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Articles in PresS. J Neurophysiol (October 8, 2014). doi:10.1152/jn.00580.2014
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Title: Suppression of putative tinnitus-related activity by extra-cochlear electrical stimulation.
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Abbreviated title: Suppression of putative tinnitus-related activity
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Norena A.J.1, Mulders W.H.A.M.2, Robertson, D.2
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NAJ, MWHAM and RD designed the study, NAJ analyzed data and made figures, NAJ,
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MWHAM and RD wrote the manuscript.
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Affiliation:
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Aix-Marseille University, Marseille, France
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The University of Western Australia, Crawley, Australia
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Aix-Marseille University, Centre Saint Charles, Case B
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3 place Victor Hugo, 13331 Marseille, France
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Email : [email protected]
Corresponding author:
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Copyright © 2014 by the American Physiological Society.
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Abstract
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Studies on animals have shown that noise-induced hearing loss is followed by an increase of
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spontaneous firing at several stages of the central auditory system. This central hyperactivity
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has been suggested to underpin the perception of tinnitus. It was shown that decreasing
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cochlear activity can abolish the noise-induced central hyperactivity. This latter result further
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suggests that an approach consisting of reducing cochlear activity may provide a therapeutic
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avenue for tinnitus. In this context, extra-cochlear electric stimulation (ECES) may be a good
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candidate to modulate cochlear activity and suppress tinnitus. Indeed, it has been shown
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that a positive current applied at the round window reduces cochlear nerve activity and can
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suppress tinnitus reliably in tinnitus subjects. The present study investigates whether ECES
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with a positive current can abolish the noise-induced central hyperactivity, i.e. the putative
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tinnitus-related activity. Spontaneous and stimulus-evoked neural activity before, during and
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after ECES was assessed from single-unit recordings in the inferior colliculus of
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anesthetized guinea pigs. We found that ECES with positive current significantly decreases
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the spontaneous firing rate of neurons with high characteristic frequencies (CF) whereas
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negative current produces the opposite effect. The effects of the ECES are absent or even
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reversed for neurons with low CFs. Importantly, ECES with positive current had only a
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marginal effect on thresholds and tone-induced activity of collicular neurons, suggesting that
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the main action of positive current is to modulate the spontaneous firing. Overall, cochlear
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electrical stimulation may be a viable approach for suppressing some forms of (peripheral-
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dependent) tinnitus.
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Keywords: plasticity; hearing loss; noise trauma; cochlear nerve; spontaneous activity;
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auditory perception
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Introduction
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Tinnitus, the auditory percept without any concomitant acoustic stimulation, is very
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prevalent in the general population and can dramatically impair the quality of life
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(McCormack et al., 2014). As tinnitus is associated with hearing loss in most, if not all, cases
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(Chung et al., 1984; Sindhusake et al., 2003), hearing loss has been suggested to act as a
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necessary cause or trigger of tinnitus. Many studies have shown that hearing loss is
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accompanied by both spontaneous and stimulus-induced hyperactivity at different levels of
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the central auditory system, i.e. the cochlear nucleus (Kaltenbach and Afman, 2000; Sumner
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et al., 2005; Bledsoe et al., 2009; Vogler et al., 2011), the inferior colliculus (Bauer et al.,
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2008; Izquierdo et al., 2008; Mulders and Robertson, 2009, 2011; Vogler et al., 2014) and
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the auditory cortex (Komiya and Eggermont, 2000; Noreña and Eggermont, 2003, 2006;
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Seki and Eggermont, 2003; Noreña et al., 2010). This increase in central neural activity
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occurs despite the reduction of spontaneous and stimulus-evoked cochlear nerve activity
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after cochlear lesions (Hartmann et al., 1984; Liberman and Dodds, 1984; Shepherd and
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Javel, 1997; Heinz and Young, 2004; Kujawa and Liberman, 2009). The spontaneous
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central hyperactivity triggered by hearing loss has been suggested to represent tinnitus-
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related activity (Kaltenbach et al., 2004; Brozoski et al., 2007; Bauer et al., 2008; Mulders
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and Robertson, 2009; Norena, 2011; Schaette and Kempter, 2012; Mulders et al., 2014).
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Interestingly, recent studies showed that the noise-induced hyperactivity recorded in
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inferior colliculus (IC) within the first few weeks after noise trauma can be abolished by
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cochlear ablation or other treatments impairing cochlear function (Mulders and Robertson,
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2009, 2011) or by activation of the efferent system (Mulders et al., 2010). These results
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show that in this time period after cochlear trauma residual spontaneous activity in the
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cochlear nerve is necessary for the persistence of the noise-induced hyperactivity in IC.
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In summary, the putative tinnitus-related activity in auditory centers (i.e. the noise-
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induced central hyperactivity) may result from the central amplification (through increase of
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neural sensitivity or gain due to hearing loss) of residual spontaneous activity provided by
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the cochlear nerve (Schaette and Kempter, 2006; Norena, 2011; Noreña and Farley, 2012).
