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Experimental Neurology
journal homepage:www.elsevier.com/locate/yexnrResearch paper
Anxiety-like behavior induced by salicylate depends on age and can be
prevented by a single dose of 5-MeO-DMT
Jessica Winne
a,b, Barbara C. Boerner
a, Thawann Malfatti
a, Elis Brisa
a, Jhulimar Doerl
c,
Ingrid Nogueira
a, Katarina E. Leão
a, Richardson N. Leão
a,c,⁎aNeurodynamics Lab, Brain Institute, Federal University of the Rio Grande do Norte, Av. Nascimento de Castro 2155, 59560-450 Natal/RN, Brazil bDevelopmental Genetics Unit, Department of Neuroscience, Uppsala University, Husarg 3, Uppsala 75234, Sweden
cNeural Development and Environment Lab, Brain Institute, Federal University of the Rio Grande do Norte, Av. Nascimento de Castro 2155, 59560-450 Natal, RN, Brazil
A R T I C L E I N F O
Keywords: Ventral hippocampus Type 2 theta Tinnitus
5-MeO-DMT and anxiety
A B S T R A C T
Salicylate intoxication is a cause of tinnitus and comorbidly associated with anxiety in humans. In a previous work, we showed that salicylate induces anxiety-like behavior and hippocampal type 2 theta oscillations (theta2) in mice. Here we investigate if the anxiogenic effect of salicylate is dependent on age and previous tinnitus experience. We also tested whether a single dose of DMT can prevent this effect. Using microwire electrode arrays, we recorded localfield potential in young (4–5- month-old) and old (11–13-month-old) mice to study the electrophysiological effect of tinnitus in the ventral hippocampus (vHipp) and medial prefrontal cortex (mPFC) in an openfield arena and elevated plus maze 1h after salicylate (300mg/kg) injection. We found that anxiety-like behavior and increase in theta2 oscillations (4–6 Hz), following salicylate pre-treatment, only occurs in young (normal hearing) mice. We also show that theta2 and slow gamma oscillations increase in the vHipp and mPFC in a complementary manner during anxiety tests in the presence of salicylate. Finally, we show that pre-treating mice with a single dose of the hallucinogenic 5-MeO-DMT prevents anxiety-like behavior and the in-crease in theta2 and slow gamma oscillations after salicylate injection in normal hearing young mice. This work further support the hypothesis that anxiety-like behavior after salicylate injection is triggered by tinnitus and require normal hearing. Moreover, our results show that hallucinogenic compounds can be effective in treating tinnitus-related anxiety.
1. Introduction
Tinnitus is a high prevalence sign that commit 11.9–30.3% of the general population (McCormack et al., 2016). Within this group, 3–3.9% of the subjects report, long-lasting, bothersome tinnitus (McCormack et al., 2016). The condition is often associated with ad-verse emotional responses, including increased stress, irritability, sleep disorders, anxiety and depression (Belli et al., 2008;Bhatt et al., 2017; Izuhara et al., 2013;Lauer et al., 2018;Pattyn et al., 2016;Winne et al., 2019). In a previous study, we have shown that salicylate, a known cause of tinnitus (Pearlman and Gambhir, 2009), induces anxiety-like behavior in mice (Winne et al., 2019). Salicylate can generate tinnitus in both humans and animals (Eggermont and Roberts, 2015) yet, few studies explore anxiety-like behavior in the salicylate tinnitus models (Lauer et al., 2018; Winne et al., 2019). It is known, however, that noise-induced tinnitus augment aggressive behavior but does not
change spatial performance (Zheng et al., 2011a, 2011b;Zhang et al., 2016).
Type 2 theta oscillation (theta2) can appear when rodents are ex-posed anxiogenic environments or in the presence of predator odor (Mikulovic et al., 2018;Sainsbury and Montoya, 1984). Anxiety in the salicylate tinnitus model also generates theta2 in the ventral hippo-campus (vHipp) during open-field test (Winne et al., 2019). Given the strong projections from the vHipp to the mPFC, theta2 generated in the vHipp could drive the mPFC, and, consequently the amygdala (Adhikari et al., 2010). In fact, theta2 coherence increases between the hippo-campus and mPFC during anxiety-like behavior (Adhikari et al., 2010). However, the effect of tinnitus-induced anxiety in the vHipp/mPFC pathway is yet to be explored.
Subjective tinnitus is primarily associated with sensorineural hearing loss (Cima et al., 2019; Moon et al., 2018; Norena and Eggermont, 2003;Weisz et al., 2006) which may be the consequence of
https://doi.org/10.1016/j.expneurol.2020.113175
Received 10 October 2019; Received in revised form 23 December 2019; Accepted 6 January 2020
⁎Corresponding author at: Neurodynamics Lab, Brain Institute, Federal University of the Rio Grande do Norte, Av. Nascimento de Castro 2155, 59560-450 Natal/
RN, Brazil.
E-mail address:[email protected](R.N. Leão).
Available online 08 January 2020
0014-4886/ © 2020 Elsevier Inc. All rights reserved.
noise trauma, ototoxic drugs, or aging. Here, we used acute salicylate injection as a tinnitus model (Rüttiger et al., 2003). Mechanisms by which salicylate induces tinnitus are unclear. Salicylate has well-es-tablished effects on cochlear function. It has been suggested that sali-cylate induces tinnitus through the activation of cochlear NMDA re-ceptors (Guitton et al., 2003). In addition, salicylate modifies the expression of the protein prestin on on the outer wall of hair cells (Yang et al., 2009;Yu et al., 2008). However, recent experiments have high-lighted direct modulation of neural activity in other auditory areas (Guitton et al., 2003;Sheppard et al., 2014;Sun et al., 2009). Salicylate can also have direct effect on limbic system neurons (Sun et al., 2009; Winne et al., 2019) and therefore could directly alter limbic function. Interestingly, inflammatory reaction can trigger anxiety (Haj-Mirzaian et al., 2016; Haj-Mirzaian et al., 2017). Hence, attenuated salicylate-triggered anxiety-like behavior in animals suffering tinnitus/hearing loss as a precondition could help to distinguish anxiety triggered by tinnitus from anxiety generated by changes in inflammatory pathways. We have previously hypothesized that salicylate induces anxiety-like behavior due to the acute generation of tinnitus (Winne et al., 2019). Hence, previous tinnitus perception and/or hearing loss could potentially minimize anxiety after acute salicylate injection, precluding a direct anxiogenic effect of the drug itself. Salicylate could also fail to trigger anxiety in older mice as several strains suffer from age-related hearing loss due to degeneration of peripheral auditory neurons (Ison et al., 2007). However, to our knowledge, no study that use acute sal-icylate injection to induce tinnitus has explored age related effects on anxiety.
