Nitric oxide increases gain in the ventral cochlear nucleus of guinea pigs with tinnitus.

Previous work has led to the hypothesis that, during the production of noise-induced tinnitus, higher levels of nitric oxide (NO), in the ventral cochlear nucleus (VCN), increase the gain applied to a reduced input from the cochlea. To test this hypothesis, we noise-exposed 26 guinea pigs, identified evidence of tinnitus in 12 of them and then compared the effects of an iontophoretically applied NO donor or production inhibitor on VCN single unit activity. We confirmed that the mean driven firing rate for the tinnitus and control groups was the same while it had fallen in the non-tinnitus group. By contrast, the mean spontaneous rate had increased for the tinnitus group relative to the control group, while it remained the same for the non-tinnitus group. A greater proportion of units responded to exogenously applied NO in the tinnitus (56%) and non-tinnitus groups (71%) than a control population (24%). In the tinnitus group, endogenous NO facilitated the driven firing rate in 37% (7/19) of neurons and appeared to bring the mean driven rate back up to control levels by a mechanism involving N-methyl-D-aspartic acid (NMDA) receptors. By contrast, in the non-tinnitus group, endogenous NO only facilitated the driven firing rate in 5% (1/22) of neurons and there was no facilitation of driven rate in the control group. The effects of endogenous NO on spontaneous activity were unclear. These results suggest that NO is involved in increasing the gain applied to driven activity, but other factors are also involved in the increase in spontaneous activity.

Nitric oxide regulates the firing rate of neuronal subtypes in the guinea pig ventral cochlear nucleus.

The gaseous free radical, nitric oxide (NO) acts as a ubiquitous neuromodulator, contributing to synaptic plasticity in a complex way that can involve either long term potentiation or depression. It is produced by neuronal nitric oxide synthase (nNOS) which is presynaptically expressed and also located postsynaptically in the membrane and cytoplasm of a subpopulation of each major neuronal type in the ventral cochlear nucleus (VCN). We have used iontophoresis in vivo to study the effect of the NOS inhibitor L-NAME (L-NG-Nitroarginine methyl ester) and the NO donors SIN-1 (3-Morpholinosydnonimine hydrochloride) and SNOG (S-Nitrosoglutathione) on VCN units under urethane anaesthesia. Collectively, both donors produced increases and decreases in driven and spontaneous firing rates of some neurones. Inhibition of endogenous NO production with L-NAME evoked a consistent increase in driven firing rates in 18% of units without much effect on spontaneous rate. This reduction of gain produced by endogenous NO was mirrored when studying the effect of L-NAME on NMDA(N-Methyl-D-aspartic acid)-evoked excitation, with 30% of units showing enhanced NMDA-evoked excitation during L-NAME application (reduced NO levels). Approximately 25% of neurones contain nNOS and the NO produced can modulate the firing rate of the main principal cells: medium stellates (choppers), large stellates (onset responses) and bushy cells (primary-like responses). The main endogenous role of NO seems to be to partly suppress driven firing rates associated with NMDA channel activity but there is scope for it to increase neural gain if there were a pathological increase in its production following hearing loss.

Reductions in cortical alpha activity, enhancements in neural responses and impaired gap detection caused by sodium salicylate in awake guinea pigs.

Tinnitus chronically affects between 10-15% of the population but, despite its prevalence, the underlying mechanisms are still not properly understood. One experimental model involves administration of high doses of sodium salicylate, as this is known to reliably induce tinnitus in both humans and animals. Guinea pigs were implanted with chronic electrocorticography (ECoG) electrode arrays, with silver-ball electrodes placed on the dura over left and right auditory cortex. Two more electrodes were positioned over the cerebellum to monitor auditory brainstem responses (ABRs). We recorded resting-state and auditory evoked neural activity from awake animals before and 2 h following salicylate administration (350 mg/kg; i.p.). Large increases in click-evoked responses (> 100%) were evident across the whole auditory cortex, despite significant reductions in wave I ABR amplitudes (in response to 20 kHz tones), which are indicative of auditory nerve activity. In the same animals, significant decreases in 6-10 Hz spontaneous oscillations (alpha waves) were evident over dorsocaudal auditory cortex. We were also able to demonstrate for the first time that cortical evoked potentials can be inhibited by a preceding gap in background noise [gap-induced pre-pulse inhibition (PPI)], in a similar fashion to the gap-induced inhibition of the acoustic startle reflex that is used as a behavioural test for tinnitus. Furthermore, 2 h following salicylate administration, we observed significant deficits in PPI of cortical responses that were closely aligned with significant deficits in behavioural responses to the same stimuli. Together, these data are suggestive of neural correlates of tinnitus and oversensitivity to sound (hyperacusis).

Neural changes accompanying tinnitus following unilateral acoustic trauma in the guinea pig.