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Two different therapeutic approaches can be derived from this view. First, normalizing
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the averaged sensory inputs to a pre-hearing loss level may restore the pre-hearing loss
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neural sensitivity and then alleviate tinnitus by reducing the central gain or reversing some
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tinnitus-related central changes. This would be achieved by customized acoustic stimulation,
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hearing aids or cochlear implants (Norena et al., 2002; Noreña and Eggermont, 2005, 2006;
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Schaette and Kempter, 2006; Noreña, 2012). Second, reducing the cochlear source of
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spontaneous activity in the auditory system may also reduce tinnitus (Mulders and
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Robertson, 2009; Norena, 2011; Mulders et al., 2014). A reduction of cochlear spontaneous
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activity can be achieved through extra- and intra-cochlear electrical stimulation with positive
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current (Konishi et al., 1970; Evans and Borerwe, 1982; Schreiner et al., 1986) and could
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account for the suppression of tinnitus reported in earlier studies using ECES with positive
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direct current (Hatton et al., 1960; Cazals et al., 1978; Portmann et al., 1979).
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The present study is aimed at improving our understanding of the tinnitus mechanisms,
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in particular the coupling between the tinnitus-related activity and the spontaneous cochlear
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activity, and at gaining further insights into the possibility of using cochlear electrical
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stimulation to suppress tinnitus in tinnitus subjects. The effects of electrical stimulation
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applied at the round window on both stimulus-induced and spontaneous single-unit activity in
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IC were investigated in anesthetized guinea pigs after noise trauma.
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Materials and Methods
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Animals
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Fifteen young adult pigmented guinea pigs of either sex were used. Animals weighed
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between 450 and 670 g at the time of electrophysiological recordings in the inferior
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colliculus. The experimental protocols were approved by the Animal Ethics Committee of
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The University of Western Australia (03/100/1007) and were carried out in accordance with
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the Guidelines from the National Health and Medical Research Council Australia regarding
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the care and use of animals for experimental procedures. All surgery was performed under
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anaesthesia and all efforts were made to minimize suffering.
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Recovery surgery for acoustic trauma and mechanical lesion of the cochlea
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Animals received a subcutaneous injection of 0.1 ml atropine sulphate (0.6 mg/ml), followed
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by an intraperitoneal injection of Diazepam (5 mg/kg). Twenty minutes later animals were
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injected intramuscularly with Hypnorm (0.315 mg/ml fentanyl citrate and 10 mg/ml
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fluanisone; 1 ml/kg). When deep anaesthesia was obtained as determined by the absence of
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the foot withdrawal reflex, the area of incision was shaved and animals were placed on a
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custom-made heating blanket in a soundproof room and mounted in hollow ear bars. A small
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opening of approximately 1 mm2 was made in the bulla to expose the cochlea and a plastic-
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insulated silver wire was placed on the round window. A compound action potential (CAP)
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audiogram (Johnstone et al., 1979) was recorded in the frequency range 4-24 kHz to assess
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the animals’ cochlear sensitivity. All sound stimuli were presented in a closed sound system
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through a ½” condenser microphone driven in reverse as a speaker (Bruel and Kjaer, type
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4134). The system was calibrated using a 1/8” condensor microphone in place of the
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tympanic membrane and an absolute sound calibrator (Bruel and Kjaer type 4231). Pure
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tone stimuli (10 ms duration, 1 ms rise/fall times) were synthesized by a computer equipped
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with a DIGI 96 soundcard connected to an analog/digital interface (ADI-9 DS, RME
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Intelligent Audio Solution). Sample rate was 96 kHz. The interface was driven by a custom-
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made computer program (Neurosound, MI Lloyd), which was also used to collect single
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neuron data during the final experiments. CAP signals were amplified, filtered (100 Hz-3 kHz
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bandpass) and recorded with a second data acquisition system (Powerlab 4SP, AD
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Instruments).
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When cochlear thresholds were within the normal range (Johnstone et al., 1979), animals
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received a unilateral acoustic trauma using the closed sound system described above.
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Acoustic trauma consisted of exposure to a continuous loud tone for 2 h (10 kHz, 124 dB
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SPL for all animals except one which was exposed to a continuous loud tone at 5 kHz, 124
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dB SPL for the same duration). During acoustic trauma animals remained under anaesthesia
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and the contralateral ear was blocked with plasticine. Immediately after acoustic trauma
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another CAP audiogram was measured to determine the magnitude of immediate hearing
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loss and the incision was sutured. Survival time varied between 2 and 3 weeks.
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Surgery for acute electrophysiological recordings
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For the non-recovery experiments animals received a subcutaneous injection with 0.1 ml
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atropine followed by an intraperitoneal injection of Nembutal (pentobarbitone sodium, 30
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mg/kg) and a 0.15 ml intramuscular injection of Hypnorm. Anaesthesia was maintained with
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full Hypnorm doses every hour and half doses of Nembutal every 2 hours. At the end of the
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experiment animals were euthanized with an injection of 0.3 ml Lethabarb (sodium
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pentobarbitone 325 mg/ml; VIRBAC).
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When deep anaesthesia was obtained as determined by the absence of the foot withdrawal
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reflex, the areas of incision were shaved and animals were placed on a custom-made
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heating blanket thermostatically controlled to maintain animal rectal temperature at (38˚C) in
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a sound proof room and artificially ventilated on carbogen (95%O2 and 5% CO2). Before
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single neuron recordings paralysis was induced with 0.1 ml pancuronium bromide (2 mg/ml
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intramuscularly). The electrocardiogram was continuously monitored. Heart rate never
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increased over pre-paralysis levels at any stage of the experiments and strong stimuli (foot
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pinch) did not alter ECG recordings. After the animals were mounted in hollow ear bars, the
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left and right cochleae were exposed by small openings in the bulla and CAP audiograms
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were recorded on both sides with an insulated silver wire placed on the round window as for
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the recovery procedures.