Recent studies evinced the potential of hallucinogens in treating mood disorders (Davis et al., 2019;Dos Santos et al., 2016; Palhano-Fontes et al., 2019;Reiche et al., 2018). Results in clinical trials were also replicated in animal models of depression (Buchborn et al., 2014). Rats submitted to olfactory bulbectomy (depression model) treated with lysergic acid diethylamide (LSD) showed near normal active avoidance (a major sign of anxiety-like behavior) and learning similar to animals treated with classical antidepressants (Buchborn et al., 2014). Davis and others described that one to four sessions of 5-MeO-DMT administration therapy lead to 79% decrease in depression and 69% in anxiety symptoms (Davis et al., 2018). Moreover, hippocampal neurogenesis, a hallmark of long-term antidepressant treatment, is also increased after a single dose of the hallucinogen 5-methoxy-N,N-di-methyltryptamine (5-MeO-DMT) (Lima da Cruz et al., 2018). Clinical assays that examined antidepressant effects of DMT analogs described no significant side effects following their administration (Davis et al., 2018;Palhano-Fontes et al., 2019). It is important to note that hallu-cinations caused by psychedelics can be aversive to certain groups of patients. (Baumeister et al., 2014). In addition, there is a lack of long-term studies assessing chronic adverse effects of hallucinogen therapy. Electrophisiological assays exploring the effect of hallucinogens in an-imal models of mood disorders are essential to assess whether anxio-lytic/antidepressant effects are accompanied by the absence of oscil-latory patterns inherent to mood disorders (e.g. theta2) and if other rhythms that encode behaviors like running, foraging etc. (e.g. type 1 theta) are not affected.
In this work we used implanted microwire arrays in young (4–5-month-old) and old (11–13-month-old) mice to assess changes in local field potentials (LFPs) triggered by tinnitus and anxiety in the ventral hippocampus (vHipp) and the medial prefrontal cortex (mPFC). In ad-dition, we tested if pre-treating mice with 5-MeO-DMT could provide resilience to anxiety comorbidily linked to tinnitus.
2. Material and methods 2.1. Mice
All procedures were approved by the Animal Ethics Committee (CEUA) of the Federal University of Rio Grande do Norte (Protocol
number 052/2015). All effort was made to minimize suffering and discomfort of animals and to reduce the number of the animals used. Young (4–5 month-old) and old (11–13 month old) C57BL/6 male mice were implanted with microwire arrays targeting the vHipp and mPFC (Winne et al., 2019). Mice were individually housed after surgeries (Adhikari et al., 2010). Electrode arrays were fabricated from insulated tungsten wires (50μm, impedance between 100 and 400KΩ, California Fine Wire). Array configuration: 6 electrodes (2 × 3, electrode spacing 200μm) targeting the ventral hippocampus and 4 electrodes (2 × 2, electrode spacing 200μm) for targeting the pre-limbic cortex. Animals were anesthetised with isoflurane 2% oxygen and placed on a heat pad maintained at 37 °C by a temperature controller (Supertec). The head was thenfixed to a stereotaxic frame and an incision was made to ex-pose the skull and three holes were drilled to implant the electrodes. Coordinates for array implantation were− 3.4 mm AP, 3.5 mm ML, 3.5 mm DV (vHipp), and 2.1 AP, 0.3 ML, 1.6 DV (mPFC). 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 before recordings. Animals were housed on a 12 h/ 12 h day/night cycle to maintain their normal biorhythms and had free access to food and water.
2.2. Auditory brainstem responses
Auditory brainstem response (ABR) was recorded in mice before and after acoustic noise trauma (see below). Mice were anesthetized with a mixture of ketamine/xylazine 90/6 mg/kg and placed in a ste-reotaxic apparatus with thermal pad to avoid heat loss. A speaker was positioned in front and 11 cm away from the head of the animal. To record the ABR, two pre-chlorinated Ag/AgCl electrodes with ~1 kΩ impedance were used, one recording electrode and one reference. Electrodes were placed subdermally; small incisions were made in the skin covering the bregma region (reference) and another in the region of lambda (recording). The ground was connected to the system ground. Sound stimulus consisted of narrow-band Gaussian white noise pulses with 3 ms duration each, presented at 10 Hz for 529 repetitions for each frequency and intensity tested. The frequency bands tested were 8 ~ 10 kHz, 9 ~ 11 kHz, 10 ~ 12 kHz, 12 ~ 14 kHz and 14 ~ 16 kHz (same as for GPIAS, see below). Pulses were presented initially at 80 dB sound pressure level (dBSPL) and decreasing in 5 dBSPL steps down to 45 dBSPL. All sound equipment was calibrated with an ultrasonic microphone (4939-A-011, Brüel and Kjær). 2.3. Tinnitus induced by salicylate
For salicylate-induced tinnitus, mice received an injection of 300 mg/kg sodium salicylate (Sigma) diluted in saline or saline (con-trol) intraperitoneal injection 1 h prior to the experiments (Brozoski and Bauer, 2016;Lauer et al., 2018;Winne et al., 2019).
2.4. Tinnitus induced by acoustic trauma
Acoustic over-exposure to cause noise-induced tinnitus were carried out in a sound shielded room, inside a sound-isolated cabinet (44x33x24cm). Mice were habituated for 5 days and were then placed inside an acrylic cylinder with restraining doors perforated at regular intervals, to maintain in front of the speaker (4 cm) during the noise-overexposure (broadband noise of 4–20 ± 1 kHz, 90 dBSPL, 90 min). This intensity is enough to generate tinnitus perception without causing permanent hearing loss (T. Malfatti, unpublished data). After the acoustic trauma, mice were kept in the sound shielded room for another hour to ensure development of tinnitus, since recent studies have shown that increased ambient noise and acoustic enrichment immediately after noise trauma prevents circuit reorganization of the inferior colli-culus and thereby can prevent noise-induced tinnitus (Sturm et al.,
2017).