Animal models of tinnitus allow us to study the relationship between changes in neural activity and the tinnitus percept. Here, guinea pigs were subjected to unilateral noise trauma and tested behaviourally for tinnitus 8 weeks later. By comparing animals with tinnitus with those without, all of which were noise-exposed, we were able to identify changes unique to the tinnitus group. Three physiological markers known to change following noise exposure were examined: spontaneous firing rates (SFRs) and burst firing in the inferior colliculus (IC), evoked auditory brainstem responses (ABRs), and the number of neurons in the cochlear nucleus containing nitric oxide synthase (NOS). We obtained behavioural evidence of tinnitus in 12 of 16 (75%) animals. Both SFRs and incidences of burst firing were elevated in the IC of all noise-exposed animals, but there were no differences between tinnitus and no-tinnitus animals. There were significant decreases in ipsilateral ABR latencies in tinnitus animals, contrary to what might be expected with a small hearing loss. Furthermore, there was an ipsilateral-contralateral asymmetry in NOS staining in the ventral cochlear nucleus (VCN) that was only apparent in tinnitus animals. Tinnitus animals had a significantly greater number of NOS-containing neurons on the noise-exposed side, whereas no-tinnitus animals did not. These data suggest that measuring NOS in the VCN and recording ABRs supplement behavioural methods for confirming tinnitus in animals, and that nitric oxide is involved in plastic neural changes associated with tinnitus.

Neural Plasticity in Tinnitus Mechanisms.

Basic work into neuroplasticity mechanisms in both invertebrate and vertebrate brains, followed by the development of the first animal model of tinnitus, and coupled with clinical studies of tinnitus, meant that, by 1990, Jastreboff [...].

Universal automated classification of the acoustic startle reflex using machine learning.

The startle reflex (SR), a robust, motor response elicited by an intense auditory, visual, or somatosensory stimulus has been widely used as a tool to assess psychophysiology in humans and animals for almost a century in diverse fields such as schizophrenia, bipolar disorder, hearing loss, and tinnitus. Previously, SR waveforms have been ignored, or assessed with basic statistical techniques and/or simple template matching paradigms. This has led to considerable variability in SR studies from different laboratories, and species. In an effort to standardize SR assessment methods, we developed a machine learning algorithm and workflow to automatically classify SR waveforms in virtually any animal model including mice, rats, guinea pigs, and gerbils obtained with various paradigms and modalities from several laboratories. The universal features common to SR waveforms of various species and paradigms are examined and discussed in the context of each animal model. The procedure describes common results using the SR across species and how to fully implement the open-source R implementation. Since SR is widely used to investigate toxicological or pharmaceutical efficacy, a detailed and universal SR waveform classification protocol should be developed to aid in standardizing SR assessment procedures across different laboratories and species. This machine learning-based method will improve data reliability and translatability between labs that use the startle reflex paradigm.

The large numbers of minicolumns in the primary visual cortex of humans, chimpanzees and gorillas are related to high visual acuity.

Minicolumns are thought to be a fundamental neural unit in the neocortex and their replication may have formed the basis of the rapid cortical expansion that occurred during primate evolution. We sought evidence of minicolumns in the primary visual cortex (V-1) of three great apes, three rodents and representatives from three other mammalian orders: Eulipotyphla (European hedgehog), Artiodactyla (domestic pig) and Carnivora (ferret). Minicolumns, identified by the presence of a long bundle of radial, myelinated fibers stretching from layer III to the white matter of silver-stained sections, were found in the human, chimpanzee, gorilla and guinea pig V-1. Shorter bundles confined to one or two layers were found in the other species but represent modules rather than minicolumns. The inter-bundle distance, and hence density of minicolumns, varied systematically both within a local area that might represent a hypercolumn but also across the whole visual field. The distance between all bundles had a similar range for human, chimpanzee, gorilla, ferret and guinea pig: most bundles were 20-45 µm apart. By contrast, the space between bundles was greater for the hedgehog and pig (20-140 µm). The mean density of minicolumns was greater in tangential sections of the gorilla and chimpanzee (1,243-1,287 bundles/mm2) than in human (314-422 bundles/mm2) or guinea pig (643 bundles/mm2). The minicolumnar bundles did not form a hexagonal lattice but were arranged in thin curving and branched bands separated by thicker bands of neuropil/somata. Estimates of the total number of modules/minicolumns within V-1 were strongly correlated with visual acuity.

Juxtacellular Labeling of Stellate, Disk and Basket Neurons in the Central Nucleus of the Guinea Pig Inferior Colliculus.

We reconstructed the intrinsic axons of 32 neurons in the guinea pig inferior colliculus (IC) following juxtacellular labeling. Biocytin was injected into cells in vivo, after first analyzing physiological response properties. Based on axonal morphology there were two classes of neuron: (1) laminar cells (14/32, 44%) with an intrinsic axon and flattened dendrites confined to a single fibrodendritic lamina and (2) translaminar cells (18/32, 56%) with axons that terminated in two or more laminae in the central nucleus (ICc) or the surrounding cortex. There was also one small, low-frequency cell with bushy-like dendrites that was very sensitive to interaural timing differences. The translaminar cells were subdivided into three groups of cells with: (a) stellate dendrites that crossed at least two laminae (8/32, 25%); (b) flattened dendrites confined to one lamina and that had mainly en passant axonal swellings (7/32, 22%) and (c) short, flattened dendrites and axons with distinctive clusters of large terminal boutons in the ICc (3/32, 9%). These terminal clusters were similar to those of cortical basket cells. The 14 laminar cells all had sustained responses apart from one offset response. Almost half the non-basket type translaminar cells (7/15) had onset responses while the others had sustained responses. The basket cells were the only ones to have short-latency (7-9 ms), chopper responses and this distinctive temporal response should allow them to be studied in more detail in future. This is the first description of basket cells in the auditory brainstem, but more work is required to confirm their neurotransmitter and precise post-synaptic targets.