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In order to make extracellular single neuron recordings in the central nucleus of the inferior
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colliculus (CNIC) a small craniotomy (approximately 4 mm2) overlying the visual cortex was
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performed. A glass-insulated tungsten microelectrode (Merrill and Ainsworth, 1972) was then
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advanced using a stepping motor microdrive (Rapidsyn) and custom-made controller along
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the dorso-ventral axis through the cortex into the CNIC contralateral to the cochlea subjected
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previously to acoustic trauma. Electrode placement in the CNIC (about 2.5 to 3mm ventral to
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the cortical surface) was indicated by the presence of strong sound-driven activity with a
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short latency (cluster onset latencies <6.5 ms) and a systematic progression from low to high
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characteristic frequencies (CF) with increasing depth. We have previously confirmed
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histologically that these response properties correlate with location of the electrode in the
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CNIC (Malmierca et al., 1995, 2008; Mulders et al., 2011). The craniotomy was covered with
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5% agar in saline to improve mechanical stability. When a single unit was isolated its CF and
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threshold at CF were determined audio-visually and depth from the cortical surface was
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recorded using methods described previously (Ingham et al., 2006; Mulders et al., 2010). In
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all neurons the spontaneous firing rate was measured for a period of 10 s as previously
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reported using an identical animal model (Mulders and Robertson, 2009; Mulders et al.,
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2010, 2011). During this measurement the polarization voltage of the speaker was turned off
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to avoid uncontrolled background noise from the sound delivery system.
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For electrical stimulation of the round window (RW) of the cochlea a plastic insulated and
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chlorided silver wire was placed on the RW using a micromanipulator. Another insulated
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chlorided wire was inserted in muscle next to the opening in the bulla as a current return
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electrode. Electrical stimulation to the electrodes was delivered via an isolated stimulator
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output (AM Systems Model 2100). When spontaneous activity of a CNIC neuron was
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determined the effects of DC electrical stimulation of the RW on the neuron’s spontaneous
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rate was measured. For this purpose histograms were constructed from 20 repetitions of 2 s
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epochs (unless otherwise specified) either without any electrical stimulation or with positive
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or negative DC current. Current intensity was varied between 25 and 100 µA in 25 µA steps.
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Electrical stimulation duration was 500 ms with a repetition rate of 2s for all neurons unless
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otherwise specified. We also measured the neural activity produced by tone stimuli (50-ms
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duration) during ECES (tone started 100 msec after the ECES onset) or without ECES.
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To assess the effects of RW stimulation on cochlear function, CAP input/output (I/O)
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functions were constructed in response to 10ms tone bursts at either 4, 8 or 16 kHz over a
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range of sound intensities in 10 dB steps, repetition period 2s. At each of the 3 frequencies
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I/O functions were measured without any electrical stimulation or after positive DC current or
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negative DC current. The CAP recordings were obtained from a different electrode than that
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used for stimulation. Electrical stimulation duration was 500 ms as for single neuron
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recordings and the tone burst was placed at either 20 or 105 ms after the end of stimulation.
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Ten stimulus repetitions were used and the averaged CAP amplitude was expressed as the
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N1-P1 amplitude.
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Data analysis
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Quantification of the ECES effect on “spontaneous” activity
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If ICAECES is the activity of inferior colliculus during ECES (with negative or positive
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current) and ICActrl the “control” activity of inferior colliculus (without ECES), then the effects
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of the ECES is calculated as follows:
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EFFECES = (ICAECES-ICActrl) / (ICAECES+ICActrl)
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ICActrl was derived from the 0 µVolt condition, so for a period of 40-60s. There is no
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effect of stimulation when the index is equal to 0 (ICAECES = ICActrl). If ICAECES is 10 times the
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ICActrl then the index is equal to 0.8. An index equal to -1 indicates complete suppression of
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neural activity produced by electrical stimulation. ICActrl was not obtained between
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stimulation periods but from a different session of recording generally carried out before the
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sessions where electrical stimulation was provided. This was to avoid comparing neural
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activity during electrical stimulation to the activity after electrical stimulation as the latter
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shows post-stimulation changes in the pattern of firing. EFFECES will be displayed as a
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function of CF and averaged into partially overlapping 1-octave frequency bands.
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Quantification of the ECES effect on stimulus-induced activity
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The stimulus-induced activity of each unit was obtained by taking the maximum firing
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rate from the running average of the post-stimulus time histograms using a 50-ms time
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window. This was similar to taking the average neural activity during the 50-ms acoustic
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stimulation; however, this method did not require knowing a priori the latency of the stimulus-
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induced responses. This activity was then averaged over all units to build the averaged rate-
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level functions. For most units, the level of the electrical current used to assess the effects of
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ECES on tone-induced activity was 25 or 50 µAmp (and -25 or -50 µAmp).
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Statistics
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The averaged effect of electric stimulation on spontaneous and stimulus-induced activity
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in IC was tested statistically using a Wilcoxon signed-rank test. Bonferroni’s correction was
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applied to adjust the p values for the number of comparisons being made.
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Results
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Figure 1 shows the CAP threshold shift (difference between CAP thresholds obtained
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after and before noise trauma) in all animals (and the mean threshold shift). Some animals
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but not all have long-term hearing loss after noise trauma in mid (≈ 12 kHz) and/or high
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frequencies (16 kHz).