2.5. Gap prepulse inhibition of acoustic startle
Gap prepulse inhibition of acoustic startle (GPIAS) test was per-formed to assess salicylate- or noise-induced tinnitus perception. Animals were placed in custom-made acrylic restraining boxes perfo-rated at regular intervals. The restraining boxes were placed in a sound-shielded custom-made cabinet (44x33x24cm) with low-intensity LED lights in a sound-shielded room. A loudspeaker (Super tweeter ST400 trio, Selenium Pro) was placed horizontally 4.5 cm in front of the re-straining cylinder, and startle responses were recorded using a digital accelerometer (MMA8452Q, NXP Semiconductors, Netherlands) mounted to the base plate of the restraining cylinder and connected to an Arduino Uno, which provides the accelerometer signal outputs to a data acquisition board (Open-ephys). Sound stimulus consisted of 60 dBSPL narrow-band Gaussian white noise (background noise); 40 ms of silence (Gap trials) or background noise (No-Gap trials); 100 ms of background noise; and 50 ms of background noise at 105 dBSPL (startle pulse). The intensity of the background noise presented was adjusted to be 10 dBSPL above the animal's hearing threshold, to assure that the animal can hear the stimulus. Trials of initial background noise were pseudo-randomized (interval of 12 to 22 s), and trials were played continuously. Frequencies between 8 and 10, 9–11, 10–12, 12–14 and 14–16, as well as 8–16 kHz were tested. The full session consisted of 18 trials per band of frequency tested, 9 Gap and 9 No-Gap trials), pre-sented pseudo-randomly. All sound equipment was calibrated with an ultrasonic microphone (4939-A-011, Brüel and Kjær).
2.6. Openfield test (OFT)
In the open-field test, each mouse received an injection of sodium salicylate diluted in saline or a control saline injection. Tests order was randomized with the second session 1 week following the first. In general, 1 h following ip injection (salicylate or saline) each mouse was placed in a rectangular openfield arena (40 cm × 32 cm × 15 cm) made of opaque white plastic for electrophysiological and video re-cordings. Two sessions of ten minutes each were recorded. At the be-ginning of each session animals were connected to the headstage and lightweight recording cable and placed individually in the center of the open field. The following behaviors were quantified: total distance travelled, and time spent in the center of the arena during the ten minutes of the test (Lopatina et al., 2014; Seibenhener and Wooten, 2015;Wang et al., 2011). Between each tested animal, the apparatus were cleaned with water. For DMT experiments, 5-MeO-DMT (sigma) wasfirst dissolved in DMSO and then in 0.9% saline (1:500) to a final dose of 20 mg/kg and injected I.P. 7 days before the re-exposed to open-filed test (4 weeks after the first open-field experiment). It is important to note that salicylate induces anxiety in the open-field even if animals are re-tested in < 15 days (Winne et al., 2019).
2.7. Elevated plus maze (EPM)
In the EPM, the mice received salicylate injection 1 h before the test and each animal were exposure only once to the EPM. The apparatus has four arms of equal dimensions (6.5 × 62 cm), two of them open and two closed (walls with 20 cm height) elevated 52 cm above of thefloor. The animals connected to the headstage and cables were placed in-dividually in the center of the EPM and allowed to explored the maze for 10 min. Total time in the open or closed arms and exploration patterns were quantified (Buccafusco and Buccafusco, 2009; Komada et al., 2008; Walf and Frye, 2007). EPM exploration sessions were synchronized with electrophysiological recordings and the apparatus were cleaned with water between each tested animal.
2.8. Histology
To verify electrode position after the experiments, animals were deeply anesthetized with ketamine/xylazine (80 mg/kg) and transcar-diac perfusion was carried out with cold saline solution followed by 4% paraformaldehyde (PFA). Brains were dissected and placed in PFA with 30% sucrose for hydration for (two days). Next brains were frozen in isopentane and sectioned on a cryostat (50μm thick coronal slices) and sections of areas of interest were processed using Nissl colorimetric staining (Paul et al., 2008). In summary, sections were placed in xylol for 10 min, gradually rehydrated through increasing concentrations of water in the alcohol, next stained with cresyl violet, and gradually dehydrated through decreasing concentrations of water in the alcohol followed by xylene andfinally mounted on glass slides using the Per-mount Per-mounting medium. Images were taken with a microscope (Zeiss, Berlin, Germany) at 10 × magnification.
2.9. Immunohistochemistry for c-Fos and analysis
For c-Fos immunohistochemistry, 60 min after the salicylate injec-tion, young mice (4–5-month-old) and old mice (11–13-month-old) were deeply anesthetized with ketamine and transcardially perfused. The brains were sectioned in 50 μm slices. Sections containing the vHipp were sequentially incubated in blocking solution (Phosphate buffer saline (PBS), 3% bovine serum albumin and 0.2% Triton-X-100. Sections were next drained and incubated in primary antibody rabbit anti-cFos (1:1000, Santa Cruz, California, USA) overnight at 4 °C. Next sections were washed 3× in PBS and incubated in secondary antibody (Goat anti-rabbit IgG 555, (1:1000, Invitrogen). Finally, sections were washed 3× in PBS and coverslipped with 10μL antifade mounting medium. Images were acquired using a conventional upright micro-scope (equipped with epifluorescence) (Zeiss) and analyzed in ImageJ (Rueden et al., 2017).
2.10. Data acquisition and statistical analysis
For each frequency tested in the Gap detection test, Gap and No-Gap trials responses were separated and the signal was filtered with a Butterworth low passfilter at 50 Hz. The absolute values of the accel-erometer axis were averaged and sliced 400 ms around the startle pulse (200 ms before and 200 ms after). The root-mean-square (RMS) of the sliced signal before the sound pulse (baseline) was subtracted from the RMS after the startle pulse (response). The GPIAS index for each fre-quency was calculated as (1-(GapRMS/NoGapRMS))*100, meaning the percentage of suppression induced by the gap.
For each intensity of each frequency tested in ABR tests, all trials were averaged,filtered using a Butterworth bandpass filter of 3rd order from 500 ~ 1500 Hz, and sliced 12 ms after the sound pulse onset. Thresholds were defined by visually inspecting the lowest intensity that can elicit a wave peak in response to sound. Python scripts for ABRs, GPIAS and sound calibration signal generation and analysis can be found athttps://gitlab.com/malfatti/SciScripts.
Localfield potentials (LFPs) were acquired by a 16-channel ampli-fier OpenEphys system (Siegle et al., 2017) with an Intan RHD head-stage set to high passfilter from 0.1 Hz during open field tests. Video was simultaneously recorded using a Basler camera (model acA1300-30um) triggered (frame by frame) by an Arduino board (precisely at 20 frames per second) also recorded using the OpenEphys. A maximum of three mice were recorded per day and the recordings were done during the night. Power spectral densities (PSD) for all channels were com-puted using the Welch method (pwelch Matlab command). A custom Matlab program (using the Matlab Imaging Processing Toolbox) was used for tracking the animal in thefield by thresholding the animal compared with the background. We calculated the coherence between mPFC and vHipp channels using the Matlab command mscohere. Data is presented by mean ± standard error of the mean (SEM). 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).
