, Aron Miranda , Thawann Malfatti Article › jspui › bitstream › 123456789 › 25918 ›...

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 Malfatti

1, João Patriota

2,

Sanja Mikulovic3, Katarina E. Leão

1 & Richardson N. Leão

1,3

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

This article is protected by copyright. All rights reserved

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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 may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/hipo.23021

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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http://github.com/cineguerrilha/Neurodynamics

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 µV

2/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 µV

2/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 µV

2/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 4-6Hz-signal 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.


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References

Adhikari A, Topiwala MA, Gordon JA. 2010. Synchronized Activity between the

Ventral Hippocampus and the Medial Prefrontal Cortex during Anxiety.

Neuron 65:257–269.

Bauer CA, Brozoski TJ, Holder TM, Caspary DM. 2000. Effects of chronic salicylate

on GABAergic activity in rat inferior colliculus. Hear Res 147:175–182.

Bender F, Gorbati M, Cadavieco MC, Denisova N, Gao X, Holman C, Korotkova T,

Ponomarenko A. 2015. Theta oscillations regulate the speed of locomotion via

a hippocampus to lateral septum pathway. Nat Commun 6:8521.

Bland BH. 1986. The physiology and pharmacology of hippocampal formation theta

rhythms. Prog Neurobiol 26:1–54.

Brozoski TJ, Bauer CA. 2016. Animal models of tinnitus. Hear Res 338:88–97.

Burgess N. 2008. Grid cells and theta as oscillatory interference: theory and

predictions. Hippocampus 18:1157–1174.

Chen G-D, Stolzberg D, Lobarinas E, Sun W, Ding D, Salvi R. 2013. Salicylate-

induced cochlear impairments, cortical hyperactivity and re-tuning, and

tinnitus. Hear Res 295:100–113.

Chen Y-C, Xia W, Chen H, Feng Y, Xu J-J, Gu J-P, Salvi R, Yin X. 2017. Tinnitus

distress is linked to enhanced resting-state functional connectivity from the

limbic system to the auditory cortex. Hum Brain Mapp 38:2384–2397.

El Idrissi A, Boukarrou L, Heany W, Malliaros G, Sangdee C, Neuwirth L. 2009.

Effects of taurine on anxiety-like and locomotor behavior of mice. Adv Exp

Med Biol 643:207–215.

Friedman M. 1940. A Comparison of Alternative Tests of Significance for the

Problem of $m$ Rankings. Ann Math Stat 11:86–92.

Fuhrmann F, Justus D, Sosulina L, Kaneko H, Beutel T, Friedrichs D, Schoch S,

Schwarz MK, Fuhrmann M, Remy S. 2015. Locomotion, Theta Oscillations,

and the Speed-Correlated Firing of Hippocampal Neurons Are Controlled by a

Medial Septal Glutamatergic Circuit. Neuron 86:1253–1264.

Gholami M, Moradpour F, Semnanian S, Naghdi N, Fathollahi Y. 2015. Chronic

sodium salicylate administration enhances population spike long-term

potentiation following a combination of theta frequency primed-burst

stimulation and the transient application of pentylenetetrazol in rat CA1

hippocampal neurons. Eur J Pharmacol 767:165–174.

Guitton MJ, Pujol R, Puel J-L. 2005. m-Chlorophenylpiperazine exacerbates

perception of salicylate-induced tinnitus in rats. Eur J Neurosci 22:2675–2678.


Acc

epte

d A

rticl

e

Hazim AI, Ramanathan S, Parthasarathy S, Muzaimi M, Mansor SM. 2014.

Anxiolytic-like effects of mitragynine in the open-field and elevated plus-

maze tests in rats. J Physiol Sci JPS 64:161–169.

Jahng JW, Lee J-H. 2015. Activation of the hypothalamic-pituitary-adrenal axis in

lithium-induced conditioned taste aversion learning. Eur J Pharmacol

768:182–188.

Kong WX, Chen SW, Li YL, Zhang YJ, Wang R, Min L, Mi X. 2006. Effects of

taurine on rat behaviors in three anxiety models. Pharmacol Biochem Behav

83:271–276.

Kramis R, Vanderwolf CH, Bland BH. 1975. Two types of hippocampal rhythmical

slow activity in both the rabbit and the rat: relations to behavior and effects of

atropine, diethyl ether, urethane, and pentobarbital. Exp Neurol 49:58–85.

Kraus KS, Canlon B. 2012. Neuronal connectivity and interactions between the

auditory and limbic systems. Effects of noise and tinnitus. Hear Res 288:34–

46.

Lubenov EV, Siapas AG. 2009. Hippocampal theta oscillations are travelling waves.

Nature 459:534–539.

McNaughton N, Gray JA. 2000. Anxiolytic action on the behavioural inhibition

system implies multiple types of arousal contribute to anxiety. J Affect Disord

61:161–176.