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As the purpose of the study was to investigate the effects of extra-cochlear electric
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stimulation (ECES) on central activity, especially on spontaneous activity, only units with
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spontaneous firing rate above 0 were selected. A more complete description on the effects
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of noise trauma on the spontaneous firing in IC has been published elsewhere (Mulders and
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Robertson, 2009, 2011, 2013; Vogler et al., 2014). 95% of units in IC have a low
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spontaneous firing rate (< 8 spikes / sec) in normal animals (Mulders and Robertson, 2009).
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Figure 2 shows the spontaneous firing rate of all the units reported in the present study as a
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function of their characteristic frequency (CF), as well as their spontaneous firing rate
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distribution. A total of 105 neurons were recorded. The distribution of units is biased towards
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CFs >3kHz as the spontaneous activity of very low CF neurons was zero or too low for the
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purpose of this study. One notes that most recordings have been made in the CF region
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affected by the acoustic trauma frequency; the averaged spontaneous activity was the
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highest in this frequency region (~ 25 spikes / sec) (Figure 1).
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Effects of cochlear electric stimulation on spontaneous collicular activity
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Figure 3 shows raw recordings for a representative unit in IC (CF=21.7 kHz) after
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trauma. This unit is hyperactive discharging with bursts of action potentials (first row). The
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effects of negative and positive currents are shown in the second and third rows,
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respectively. During current application, the negative current produces a strong increase of
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the discharges whereas the positive current abolishes completely neural activity. The post-
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stimulation periods were also dramatically modified: stimulations with negative current are
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followed by a strong suppression of neural activity, whereas a rebound of activity is observed
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after the stimulations with positive current. Figure 4 and 5 show the firing rate as a function
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of time in additional examples. These examples illustrate further the effects of ECES on
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neural activity in IC and also the important changes in the dynamic of the neural activity.
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Indeed, the ECES modulates central activity during and after the stimulation. These effects
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are particularly striking in the example shown in column 1 of Figure 5. Note however that
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positive current suppresses the activity in some units with only a moderate post-stimulation
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rebound of activity (Figure 4, column 3). Some units (generally with low CF) showed a
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reversed pattern of effects, with negative current being suppressive and positive current
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being excitatory (Figure 5, column 3). It is clear from the examples shown above that the
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post-stimulation effects could be much longer than the 2-sec inter-stimulation interval used in
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this study. Figure 6 shows an example of a unit discharging with bursts in which two different
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time intervals (2 and 5 sec) between the electric stimulations were used. The negative
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stimulation had only a modest effect on the discharges when provided every 2 sec, and
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triggered the burst of discharges when provided every 5 sec. The positive stimulation had a
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strong suppressive effect for the two inter-stimulus intervals (at least neural activity was
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absent during stimulation). Although we did not investigate the effects of varying
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systematically the inter-stimulation interval, it is interesting to note that the effects of electric
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stimulation seem to depend on a complex relationship between the discharge properties of
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neurons and the characteristics (polarity and temporal sequence) of electrical stimulation.
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The top panel of Figure 7 shows the effects of ECES as a function of CF for all units
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(see methods for the calculation of the ECES effect). The bottom panel of Figure 7 shows
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the ECES effects averaged over partially overlapping 1-octave frequency bands (the center
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frequencies of the bands are indicated in the x-axis). The averaged responses were
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compared statistically to 0 (Wilcoxon signed-rank test). Differences were considered
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significant at a level of 0.001 (Bonferroni’s correction, 6 frequency bands * 8 current
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conditions = 48 comparisons). For the negative current, the only differences which crossed
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the statistical threshold were those for the 25 µAmp and the frequency bands centred on 14
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and 20 kHz. For the positive current, the differences were statistically significant for the
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stimulation amplitude 25, 50 and 75 µAmp and the frequency bands centred on 10, 14 and
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20 kHz. The difference was also significant for the stimulation amplitude 50 µAmp and the
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frequency band centred on 7 kHz.
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Figure 8 shows the effects of electrical stimulation over time and averaged over units
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with a CF within 1 octave around 20 kHz (where the effects of electrical stimulation is
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maximal). One observes that the suppression of activity with positive current (bottom panel)
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is very rapid after the beginning of the stimulation and lasts as long as the stimulation is
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applied. When the stimulation is turned off, the activity increases over time and remains
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elevated (compared to the background activity) between the stimulation periods. The
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reversed pattern is observed for the negative current (top panel): neural activity is
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maintained at a high level as long as the negative current is provided (without any sign of
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neural adaptation) over the 500-ms period of stimulation. After the stimulation, the neural
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activity comes down to well below the background activity.
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Effects of cochlear electric stimulation on stimulus-induced collicular activity
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The results presented above show that positive current is able to reduce significantly the
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spontaneous activity in the auditory centers. While these results are very promising
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regarding the potential for therapeutic developments, this approach would be viable only if
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the positive current does not produce significant hearing loss. In order to investigate whether
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positive current abolished hearing at the current levels used in this study, stimulus-induced
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activity was recorded in IC while electrical stimulation was provided at the round window
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(see methods). Figure 9 shows the post-stimulus time histograms for 4 different units
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recorded with and without concomitant electrical stimulation. One notes that the negative
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current enhances the amplitude of stimulus-induced responses, whereas the positive current
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reduces it. Figure 10 shows the rate-level functions (top panel) and the ECES effects on the
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stimulus induced activity (subtraction from the condition without electric stimulation) (bottom
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panel) averaged over units with CF above 8 kHz. The averaged differences were compared
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statistically to 0 (Wilcoxon signed-rank test). Differences were considered significant if
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p<0.004 (after Bonferroni’s correction, 12 comparisons). The negative current induced a
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significant increase of activity at 0 dB SL. The positive current induced a significant decrease
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of activity at 0, 10 and 20 dB SL. One can observe that the rate-level function with positive
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current corresponds roughly to the rate-level function without stimulation shifted by 10 dB.