The expression of c-Fos in the ventral hippocampus of animals of both groups was quantified by optical densitometry using ImageJ,
Fig. 1. Auditory brainstem responses (ABRs) and Gap prepulse inhibition of acoustic startle (GPIAS) tests of young and old mice. A. Representative auditory brainstem responses (ABRs, left) from an young animal in response to 14-16 kHz sound pulses. Traces represent average of responses to 529 sound pulses for each intensity. Response peaks with at least 1SD amplitude can be detected for all intensities tested, which indicates that the hearing threshold in this example is < 50dBSPL. Right, hearing thresholds for 10 old and 10 young animals for different narrowband frequencies tested. As expected, old animals showed increase in hearing threshold for higher frequencies (12-14KHz and 14-16KHz; p = .02556 and 0.00602, respectively, Student's t-test). B. GPIAS paradigm and schematic responses for stimulus without gap (left top) and with gap (left bottom) from a normal hearing mouse. Group responses of GPIAS index (1-(Gap/noGap)*100) separated by frequency of old and young animals (middle). Group GPIAS index of the most affected frequency (the least GPIAS suppression as the gap is not perceived well– indication of tinnitus perception) for each animal (right). C. Example traces for an animal with salicylate-induced tinnitus (left). Group responses show no significant difference between performance after saline and salicylate injection for any frequency tested (middle), but group responses of the most affected frequency for each animal showed that after salicylate injection there was a decrease in gap detection performance (p = .00521, Student's t-test, Bonferroni-corrected for 6 comparisons). D. After noise overexposure, young animals group responses had no decrease in GPIAS performance for any tested frequency comparing to the same group of non-exposed young animals in B, but group responses of the most affected frequency for each animal showed a significant decrease for noise-exposed animals (p = .02104, Student's t-test, Bonferroni-corrected for 6 comparisons).
following a protocol adapted from (Biran et al., 2005; Budoff et al., 2019). Mann-Whitney U tests were performed after a Kolmogorov-Smirnov test that demonstrated the necessity of non-parametric ana-lysis in this case (statistically significant difference is indicated for p < .05).
3. Results
3.1. Auditory brainstem responses, gap detection and c-Fos immunohistochemistry
We first analyzed auditory brainstem responses (ABRs) in young (age 4–5 month) and old (age 11–13 month) as the C57Bl/6 J mouse line is known to develop age-related hearing loss (Ison et al., 2007). As expected, old animals showed an increased hearing threshold to higher frequencies (Fig. 1A), specifically in response to frequencies of 12–14 kHz and 14–16 kHz (64.5 ± 4.0 dBSPL and 67.0 ± 3.6 dBSPL, respectively) when compared to young animals (53.3 ± 2.1 dBSPL and 49.4 ± 2.0 dBSPL; p = .02556 and 0.00602, Student's t-test, Bonfer-roni-corrected for 5 comparisons,Fig. 1A). Next, we wanted to test if old animals have an increased tinnitus perception comparing to young mice, and here we evaluated tinnitus-like behavior using a gap detec-tion test. The GPIAS index shows the level of suppression (in percent) generated by the silent gap. Low GPIAS index indicates that the animal does not perceive the gap, which may be due to a persistent tinnitus in the same frequency as the background noise band. We did notfind any significant difference in suppression of the startle response by the silent gap related to any specific frequency between old (n = 5) and young (n = 5) mice (Fig. 1B, middle), however when grouping the most af-fected frequency for each animal a trend of decrease in GPIAS sup-pression was seen in the old mice (Fig. 1B, right, p = .05209, Student's t-test, Bonferroni-corrected for 6 comparisons, Fig. 1B). This suggests that old mice tend to have a decreased gap suppression, which may indicate age-related tinnitus perception, compared to young animals.
We then investigated whether salicylate (300 mg/kg, i.p.) and noise overexposure (broadband noise of 4–20 ± 1 kHz, 90 dBSPL, 90 min) produced changes in gap startle suppression. The GPIAS index for the most affected frequency showed a significant decrease in suppression for the salicylate-injected group compared to saline-injected group (p = .00521, Student's t-test, Bonferroni-corrected for 6 comparisons; Fig. 1C, right). Similarly, young mice showed a significant decrease in startle suppression following noise overexposure when compared to non-exposed young animals (p = .02104, Student's t-test, Bonferroni-corrected for 6 comparisons;Fig. 1D, right) for the most affected fre-quency, but not for any particular frequency band (Fig. 1D, center). Taken together, this data supports the notion that both salicylate and noise overexposure can cause tinnitus, but that the frequency of tinnitus perception is different among individual animals (Longenecker and Galazyuk, 2016)
We next used c-Fos immunohistochemistry to investigate whether salicylate differentially affect neuronal activity of cells in the ventral hippocampus of young (age 4–5-month, n = 3) and old mice (age 11–13-month, n = 3,Fig. 2A). Neurons with increased activity were detected by cFos-immunoreactivity within the ventral CA1 region. Animals sacrificed 1 h following salicylate injection showed no statis-tical difference in staining density median between the vHipp of young mice (21.06 ± 7.43 A.U) and old mice (16.64 ± 7.55 A.U, p = .059, Fig. 2B). These results suggest that salicylate injection have similar immediate effects on overall neuronal activity in the vHipp of young and old mice.
3.2. Openfield test
Anxiety-like behavior in animals of different ages was assessed using the openfield test (young mice: 4–5-month-old, n = 10 and old mice: 11–13-month-old, n = 7). Animals were placed in an open field
arena 1 h after i.p. injection of saline or salicylate (Fig. 3A). Following the salicylate injection, young animals spent significantly less time in the center of the arena compared to the saline group (mean center time, saline group: 192.61 ± 44 s; salicylate group: 72.41 ± 34.74 s, for a 10 min session, p = .003, n = 10 mice,Fig. 3C). In addition, these mice showed decreased locomotion (mean travelled distance saline group: 41.11 ± 10.79 m; salicylate group 21.24 ± 5.78 m, p = .0001, Fig. 3C). When testing old animals, salicylate injected mice spent more time in the center (saline group: 192.61 ± 58.26 s; salicylate group: 246.71 ± 76.72 s, p = .04, n = 7 mice,Fig. 3D). Locomotion of old animals showed no difference between saline and salicylate groups (total travelled distance saline group: 54.84 ± 9.53 m; salicylate group: 41.87 ± 6.2 m, p = .14,Fig. 3D). As reduced time in the center and reduction in exploration indicates anxiety-like behavior and our results suggest that acute salicylate administration produce anxiety-like behavior preferentially in younger mice.