Montoya CP, Heynen AJ, Faris PD, Sainsbury RS. 1989. Modality specific type 2

theta production in the immobile rat. Behav Neurosci 103:106–111.

Nakajima S. 2018. Clay eating attenuates lithium-based taste aversion learning in rats:

A remedial effect of kaolin on nausea. Physiol Behav 188:199–204.

Newman EL, Gillet SN, Climer JR, Hasselmo ME. 2013. Cholinergic Blockade

Reduces Theta-Gamma Phase Amplitude Coupling and Speed Modulation of

Theta Frequency Consistent with Behavioral Effects on Encoding. J Neurosci

33:19635–19646.

O’Keefe J. 1993. Hippocampus, theta, and spatial memory. Curr Opin Neurobiol

3:917–924.

Patel J, Fujisawa S, Berényi A, Royer S, Buzsáki G. 2012. Traveling theta waves

along the entire septotemporal axis of the hippocampus. Neuron 75:410–417.

Pattyn T, Van Den Eede F, Vanneste S, Cassiers L, Veltman DJ, Van De Heyning P,

Sabbe BCG. 2016. Tinnitus and anxiety disorders: A review. Hear Res

333:255–265.

Pearlman BL, Gambhir R. 2009. Salicylate intoxication: a clinical review. Postgrad

Med 121:162–168.


Acc

epte

d A

rticl

e

Puel J-L, Guitton MJ. 2007. Salicylate-induced tinnitus: molecular mechanisms and

modulation by anxiety. Prog Brain Res 166:141–146.

Sainsbury RS, Montoya CP. 1984. The relationship between type 2 theta and

behavior. Physiol Behav 33:621–626.

Scheffer-Teixeira R, Belchior H, Caixeta FV, Souza BC, Ribeiro S, Tort ABL. 2012.

Theta phase modulates multiple layer-specific oscillations in the CA1 region.

Cereb Cortex N Y N 1991 22:2404–2414.

Schmidt B, Hinman JR, Jacobson TK, Szkudlarek E, Argraves M, Escabí MA,

Markus EJ. 2013. Dissociation between dorsal and ventral hippocampal theta

oscillations during decision-making. J Neurosci Off J Soc Neurosci 33:6212–

6224.

Seibenhener ML, Wooten MC. 2015. Use of the Open Field Maze to measure

locomotor and anxiety-like behavior in mice. J Vis Exp JoVE:e52434.

Seidenbecher T, Laxmi TR, Stork O, Pape H-C. 2003. Amygdalar and hippocampal

theta rhythm synchronization during fear memory retrieval. Science 301:846–

850.

Siegle JH, López AC, Patel YA, Abramov K, Ohayon S, Voigts J. 2017. Open Ephys:

an open-source, plugin-based platform for multichannel electrophysiology. J

Neural Eng 14:045003.

Spencer CM, Eckel LA, Nardos R, Houpt TA. 2012. Area postrema lesions attenuate

LiCl-induced c-Fos expression correlated with conditioned taste aversion

learning. Physiol Behav 105:151–160.

Su Y-Y, Luo B, Wang H-T, Chen L. 2009. Differential effects of sodium salicylate on

current-evoked firing of pyramidal neurons and fast-spiking interneurons in

slices of rat auditory cortex. Hear Res 253:60–66.

Tendler A, Wagner S. 2015. Different types of theta rhythmicity are induced by social

and fearful stimuli in a network associated with social memory. eLife

4:e03614.

Wells CE, Amos DP, Jeewajee A, Douchamps V, Rodgers J, O’Keefe J, Burgess N,

Lever C. 2013. Novelty and anxiolytic drugs dissociate two components of

hippocampal theta in behaving rats. J Neurosci Off J Soc Neurosci 33:8650–

8667.

Wu H, Xu F-L, Yin Y, Da P, You X-D, Xu H-M, Tang Y. 2015. Salicylate-induced

changes in immediate-early genes in the hippocampal CA1 area. Mol Med

Rep 12:1625–1630.

Yeung M, Treit D, Dickson CT. 2016. Ventral hippocampal histamine increases the

frequency of evoked theta rhythm but produces anxiolytic-like effects in the

elevated plus maze. Neuropharmacology 106:146–155.


Acc

epte

d A

rticl

e

Zhang G-W, Sun W-J, Zingg B, Shen L, He J, Xiong Y, Tao HW, Zhang LI. 2018. A

Non-canonical Reticular-Limbic Central Auditory Pathway via Medial

Septum Contributes to Fear Conditioning. Neuron 97:406-417.e4.

Zöger S, Svedlund J, Holgers KM. 2001. Psychiatric disorders in tinnitus patients

without severe hearing impairment: 24 month follow-up of patients at an

audiological clinic. Audiol Off Organ Int Soc Audiol 40:133–140.


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