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The control neural activity at 0 dB SL was not significantly different from the neural activity at
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10 dB SL during positive stimulation. This result indicates that the positive stimulation may
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produce an auditory threshold shift by about 10 dB, at most.
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Effects of cochlear electric stimulation on cochlear activity
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To investigate the post-stimulatory effects of ECES on tone-evoked activity in the
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periphery, CAPs obtained from different stimulus levels and at two delays after the end of
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ECES (20 and 105 msec) were recorded in two animals (Figure 11). The results indicate that
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positive currents are followed by an increase of CAP amplitude. It is notable that at these
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short time delays, however, spontaneous single-unit activity is still reduced in IC (Figure 8).
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Discussion
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The present study was aimed at investigating whether cochlear electrical stimulation can
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reduce the spontaneous activity in the IC. The results show that positive direct current
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applied at the round window can dramatically reduce the noise-induced central hyperactivity,
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for units with high CF, while it generally does not change the firing (or produces the opposite
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effect) for neurons with low CF. Negative direct current applied at the round window
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produces the opposite. Moreover, cochlear electrical stimulation can marginally modulate
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tone-induced activity at low level of acoustic stimulation but not at moderate levels. This
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approach could lead to important developments to suppress tinnitus provided that reduction
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of cochlear activity can be achieved from charge-balanced electrical stimulation.
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Effects of ECES on the spontaneous firing of cochlear fibers
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A stimulating electrode placed on the round window produces a current flow along the
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scala tympani that spreads over (and affects) a large cochlear region (from the base or high-
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CF region to the mid-cochlear or mid-CF region) (Clopton and Spelman, 1982; Spelman et
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al., 1982; Hartmann et al., 1984; van den Honert and Stypulkowski, 1984; Ho et al., 2004;
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Micco and Richter, 2006). Cochlear electrical stimulation can affect the spontaneous
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discharges of cochlear fibers through presynaptic and postsynaptic mechanisms.
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Cochlear fibers have spontaneous firing in the absence of sound which is related to the
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(presynaptic) spontaneous release of neurotransmitters by the inner hair cells (IHCs)
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(Liberman and Dodds, 1984). The rate of the spontaneous release of neurotransmitters
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depends on the IHC membrane potential which in turn controls the opening probability of the
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voltage-gated calcium channels (Hudspeth, 1985; Pickles, 1985). The intracellular influx of
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calcium then catalyzes the fusion of synaptic ribbon to the plasma membrane allowing
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exocytosis (Moser et al., 2006). ECES could affect the IHC membrane potential (and
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therefore the rate of spontaneous discharges) by 1) a direct effect of the injected current on
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the IHC membrane potential and 2) an effect on the endocochlear potential and hence on
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the standing current through the IHCs (Nuttall, 1985; Russell and Kössl, 1991; Zeddies and
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Siegel, 2004). A modification of the cochlear potential could be mediated by a direct effect of
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injected current on the scala media boundaries. Also, slow length changes of OHCs can
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affect the endocochlear potential through the opening probability of their mechano-electric
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channels. Namely, elongation of OHCs, for instance, is accompanied by a closure of the
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mechano-electric channels and a reduction of the current flow through the OHC. This may
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then result in an increase of the endocochlear potential (Javel and Shepherd, 2000; O’Beirne
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and Patuzzi, 2007). One notes that ECES may also affect the spontaneous firing of cochlear
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fibers through a change in the membrane potential of the efferent nerve terminals and a
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resulting hyperpolarization of OHCs (Wiederhold and Kiang, 1970; Guinan and Gifford,
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1988).
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It is also likely that ECES alters the spontaneous firing of cochlear fibers through
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postsynaptic effects of electrical stimulation, i.e. direct modification of the membrane
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potential of cochlear fibers and the opening probability of voltage-dependent sodium
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channels present in the peripheral and central regions of the cochlear fibers (Javel and
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Shepherd, 2000; Hossain et al., 2005).
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When considering the possible mechanisms of the effects seen with positive and
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negative current, theoretical predictions are complicated. Nevertheless, it is reasonable to
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suggest that for hair cells and cochlear fibers at the base of the cochlea i.e. near the round
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window electrode, a positive current, by producing a movement of negative charges away
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from the close environment of the cells, would result in a hyperpolarization of their
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membrane potentials. For IHCs and the cochlear nerve fibers this would produce a reduction
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of the spontaneous firing. For OHCs, on the other hand, this would increase the length of the
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cells (Brownell et al., 1985; Ashmore, 1987), closing the mechano-electric channels,
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reducing the shunt of strial current through OHCs and thereby increasing the endocochlear
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potential (O’Beirne and Patuzzi, 2007). The increase of the endocochlear potential can
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produce an increase of the standing transducer current through the IHCs, depolarizing the
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IHCs, triggering glutamate release and thereby increasing the firing rate of cochlear fibers
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(Sewell, 1984).