The resilience of old mice in displaying anxiety-like behavior fol-lowing salicylate treatment could be explained by hearing loss (and possible pre existing tinnitus). In a reversed scenario, young animals with tinnitus as a (precondition induced by noise trauma) would not show anxiety-like behavior after salicylate injection (in case anxiety is directly related to tinnitus perception rather than salicylate itself). We, therefore, injected salicylate in animals previously exposed to acoustic trauma with signs of tinnitus screened by GPIAS followed by the open field test (n = 9 mice,Fig. 3B). Young mice with preconditioned tin-nitus (acoustic trauma) injected with salicylate spent more time in the center than saline treated mice (time in the center acoustic trauma/ saline: 198.63 ± 80.579 s; acoustic trauma/salicylate: 304.68 ± 73.48 s, p = .04,Fig. 3E). However, acoustic trauma ani-mals showed less locomotion following salicylate injection (mean tra-velled distance acoustic trauma/saline: 55.10 ± 17.40 m; acoustic trauma/salicylate: 39.29 ± 10.51 m, p = .01, n = 9 mice,Fig. 3E). These findings showed that salicylate conversely increased the time spent in the center, indicating reduced anxiety. However, due to less distance travelled, this could also indicate a hypolocomotion rather than reduced anxiety in the acoustic trauma + salicylate animals.
Next we compared the age-effect of salicylates by comparing old mice versus young mice post-acoustic trauma (Fig. 3F). Interestingly, old animals treated with salicylate showed similar values to young acoustic trauma animals administered salicylates when comparing mean time in the center of the arena (acoustic trauma salicylate group: 304.68 ± 73.48 s; old salicylate group: 246.71 ± 76.72 s in a 10 min session, p = .34, n = 9 and 7 mice respectively,Fig. 3F) and loco-motion (total travelled distance post-acoustic trauma animals after salicylate 39.29 ± 10.51 m, old mice after salicylate: 41.87 ± 6.2 m; p = .48,Fig. 3F). These results show that, similar to old mice, young mice that had a previous tinnitus perception are less prone to display anxiety-like behavior after salicylate injection cause (when time in center and distance travelled is taken into account). As salicylate alone in young mice cause less time spent in the center and less distance travelled (Fig. 3C) these results could indicate that previously altered auditory perception caused by noise trauma masks salicylate-induced tinnitus effects on anxiety.
3.3. Type 2 theta and slow gamma oscillations in openfield
In addition to behavioral data, LFP was recorded in young mice (n = 10 mice) and old mice (n = 7 mice). We histologically and electrophysiologically confirmed the electrode placement at the radial stratum CA1 (SR) (Schomburg et al., 2014) (Fig. 4A and B). Theta os-cillations were separated in 2 bands, 4–6 Hz and 7–10 Hz, for type 2 and type 1 theta bands (Kramis et al., 1975;Sainsbury and Montoya, 1984;Montoya et al., 1989). Increased gamma oscillations were also observed in the power spectrum density (PSD) plots. These oscillations are classically separated in 2 bands, ~30–60 Hz (slow gamma) and ~65–100 Hz (fast gamma) (Colgin, 2015;Hsiao et al., 2016). In young
animals, during free exploration in the openfield following salicylate injection, power spectrum analysis revealed an increase in amplitude of frequency between 4 and 6 Hz and 30–60 Hz (slow gamma) in com-parison to injection of saline. Mean 4–6 Hz power was equal to 2335.98 ± 888.53 μV2 /Hz for the saline group and 3211.33 ± 930.56μV2/Hz for the salicylate group (p = .01,Fig. 4C) and mean 30–60 Hz power was equal to 75.16 ± 41.44 μV2/Hz for the saline group and 107.16 ± 58.83 μV2 /Hz for the salicylate group (p = .004,Fig. 4D). However, in old mice, salicylate injections did not cause a significant difference in the mean power of theta2 (4–6 Hz; 2258.48 ± 1161.61 μV2 / Hz for saline solution and 2344.78 ± 1061.01μV2/ Hz for salicylate, p = .496,Fig. 4E) or mean power of slow gamma (30–60 Hz; 108.44 ± 34.37 μV2/ Hz for saline solution and 118.55 ± 43.86μV2/ Hz for salicylate, p = .38,Fig. 4F). These data indicate that salicylate modulates circuits associated with type 2 theta and slow gamma oscillation in the ventral hippocampus specifically in young mice naive to tinnitus.
3.4. Complementary appearance of theta2 and slow gamma in the elevated plus maze
To test if theta2 and slow gamma encode different states of the animal in anxiogenic environments, we use the elevated plus maze test (EPM) for a clear separation of perception of safety (closed arms) or eminent danger (open arms) during salicylate-induced tinnitus. Salicylate injected young mice (n = 6) spend significantly less time in the open arms (117 ± 55, 8 s) compared in the close arms (459 ± 73.8 s), p = .001, (Fig. 5A) of the EPM. However, old mice (n = 6) treated with salicylate did not show significant difference in time spent in the open arms compared with closed arms (258.54 ± 55.67 s for opened arms and 323.09 ± 90.81 s for closed arms, p = .42,Fig. 5B). LFP recordings during the exploration in the EPM showed that in salicylate-treated young mice an increase in theta2 amplitude (4–6 Hz) in the closed arms in comparison to the open arms as shown by power spectrum analysis (mean 4–6 Hz power was equal to 3582,02 ± 1654,33 μV2 /Hz in the closed arms and 1869,56 ± 993,51μV2/Hz in the open arms, p = .008,Fig. 5C). On the other hand, there was a significant increase in the mean 30–60 Hz oscillation (slow gamma) power in the open arms of the EPM (mean
30–60 Hz power was equal to 154.96 ± 73.09 μV2/Hz for the closed arms and 243.87 ± 106.20 μV2/Hz for the open arms, p = .047, Fig. 5C). However, in old mice injected with salicylate showed no sig-nificant difference in the mean power of 4–6 Hz (1901.93 ± 1206.79 μV2 / Hz for closed arms and 1847.99 ± 1255.21μV2/ Hz for open arms, p = .165,Fig. 5D) and 30–60 Hz between opened and closed arms (123.14 ± 27.47 μV2/ Hz for closed arms and 149.99 ± 52.80μV2/ Hz for open arms, p = .165, Fig. 5D). Taken together, this data shows that old mice do not show “anxiety-signaling” theta2 after salicylate injection and theta2 and slow gamma encode different states in anxiogenic environments.
3.5. Complementary theta2 and slow gamma in the openfield
Anxious-like young mice displayed higher theta2 power“safer” re-gions of the EPM (closed arms) while slow gamma power increased in the most anxiogenic regions (open arms). To further demonstrate the complementary appearance of these two rhythms, we investigated if young animals (n = 10) presented similar results when exploring the center (“anxiogenic” area) and border (“safer” area) of the open field. Similar to the EPM, after salicylate injections, mice in the openfield arena showed a significant increase in the mean 4–6 Hz oscillation power in the border (2639.38 ± 950.71μV2/Hz) in compared the center (2223.51 ± 854,21 μV2 /Hz) of the open field (p = .006, Fig. 5E). We have also found a significant increase in the mean 30–60 Hz oscillation power in the center compared of the border (mean 30-60 Hz power was equal to 134.87 ± 27.0.07μV2/Hz and in the center and 114.52 ± 28.69 μV2 /Hz in the border and, p = .01, Fig. 5E). Taken together, these results show that theta2 and slow gamma oscillations increase in the vHipp and after salicylate injections in anxiety tests in a complementary manner.
