP recordings from one channel from the same animal for salicylate (red) and salicylate+atropine (green). B. Mean power for salicylate (red) compared to
salicylate+atropine (black). C. Boxplots showing the total power for the saline and the
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salicylate+atropine groups for 4-7Hz oscillation (type 2 theta) and for 7-10Hz oscillation (type 1 theta). *p<0.05.
Figure 6. Type 2 theta in salicylate animals is inversely related to movement. A.
Graph showing instantaneous velocity (top), and instantaneous amplitude of 4-6Hz theta (middle) and 7-10Hz theta (bottom) for one animal during a recording session.
B. Relationship between 4-6Hz theta power versus locomotion speed (top) and
7-10Hz theta and speed (bottom). C. Mean correlation coefficient correlation of 4-6Hz theta and speed (left) and 7-10Hz theta and speed (right). *p<0.05.
Figure 7. Salicylate administration increases type 2 theta in the dorsal hippocampus. A. left: Schematic of the localization of electrodes in the dorsal
hippocampus (*). Right: Bright field image showing positioning of electrode in
stratum radiatum (arrow) of the dorsal hippocampus. B. Example of cross-frequency
modulation analysis for one channel used in combination with histology to select the electrodes in the stratum radiatum. C. Example of a LFP recording from one animal after saline (top) and salicylate (bottom) injection. D. Mean power spectral density plots for salicylate (red) and saline (black) treated animals. Red arrow shows 4-6Hz peak after salicylate injection. E. Boxplots for 4-6Hz and 7-10Hz power between saline and salicylate. *p<0.05.
Figure 8. Salicylate induced theta shows little ventral and dorsal hippocampus correlation. A. Mean dorsal/ventral coherence of control (black) and salicylate
(grey). Red arrow shows 4-6Hz coherence peak after salicylate injection. B. Boxplot summarising coherence for 4-6Hz (left) and 7-10Hz (right) for saline and salicylate-treated animals.
Figure 9. Salicylate abolishes the linear trend between running speed and theta frequency. A. Examples of spectrograms of an animal placed on a treadmill
(treadmill speed is shown in the top graph) treated with saline (middle panel) or with salicylate (bottom panel) recorded in SR of DHipp. B. Running speed vs. fast theta frequency of the animal in (A) in the treadmill. C. Mean correlation coefficient between fast theta frequency and running speed for saline and salicylate treated animals, *p=0.02.
Accepted
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Accepted
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0 100 200 300 400 500 600 0 10 20 30 Time (s) Speed (cm s −1) 0 100 200 300 400 500 600 0 10 20 30 Speed (cm s −1) Time (s)
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Group 1 Group 2 Injection Record Saline Salicylate Injection Record Salicylate Saline 60 minutes 60 minutes 1 week
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Distance traveled (m) Time in center (s)
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salicylatesalicylate+taurine salicylatesalicylate
+taurine
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* * LiCl 60 0 60 0 300 0 300 0 salicylate LiCl salicylate ** *Distance traveled (m) Time in center (s)
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Salicylate Saline 0.2mV 0.5s Ventral CA1 SR Amplitude Frequency (Hz) Phase Frequency (Hz) 5 10 15 20 50 100 150 200 0 Saline Salicylate 0.0017 0 Modulation Index
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Power (µV 2/Hz) 20 0 0 50 100 Frequency (Hz) 20 0 0 50 100 Frequency (Hz) Time (s) 5000 0 Saline SalicylateE
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Franzon et al., Figure 3
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200 µm Salicylate Saline 7000 Power (mV 2/Hz -1) Power (mV 2/Hz -1) ** * 4-6Hz 7-10Hz 0 5 10 15 20 0 3000 6000 Power (mV 2 /Hz -1 ) Frequency (Hz) Salicylate Saline 0 7000Accepted
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Power (mV 2 /Hz -1 ) Frequency (Hz) 4-6Hz 7-10Hz Mean Power (mV 2/Hz -1) Mean Power (mV 2/Hz -1) 0 5 10 15 20 0 1000 2000 3000 4000 5000 0 5 10 15 20 0 1000 2000 3000 4000 5000 Power (mV 2 /Hz -1 ) Frequency (Hz) 0 3000 6000 0 3000 6000 0 3000 6000 0 3000 6000 Mean Power (mV 2/Hz -1) Mean Power (mV 2/Hz -1) 4-6Hz 7-10Hz Saline Saline Saline Saline Lithium Lithium Taurine+ Salicylate Taurine+ Salicylate Saline Taurine+Salicylate Saline Lithium
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Franzon et al., Figure 4
This article is protected by copyright. All rights reserved
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0 5 10 15 20 0 1000 2000 3000 4000 5000 6000 Power (mV 2/Hz -1) Frequency (Hz) 0.25mV 500ms
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Salicylate+Atropine 1000 2000 3000 4000 5000 6000 7000 0 1000 2000 3000 4000 5000 6000 7000 4-6Hz 7-10Hz 0 * Power (mV 2/Hz -1) Power (mV 2 /Hz -1 )C
Franzon et al., Figure 5
This article is protected by copyright. All rights reserved
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x105 x105 0
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0.6 0.4 0.2 -0.2 -0.4 -0.6 4-6Hz 7-10Hz Time (s) Time (s) Time (s) 0 100 200 300 400 500 600 0 10 20 Velocity 0 100 200 300 400 500 600 0 1 2 3 7-10Hz Theta 0 100 200 300 400 500 600 0 1 2 3 4-6Hz Theta Speed (cm s -1) Power (µV 2/Hz) Power (µV 2/Hz) 7-10Hz 4-6Hz 0 2 4 6 8 10 0 2 4 6 8 10 x105 x105 2.5 2 1.5 1 0.5 2.5 2 1.5 1 0 0.5 Power (µV 2/Hz) Power (µV 2/Hz) Speed (cm s-1) Speed (cm s-1) Correlation CoefficientFranzon et al., Figure 6
*
This article is protected by copyright. All rights reserved
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0.4mV 0.5s Salicylate Control Dorsal CA1 SR 200 µm Frequency (Hz)
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Franzon et al., Figure 7
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Power ( PV 2/Hz -1) 0 5 Power ( PV 2/Hz -1) 4-6Hz 7-10Hz Power ( PV 2/Hz -1) Amplitude Frequency (Hz) Phase Frequency (Hz)5 10 15 20 50 100 150 200 0.0017 0 Modulation Index
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0 5 10 15 20 0 0.2 0.4 0.6 0.8 Coherence Frequency (Hz) 0.2 0.3 0.4 0.5 0.6 0.7 Mean Coherence Mean Coherence 0.2 0.3 0.4 0.5 0.6 0.7
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Frequency (Hz)