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Overall, however, the net effect of ECES with positive current on cochlear nerve activity
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is a suppression (Tasaki and Fernandez, 1952; Konishi et al., 1970; Evans and Borerwe,
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1982; Schreiner et al., 1986; Miller et al., 2001). All the effects discussed above are
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expected to be reversed for negative currents (Konishi et al., 1970; Evans and Borerwe,
387
1982; Schreiner et al., 1986).
388
389
Effects of extra-cochlear electrical stimulation on auditory centers
390
The present study demonstrates that the spontaneous activity in the IC of animals that
391
have been subjected to a prior cochlear trauma is tightly coupled to the activity in the
392
cochlea. In other words, any modulation of the spontaneous activity in the cochlear nerve
393
(including a reduction of activity) simply propagates through the central auditory system, at
394
least up to the IC. This is consistent with an earlier study showing that decreasing the resting
395
cochlear activity is accompanied by a reduction of the noise-induced hyperactivity in the IC
396
(Mulders and Robertson, 2009). All animals used in this study were exposed to noise trauma
397
as it was the sine quo none condition to get “enough” central spontaneous activity to test our
398
hypothesis. In unexposed control animals, under our anesthesia regime, we have shown that
399
spontaneous activity in IC neurons is very low (Mulders and Robertson, 2009). As a
400
consequence of this experimental design, we cannot say that ECES with positive current can
401
suppress the central spontaneous activity in control (non-exposed) animals. Moreover, we
402
cannot differentiate the neurons with elevated firing rate due to the noise trauma from
403
neurons that had high firing rate before the trauma and that maintained high level of firing
404
after trauma. This does not change the main conclusion of our study that ECES with positive
405
current can suppress all central spontaneous activity, including the noise-induced central
406
hyperactivity. We speculate that ECES with positive current can reduce the spontaneous
407
firing of hyperactive and non-hyperactive neurons in IC as long as they receive strong inputs
408
from the cochlear region affected by the ECES (from the base to the mid-region of the
409
cochlea corresponding to around 5 kHz).
16
410
The effects of ECES are stronger on high CF neurons than on low CF neurons,
411
presumably due to the position of the stimulating electrode close to the base of the cochlea
412
(corresponding to high CFs). One explanation could be that the current density may
413
decrease as the current passes through the cochlea. The effects of ECES can be opposite
414
for low-CF fibers compared to those for high-CF fibers. One notes, however, that while this
415
reversal can be very clear for some units, the changes for low-CF units are not statistically
416
significant when the recordings from low-CF units are pooled. This indicates that ECES
417
produces variable results for low-CF units. It is unknown whether this reversal for low-CF
418
units results from an action of electrical stimulation directly on the cochlear apex or indirectly
419
through cross-frequency interactions within the auditory centers. The large variability for low-
420
CF units indicate that the reversal of effects may result from central mechanisms, i.e. lateral
421
inhibition (Yang et al., 1992; LeBeau et al., 2001; Wu et al., 2008; Catz and Noreña, 2013).
422
Namely, a decrease of cochlear activity produced by ECES at the cochlear base may
423
produce an increase of neural firing in auditory centers through a release from central
424
inhibition for units with relatively low CF, and conversely for ECES causing an increase in
425
cochlear activity.
426
Regarding the modulation of tone-induced activity in IC produced by ECES, electrical
427
stimulation of the cochlea has been shown to strongly affect the cochlear microphonic and
428
tone-induced activity of the cochlear fibers (1952, 1952; Teas et al., 1970; Nuttall, 1985).
429
The direction of the changes (increase vs. decrease) depends on the current polarity (Tasaki
430
and Fernandez, 1952; Konishi et al., 1970; Teas et al., 1970; Nuttall, 1985). Interestingly,
431
while electrical stimulation changed the cochlear activity produced by relatively low-level
432
tones, it did not change the maximum firing rate (Teas et al., 1970). This result is broadly
433
consistent with our findings.
434
Finally, strong post-stimulation effects are sometimes observed in the IC: on average,
435
positive current is followed by a delayed rebound of activity, whereas negative current can
436
produce a long-lasting reduction of activity. It is unclear whether these post-stimulation
437
effects result from the cochlea and/or the auditory centers. Prolonged direct stimulation can
17
438
have long-lasting effects on the cochlea (Schreiner et al., 1986). Part of the post-stimulation
439
effects observed in IC may then result from the cochlea. Our CAP data obtained in two
440
animals are, however, only partially consistent with the spontaneous activity recorded in IC.
441
Indeed, while the collicular spontaneous activity remains decreased for up to 400 msec after
442
stimulation with positive current, the CAP amplitude is increased shortly (20 and 105 msec)
443
after the end of the stimulation. This suggests that some central mechanisms may be
444
involved in the rebound of activity observed after stimulating with positive current.
445
446
Implications for understanding tinnitus mechanisms and tinnitus suppression
447
ECES with positive current has been found to suppress the tinnitus percept in tinnitus
448
subjects (Hatton et al., 1960; Cazals et al., 1978; Portmann et al., 1979; Aran and Cazals,
449
1981). Our results suggest that tinnitus suppression produced by ECES with positive current
450
is related to the unspecific suppression of the central spontaneous activity of central
451
neurons, including hyperactive neurons (neurons supposed to propagate the tinnitus-related
452
activity). In this context, one proposes that ECES can be a very promising approach for
453
suppressing peripheral-dependent tinnitus, i.e. tinnitus linked to the residual spontaneous
454
firing in the cochlea (Mulders and Robertson, 2009; Norena, 2011).