3.6. mPFC oscillations and coherence with vHipp in salicylate-induced anxiety
As the old animal group did not show anxiety-like behavior, further experiments only explore effect of salicylate on theta and gamma os-cillations in the mPFC of young animals (n = 10). We used LFP and post-hoc histology to confirm the placement of electrodes mPFC (Winne
Fig. 2. c-Fos expression after salicylate injection is similar in young and old mice. A. Experimental protocol for c-Fos immunohistochemistry. B. c-Fos expression observed in brain tissue from young and old animals after salicylate injection. C. Boxplot summarizing c-Fos-immunostaining staining density, *p < .05, **p < .01.
et al., 2019), (Fig. 6A and B). In the openfield, power spectrum analysis revealed an increase of frequency peak between 4 and 6 Hz and 30–60 Hz when animals receive salicylate injection in comparison to injection of saline. Mean 4–6 Hz power was equal 758.63 ± 231.60 μV2 /Hz for the saline group and 898.23 ± 268.64μV2/Hz for the salicylate group (p = .008,Fig. 6C) and mean 30-60 Hz power was equal to 32.62 ± 6.52μV2/Hz for the saline group and 42.67 ± 9.19 μV2 /Hz for the salicylate group (p = .00002,Fig. 6D). In the elevated plus maze test, when previously injected with salicylate, the power spectrum analysis revealed an in-crease in amplitude of frequency between 4 and 6 Hz in the closed arms (587.70 ± 183.50 μV2 /Hz) in comparison to the open arms
(376.20 ± 151.21μV2/Hz), p = .0004,Fig. 6E. There was a sig-nificant increase in the mean 30–60 Hz oscillation power in the open arms of the EPM (42.38 ± 20.87μV2/Hz) in comparison to the open arms (32.29 ± 15.53μV2/Hz, p = .03,Fig. 6F). We then asked if the mPFC and ventral hippocampus show coherent salicylate induced theta2 oscillation in young animals (n = 10). We measure coherence in theta oscillations from pairs of electrodes implanted in the mPFC and vHipp (Fig. 7A). Ventral hippocampus/mPFC theta2 coherence was significantly higher in the salicylate condition (mean coherence for the saline group was equal to 0.28 ± 0.12 for the saline group and 0.51 ± 0.06 for the salicylate group, p = .0001, n = 10,Fig. 7B). Thus, coherence analysis indicates that salicylate-induced theta
Fig. 3. Decreased locomotion and increase in anxiety-like behavior induced by salicylate depends on age. A. Experimental protocol for exposure of the animals to openfield under salicylate and saline effect. B. Experimental protocol for induction of acoustic trauma. C. Young animals: Example plots of locomotion in the arena (left) for saline (top) and salicylate (bottom). Purple and pink points highlight the movement at the center and border area, respectively. Boxplot showing mean total distance travelled for mice treated with saline and salicylate showing reduced locomotion activity for salicylate group (top). Boxplot of total time spent in center for mice treated with saline and salicylate indicating increased anxiety-like behavior (bottom). *p < .05, **p < .01. D. Old animals: Example plots of locomotion in the arena (right) for saline (top) and salicylate (bottom). (top) Boxplot showing mean total distance travelled for mice treated with saline or salicylate and (bottom) total time spent in center for mice treated with saline and salicylate. E. Acoustic trauma: Boxplot showing mean total distance travelled for mice treated with saline and salicylate showing increase locomotion activity for salicylate group (left). Boxplot of total time spent in center for mice treated with saline and salicylate indicating decrease anxiety-like behavior (right). *p < .05, **p < .01. F. (left) Boxplot showing mean total distance travelled for mice post acoustic trauma and old animals treated with salicylate (right) and total time spent in center arena by these animals. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)
oscillation is efficiently transmitted from the ventral hippocampus to the mPFC.
3.7. Behavior and LFP recordings in mice pretreated with 5-MeO-DMT Lastly, we investigated whether a single dose of 5-MeO-DMT would have a short-term anxiolytic effect (4 days after DMT injection) on the behavior of young mice (n = 8) after salicylate injection (Fig. 8A, B). We found that 20 mg/kg 5-MeO-DMT reversed the salicylate anxiogenic effect (Fig. 8C). The animals treated with DMT four days prior to the test showed increase locomotion after salicylate administration (1 h prior to test) when compared to animals exposed to salicylate without previous DMT treatment (total distance walked 22.52 ± 6.07 m and 40.00 ± 14.82 m for salicylate and salicylate after DMT respectively, p = .04,Fig. 8C). Pre-treatment with DMT also caused the animals to
spend more time in the center of the arena (69.06 ± 34.03 s for sal-icylate and 120.29 ± 44.29 s salsal-icylate after DMT, respectively, p = .001,Fig. 8C). In addition to reversing the anxiogenic effect of salicylate, mean theta2 power was also significantly reduced in animals pre-treated with DMT (1959.16 ± 673.22μV2/Hz for the salicylate post DMT group and 3337.53 ± 1081.43μV2/Hz for the salicylate group, p = .01, n = 8,Fig. 8D). The mean of 30–60 Hz of slow gamma power was also significantly reduced (117.52 ± 43.25 μV2/Hz for salicylate post DMT group and 137.93.43 ± 53.94 μV2/Hz for the salicylate group, p = .04, n = 8,Fig. 8D). These results demonstrate that pre-treatment with DMT can prevent anxiety related changes in the type 2 theta and slow gamma oscillation in the ventral hippocampus in young mice produced by salicylate.