455
Since we did not try to reveal tinnitus in noise-exposed animals, the assumption that the
456
tinnitus percept is related to the noise-induced hyperactivity is necessarily speculative (see,
457
for instance, Coomber et al., 2014 and Kalappa et al., 2014, for conflicting views). One
458
notes, however, that it would have been challenging to objectify tinnitus reduction or
459
suppression while electrical stimulation was provided at the round window (i.e. which is the
460
ultimate goal of the present study). In this context, we felt that we should first test our
461
hypothesis (effects of ECES on central hyperactivity) without checking for tinnitus.
462
Peripheral-dependent tinnitus should be abolished by cochlear nerve section and indeed
463
cochlear nerve section can lead to tinnitus suppression (House and Brackmann, 1981; Barrs
464
and Brackmann, 1984; Silverstein et al., 1986; Pulec, 1995). As most tinnitus subjects have
465
relatively good hearing (Pan et al., 2009; Sereda et al., 2011) with functional IHCs and
18
466
consequently residual spontaneous firing in the cochlear nerve, the peripheral-dependent
467
tinnitus may be a relatively prevalent form of tinnitus. Any therapeutic approach aimed at
468
suppressing tinnitus should not alter auditory thresholds, even temporarily. Here we show
469
that ECES is able to reduce the putative tinnitus-related activity while hearing thresholds are
470
only modestly affected.
471
On the other hand, other forms of tinnitus such as “centralized” tinnitus (forms of tinnitus
472
that become independent of the residual spontaneous activity in the cochlear fibers), should
473
not respond to this treatment (Mulders and Robertson, 2011). Centralized tinnitus should be
474
unaffected by cochlear nerve section and indeed cochlear nerve section is sometimes
475
inefficient to suppress tinnitus (House and Brackmann, 1981; Barrs and Brackmann, 1984;
476
Silverstein et al., 1986; Pulec, 1995). However, ECES with positive current has been shown
477
to suppress tinnitus in subjects with severe hearing loss and chronic tinnitus (Cazals et al.,
478
1978). This result suggests that some forms of chronic tinnitus may remain dependent on
479
cochlear residual activity. Whether tinnitus is peripheral-dependent or not may depend on
480
the cochlear residual activity and therefore on the extent of cochlear damages. One notes
481
that the susceptibility of the cochlea to various stressors can vary as a function of the context
482
and among individuals and species (Turner et al., 2005; Canlon et al., 2007; Graham et al.,
483
2011).
484
Electric stimulation used in cochlear implants is well known to reduce tinnitus (Okusa et
485
al., 1993; Ito and Sakakihara, 1994; Rubinstein et al., 2003; Quaranta et al., 2004; Zeng,
486
2004; Baguley and Atlas, 2007; Chang and Zeng, 2012). The approach tested in this study
487
is however very different from stimulating the cochlea with cochlear implants. Cochlear
488
implants are meant to restore hearing and therefore to produce excitation of the cochlear
489
fibers. The tinnitus reduction produced by cochlear implants could then be achieved through
490
masking, residual inhibition or reduction of central gain (Norena, 2011; Noreña, 2012). Our
491
approach, instead, is aimed at suppressing the cochlear driver of tinnitus by hyperpolarizing
492
inner hair cells and/or cochlear fibers.
19
493
Finally, while this study suggests that electrical stimulation can be a promising approach
494
to suppress peripheral-dependent tinnitus, it is important to note that it cannot be used as
495
such in tinnitus subjects. Indeed, direct current can be harmful for biological tissues by
496
producing free radicals, changing the pH of the medium around the electrode and leading to
497
electrode corrosion among other electrochemical reactions (Shepherd et al., 1991, 1999;
498
Merrill et al., 2005). The main challenge faced by this approach is therefore to achieve
499
robust suppression of cochlear activity from a charge-balanced electrical stimulation.
500
501
Acknowledgements
502
This work was supported by the Tinnitus Research Initiative and by L’Agence Nationale de la
503
Recherche Grant ANR-2010-JCJC-1409-1 and the Action on Hearing Loss (G55). The
504
authors wish to thank Olivier Macherey, Yves Cazals and Fan-Gang Zeng for valuable
505
comments on a previous version of the manuscript.
506
507
20
508
Legends
509
Figure 1
510
CAP threshold shift obtained in all animals as a function of frequency (thin lines) and the
511
averaged CAP threshold shift (thick line). The dashed line corresponds to the threshold shift
512
obtained in an animal exposed to 5-kHz tone. All other animals have been exposed to a 10-
513
kHz tone.
514
515
Figure 2
516
Middle panel: spontaneous activity (SA) (after noise trauma) as a function of the
517
characteristic frequency (CF) for all recorded neurons in the present study. The gray line
518
represents a running average of SA as a function of CF. A peak of SA is present slightly
519
above the trauma frequency (10 kHz). Top panel: distribution of CF. Right panel: distribution
520
of SA.