Fig. 4. Salicylate increases type 2 theta and gamma rhythms in the ventral hippocampus depends on age. A. (left) Schematic drawing of the localization of electrodes in the ventral hippocampus (*). (right) Brightfield image showing positioning of electrode in stratum radiatum (arrow) on the vHipp. B. Representable example of a raw trace signal from one channel from the same animal for saline (top) and salicylate (bottom). C. Young animals: Mean power spectrum density for all animals showing an increase in type 2 theta (4–6 Hz) in salicylate (red) compared to saline (gray). Boxplots showing the mean power was significantly different between saline and the salicylate group for 4–7 Hz oscillations (type 2 theta), *p < .05, **p < .01, n = 10 mice. D. Young animals: Mean power spectrum density for all animals showing an increase increase in gamma (30–60 Hz) in salicylate (red) compared to saline (gray). Boxplots showing the mean power was significantly different between saline and the salicylate group for gamma oscillations, *p < .05, **p < .01, n = 10 mice. E. Old animals: Mean power spectrum density for saline (gray) vs. salicylate (blue) for 4–7 Hz oscillations (type 2 theta). Boxplots showing the mean power for saline and salicylate groups for 4–7 Hz oscillation (type 2 theta), *p < .05, **p < .01. F. Old animals: Mean power spectrum density for saline (gray) vs. salicylate (blue) for 30–60 Hz oscillations. Boxplots showing the mean power for saline and salicylate groups for gamma oscillations, *p < .05, **p < .01. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)
4. Discussion
In this work, we show that anxiety-like behavior observed in
animals acutely exposed to salicylate only occurs in animals naive to tinnitus. In addition, we describe that during anxiety both theta2 and slow gamma can serve as biomarkers encoding safety and danger,
Fig. 5. Salicylate increases type 2 theta and gamma rhythms in different arms of elevated plus maze and depends of age. A. Young animals: Example plots of locomotion in the elevated plus maze for salicylate group (left). Boxplot showing total time spent in closed and opened arms for young mice treated with salicylate, *p < .05, **p < .01, ***p < .001. B. Old animals: Example plots of locomotion in the elevated plus maze for salicylate group (left). Boxplot showing total time spent in closed and opened arms for old mice treated with salicylate. C. Mean power spectrum density for salicylate young group showing an increase in type 2 theta (4–6 Hz) in closed arms (gray) compared to opened arms (red). Boxplots showing that mean power was significantly different between closed arms and opened arms for 4–7 Hz oscillations (type 2 theta), *p < .05, **p < .01 (left). Mean power spectrum density in the closed arms (gray) vs. opened arms (red) for 30–60 Hz oscillations (gamma). Mean power was significantly different between closed arms and the opened arms for 30-60 Hz oscillations (gamma) *p < .05, **p < .01 (right). D. Mean power spectrum density for salicylate old mice showing type 2 theta (4–6 Hz) in closed arms (gray) compared to opened arms (red). Boxplots showing mean power between closed arms and opened arms for 4–7 Hz oscillations (type 2 theta). Mean power spectrum density in the closed arms (gray) vs. opened arms (red) for 30–60 Hz oscillations (gamma). Mean power between closed arms and the opened arms for 30-60 Hz oscillations (gamma) *p < .05, **p < .01 (right). E. Mean power spectrum density in the border (gray) vs. center (red) in the openfield for 4–7 Hz and 30–60 Hz oscillations. Boxplots showing mean power between border and center for 4–7 Hz oscillations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
respectively. These two rhythms were augmented in both the vHipp and mPFC. However, only theta2 was coherent between these two regions. Lastly, we demonstrate that a single, pre-salicylate, injection of 5-MeO-DMT provides resilience to the anxiety generate by tinnitus in the sal-icylate animal model.
Our GPAS experiments show that salicylate induced tinnitus do not show specific frequency responses across different subjects. Salicylate can cause a variety of types tinnitus, especially in relation to the center frequency of tinnitus (~7 kHz,Jastreboff et al., 1988; ~10 kHz,Guitton et al., 2003 and Zheng et al., 2006; ~15 kHz, Bauer et al., 1999; ~16 kHz,Yang et al., 2007,Kizawa et al., 2010and Ralli et al., 2010; ~12, ~16, ~20 and ~24 kHz,Su et al., 2012; broadband noise-like, Rüttiger et al., 2003andTurner and Parrish, 2008; or BBN-like 2 h after and 8–10 kHz 5 h after salicylate injection,Berger et al., 2013). Still
there is variability in tinnitus-like behavior observed when different animals are subjected to the same acoustic trauma (noise-induced tin-nitus), showing altered Gap-detection in response to different fre-quencies (Coomber et al., 2014). Therefore, it is not possible to predict in which frequency each animal will perceive tinnitus.
Salicylate poisoning is a known cause of tinnitus in humans (Pearlman and Gambhir, 2009;Puel and Guitton, 2007) but the drug has diverse effects in non-auditory neurons (Pearlman and Gambhir, 2009;Wu et al., 2015). Hence, anxiety-like behavior found in animals treated with salicylate could be a direct effect of the compound on limbic neurons. However, young animals subjected to acoustic trauma or older animals suffering of age-related hearing loss showed no signs of anxiety after salicylate injection. Besides, there was no difference in the c-Fos expression in the vHipp of young versus old animals following
Fig. 6. Salicylate increases type 2 theta and gamma rhythms in the prelimbic cortex: A. left: Schematic drawing of the localization of electrodes in the prelimbic cortex (*). Right: Brightfield image showing positioning of electrode in the PL cortex. B. Example of a LFP recording in the PL cortex from one animal after saline (top) and salicylate (bottom) injection. C. Mean power spectrum density for all animals showing an increase in type 2 theta (4–6 Hz) following salicylate (purple) compared to saline (gray) (n = 10) administration. Boxplots showing that mean power was significantly different between saline and the salicylate group for 4–7 Hz oscillations (type 2 theta), *p < .05, **p < .01. D. Mean power spectrum density for saline (gray) vs. salicylate (purple) for 30–60 Hz oscillations (gamma). Mean normalized power was significantly different between saline and the salicylate group for 30-60 Hz oscillations (gamma) *p < .05, **p < .01. E. Mean power spectrum density for all animals showing an increase in type 2 theta (4–6 Hz) in closed arms (gray) compared to opened arms (purple) in salicylate group (n = 10). Boxplots showing that mean power was significantly different between closed arms and opened arms for 4–7 Hz oscillations (type 2 theta), *p < .05, **p < .01. Mean power spectrum density in the closed arms (gray) vs. opened arms (purple) for 30–60 Hz oscillations (gamma). Mean normalized power was significantly different between closed arms and the opened arms for 30-60 Hz oscillations (gamma) *p < .05, **p < .01.
salicylate injections. Old mice injected with salicylate, however, stayed longer in the center of thefield and showed greater locomotion than salicylate-treated young mice. A recent study has shown that old and young normal male animals show no difference in performance in EPM and open-field tests (Nolte et al., 2019). Thus, it seems that previous tinnitus perception and/or hearing loss protect animals from anxiogenic effects of salicylate. Our data also strengthen the hypothesis that after salicylate injection, tinnitus perception rather than the drug itself cause anxiety.