521
522
Figure 3
523
Raw neural recordings (after filtering – see methods) as a function of time for a given neuron
524
in three conditions. Top panel: spontaneous activity without electrical stimulation. Middle
525
panel: effects of an electric stimulation with a negative current. Bottom panel: effects of an
526
electric stimulation with a positive current. The time windows of electrical stimulation (500-
527
ms duration) are delimited by vertical dotted lines. Inserts in the middle and low panels
528
indicate the pattern of the electric stimulation. Spontaneous firing is completely abolished
529
during the stimulation with the positive current, whereas firing rate is greatly enhanced by the
530
stimulation with the negative current. On that particular example, one also notes a post-
531
stimulation effect consisting of an enhanced firing rate or “activity rebound” after the
532
stimulation with the positive current and a long-lasting suppression of the spontaneous firing
533
after the stimulation with the negative current.
534
535
Figure 4
21
536
Firing rate as a function of time for three different neurons in the inferior colliculus. Firing rate
537
was obtained from the running average of raw recordings using a 50-ms time window. Top
538
panels: spontaneous activity without electrical stimulation. Middle panels: effects of an
539
electric stimulation with a negative current. Bottom panel: effects of an electric stimulation
540
with a positive current. The stimulation with positive current reduces or abolishes neural
541
firing, whereas the stimulation with negative current produces an increase of firing. One
542
notes that positive currents can change dramatically the pattern of spontaneous firing of
543
collicular neurons (first column). The spontaneous activity (SA) and the characteristic
544
frequency CF of each unit is indicated at the top of the panels. The time windows of
545
electrical stimulation (500-ms duration) are delimited by vertical dotted lines. Inserts in the
546
second and third rows indicate the pattern of the electric stimulation.
547
548
549
Figure 5
550
Same as Figure 4 for three additional examples. The example shown in the third column
551
shows the reversed effects of electrical stimulation: positive current enhances the firing rate
552
whereas negative current reduces it. This particular unit has low CF (3.7 kHz).
553
554
Figure 6
555
Firing rate as a function of time for a given neuron in the inferior colliculus for different time
556
sequences. Firing rate was obtained from the running average of raw recordings using a 50-
557
ms time window. First (top) row: spontaneous activity without electrical stimulation. Second
558
row: effects of an electric stimulation with a negative current provided with an inter-stimulus
559
interval of 1500 ms. Third row: effects of an electric stimulation with a negative current
560
provided with an inter-stimulus interval of 4500 ms. Fourth row: effects of an electric
561
stimulation with a positive current provided with an inter-stimulus interval of 1500 ms. Fifth
562
row: effects of an electric stimulation with a positive current provided with an inter-stimulus
563
interval of 4500 ms. The time windows of electrical stimulation (500-ms duration) are
22
564
delimited by vertical dotted lines. Inserts in the second, third, fourth and fifth rows indicate
565
the pattern of the electric stimulation.
566
567
Figure 7
568
Effects of extra-cochlear electrical stimulation as a function of characteristic frequency (CF)
569
for all recorded neurons (top panel). Effects of extra-cochlear electric stimulation are
570
averaged over partially overlapping 1-octave frequency bands (the center frequency of the
571
bands are indicated in the x-axis). Positive values indicate an increase of firing whereas
572
negative values indicate a decrease of firing (see methods). One observes that negative
573
current enhances the firing rate for neurons with high CF and reduces it for neurons with low
574
CF; the effects are reversed for positive stimulation. The reversal of the effect is observed for
575
CF roughly comprised between 5 and 10 kHz.
576
577
Figure 8
578
Averaged effects of extra-cochlear electrical stimulation with positive or negative currents as
579
a function of time for neurons with CF between 14 and 28 kHz (i.e., where the effects of the
580
electrical stimulation were maximal – see figure 8). The time windows of electrical
581
stimulation (500-ms duration) are delimited by vertical dotted lines. Inserts above panels
582
indicates the pattern of the electric stimulation. The peaks at the onset and offset of the
583
electrical stimulation are due to the electrical artifacts generated when the stimulation is
584
turned on and off.
585
586
Figure 9
587
Post-stimulus time histogram for four different neurons in the inferior colliculus without (black
588
dotted line), during stimulation with negative (gray lines) or positive current (black lines).
589
Acoustic stimuli were 50-ms long and started at time = 0 ms. The tone levels (in dB
590
attenuation) used are indicated in each panel. One notes that the spontaneous activity
591
(time<0ms) was enhanced or reduced during negative or positive stimulation, respectively.
23
592
Tone-induced activity followed the same trend. The threshold for tone-induced activity is
593
reduced by concomitant negative stimulation (column 1) or is enhanced by positive
594
stimulation (columns 2-4). The CFs of each unit are indicated at the top of each column.
595
596
Figure 10
597
Effects of electrical stimulation on the stimulus-induced activity of collicular units with
598
characteristic frequency above 8 kHz. Top panel: averaged neural activity as a function of
599
stimulus level without electrical stimulation (black dotted line), during negative (gray line) or
600
positive (black line) stimulation. Bottom panel: averaged difference between the stimulus-
601
induced activity obtained during electrical stimulation and without electrical stimulation for
602
units with characteristic frequency above 8 kHz. Negative values indicate a decrease of
603
neural activity during electrical stimulation compared to the control condition, whereas
604
positive values indicate an increase of activity during electrical stimulation compared to the
605
control condition.
606
607
Figure 11
608
Peak–to-peak amplitude of compound action potential (CAP) induced by different tone burst
609
frequencies and at different delays after electrical stimulation as a function of stimulus level.
610
At the two delays (20 and 105 ms), the amplitude of CAP is enhanced after the positive
611
stimulation. Dotted lines (control, no electric stimulation), gray lines (negative stimulation)
612
and black line (positive stimulation).
613
24
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