Our previous work show that anxiety-like behavior elicits theta2 oscillation in the vHipp (Winne et al., 2019). Here, we show that the mPFC show coherent theta2 with vHipp. Other studies described syn-chrony between vHipp to mPFC is observed in anxiety-related and spatial representations of aversion (Adhikari et al., 2010; Padilla-Coreano et al., 2016). As a significant anxiety-like behavior was only observed in normal-hearing (young) animals, theta2 was not observed in old mice. Another importantfinding of this paper is the increase in slow gamma in both vHipp and mPFC when animals display anxiety-like behavior. Interestingly, we found that these two rhythms encode different states (theta2: safety/slow gamma: danger). We have pre-viously suggested that theta2 could encode safety in anxiogenic situa-tions (Mikulovic et al., 2018). Here, we not only confirm this idea but also describe another rhythm that is triggered by eminent danger.
Fig. 7. Salicylate increases type 2 theta coherence between vHipp and mPFC. A. Mean vHipp/mPFC coherence of control (gray) and salicylate (green). B. Boxplot summarizing coherence for 4–6 Hz for saline and salicylate-treated animals, *p < .05, **p < .01. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)
Fig. 8. Anxiety-like behavior induced by salicylate can be prevented by a single dose of 5-MeO-DMT: A. Experimental protocol for exposure of the animals to the openfield under salicylate effect before and after a single dose of DMT. B. Example of a LFP recording in the vHipp from one animal after salicylate (top) and salicylate post DMT (bottom). C. Example plots of locomotion in the arena (left) for saline (top) and salicylate (bottom). Dark green and light green points highlight the movement at the center and border area, respectively. D. Boxplot showing mean total distance travelled for mice treated with salicylate and salicylate post DMT showing increased locomotion activity for salicylate post DMT group (left). Boxplot of total time spent in center for mice treated with salicylate and salicylate post DMT indicating decrease anxiety-like behavior for salicylate post DMT group (right). *p < .05, **p < .01. E. Mean power spectrum density for salicylate (gray) vs. salicylate post DMT (Yellow) for 4–6 Hz oscillations (type 2 theta). Mean normalized 4-6 Hz and power between salicylate and salicylate post DMT. *p < .05 **p < .01. F. Mean power spectrum density for salicylate (gray) vs. salicylate post DMT (Yellow) for 30–60 Hz oscillations (gamma). Boxplots showing that mean power was significantly different between saline and the salicylate group for 30–60 Hz oscillations (gamma). *p < .05, **p < .01. (For interpretation of the references to colour in thisfigure legend, the reader is referred to the web version of this article.)
Specifically, both theta2 and slow gamma are markers of anxiety but while slow gamma encodes danger, theta2 appears in safer regions in anxiogenic situations.
Changes in theta2 could be explained by aging as its power can decrease in older animals (Abe and Toyosawa, 1999). However, older animals did not show increased power of slow gamma oscillations after salicylate injection in both open arms and center of openfield. In ro-dents, changes in slow hippocampal gamma rhythms are generally as-sociated, among other things, with slower running speed or increase in synchronization between CA3 and the cingulate cortex during im-mobility (Ahmed and Mehta, 2012;Buzsáki, 1986;Buzsáki and Wang, 2012;Zheng et al., 2015). Other studies showed that alteration gamma with anxiety behaviors (Batashvili et al., 2019;Cruces-Solis et al., 2019; Zhou et al., 2018). Nevertheless, to our knowledge, there is no study showing a systematic effect of aging on magnitude of gamma power, except when degeneration is present (Etter et al., 2019;Goutagny et al., 2013). The lack of theta2 in animals treated with 5-MeO-DMT asso-ciated to the lack of anxiety-like behavior also suggest that lack of anxiety rather than aging explain the absence of theta2 in old animals. The mechanisms by which hallucinogenic substances act in mood disorders are largely unknown. Neuroimaging studies suggest that treatment with psychedelics increases the connectivity between typi-cally ‘unconnected’ brain areas (Carhart-Harris et al., 2016; Kaelen et al., 2016;Tagliazucchi et al., 2016). Psychedelics affects neuroplas-ticity. Synaptically, 5-MeO-DMT alters frequency and amplitude of excitatory postsynaptic currents (EPSCs) in the prefrontal cortex (PFC) of rats that lasted long after release of the drug by the body (Ly et al., 2018). Also, neurogenesis (augmented by 5-MeO-DMT treatment), may also play a role in the anxiolytic effect of this compound (Lima da Cruz et al., 2018). Classic pharmacology works showed that 5-MeO-DMT acts as a non-selective HT agonist, and active at both the HT1A and 5-HT2A receptors (Calvey, 2018). But an important study showed that it can also modulate the sigma-1 receptor and therefore, indirectly affect Na+ and K+ channels (Fontanilla et al., 2009). Adding to the com-plexity of the mechanisms in which psychedelics affects the anxious brain, specific agonists of 5HT receptors do not seem to provide any beneficial effects in mood disorders (Sakloth et al., 2019).
To our knowledge, this is the first study that demonstrate that hallucinogen treatment can prevent anxiety triggered by tinnitus. Our data confirm that a pre-treatment with hallucinogen 5-MeO-DMT con-fers resilience to anxiety following salicylate intoxication. A single in-traperitoneal dose of 5-MeO-DMT was efficient to prevent changes in animal behavior caused by acute salicylate injection in the openfield and elevated plus maze and theta2 increase several days after treatment with the psychedelic compound. The absence of theta2 or slow gamma in animals resilient to tinnitus-induced anxiety further support the idea of resilience rather than a careless stereotypical behavior caused by the 5-MeO-DMT treatment. This study further evince the therapeutic po-tential of hallucinogenic substances in mood disorders. Future work should assess how long 5-MeO-DMT injections can protect animals from anxiety related to tinnitus perception.
In summary, this work supports our previous hypothesis that an-xiety-like behaviors triggered by salicylate is due to tinnitus perception rather than a direct effect of the drug. We also show that com-plementary theta2 and slow gamma are markers of anxiety and that these rhythms signal low and high danger, respectively, in anxious animals. Moreover, the present study shows an anxiolytic effect of 5-MeO-DMT pre-treatment for tinnitus-triggered anxiety. We did not in-vestigate here whether animals with a fourday prior injection of 5-MeO-DMT are less prone to suffer from tinnitus, but we are currently testing this hypothesis.
Author contributions
JW and RNL wrote the paper with input from KEL. JW and RNL designed the experiments and collected data. BCB, TM and IN collected
gap detection and ABR data with input from KEL. EB participated in DMT data collection. JD and EB collected c-Fos data.
Funding sources
This work is supported by the American Tinnitus Association and the Brazilian National Council for Scientific and Technological Development.
Declaration of Competing Interest
The authors declare no conflict of interest. References
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