Salicylate decreases the spontaneous firing rate of guinea pig auditory nerve fibres.

Tinnitus has similarities to chronic neuropathic pain where there are changes in the firing rate of different types of afferent neurons. We postulated that one possible cause of tinnitus is a change in the distribution of spontaneous firing rates in at least one type of afferent auditory nerve fibre in anaesthetised guinea pigs. In control animals there was a bimodal distribution of spontaneous rates, but the position of the second mode was different depending upon whether the fibres responded best to high (> 4 kHz) or low (≤4 kHz) frequency tonal stimulation. The simplest and most reliable way of inducing tinnitus in experimental animals is to administer a high dose of sodium salicylate. The distribution of the spontaneous firing rates was different when salicylate (350 mg/kg) was administered, even when the sample was matched for the distribution of characteristic frequencies in the control population. The proportion of medium spontaneous rate fibres (MSR, 1≤ spikes/s ≤20) increased while the proportion of the highest, high spontaneous firing rate fibres (HSR, > 80 spikes/s) decreased following salicylate. The median rate fell from 64.7 spikes/s (control) to 35.4 spikes/s (salicylate); a highly significant change (Kruskal-Wallis test p < 0.001). When the changes were compared with various models of statistical probability, the most accurate model was one where most HSR fibres decreased their firing rate by 32 spikes/s. Thus, we have shown a reduction in the firing rate of HSR fibres that may be related to tinnitus.

Gap-induced inhibition of the post-auricular muscle response in humans and guinea pigs.

A common method for measuring changes in temporal processing sensitivity in both humans and animals makes use of GaP-induced Inhibition of the Acoustic Startle (GPIAS). It is also the basis of a common method for detecting tinnitus in rodents. However, the link to tinnitus has not been properly established because GPIAS has not yet been used to objectively demonstrate tinnitus in humans. In guinea pigs, the Preyer (ear flick) myogenic reflex is an established method for measuring the acoustic startle for the GPIAS test, while in humans, it is the eye-blink reflex. Yet, humans have a vestigial remnant of the Preyer reflex, which can be detected by measuring skin surface potentials associated with the Post-Auricular Muscle Response (PAMR). A similar electrical potential can be measured in guinea pigs and we aimed to show that the PAMR could be used to demonstrate GPIAS in both species. In guinea pigs, we compare the GPIAS measured using the pinna movement of the Preyer reflex and the electrical potential of the PAMR to demonstrate that the two are at least equivalent. In humans, we establish for the first time that the PAMR provides a reliable way of measuring GPIAS that is a pure acoustic alternative to the multimodal eye-blink reflex. Further exploratory tests showed that while eye gaze position influenced the size of the PAMR response, it did not change the degree of GPIAS. Our findings confirm that the PAMR is a sensitive method for measuring GPIAS and suggest that it may allow direct comparison of temporal processing between humans and animals and may provide a basis for an objective test of tinnitus.

Gap-induced reductions of evoked potentials in the auditory cortex: A possible objective marker for the presence of tinnitus in animals.

Animal models of tinnitus are essential for determining the underlying mechanisms and testing pharmacotherapies. However, there is doubt over the validity of current behavioural methods for detecting tinnitus. Here, we applied a stimulus paradigm widely used in a behavioural test (gap-induced inhibition of the acoustic startle reflex GPIAS) whilst recording from the auditory cortex, and showed neural response changes that mirror those found in the behavioural tests. We implanted guinea pigs (GPs) with electrocorticographic (ECoG) arrays and recorded baseline auditory cortical responses to a startling stimulus. When a gap was inserted in otherwise continuous background noise prior to the startling stimulus, there was a clear reduction in the subsequent evoked response (termed gap-induced reductions in evoked potentials; GIREP), suggestive of a neural analogue of the GPIAS test. We then unilaterally exposed guinea pigs to narrowband noise (left ear; 8-10 kHz; 1 h) at one of two different sound levels - either 105 dB SPL or 120 dB SPL - and recorded the same responses seven-to-ten weeks following the noise exposure. Significant deficits in GIREP were observed for all areas of the auditory cortex (AC) in the 120 dB-exposed GPs, but not in the 105 dB-exposed GPs. These deficits could not simply be accounted for by changes in response amplitudes. Furthermore, in the contralateral (right) caudal AC we observed a significant increase in evoked potential amplitudes across narrowband background frequencies in both 105 dB and 120 dB-exposed GPs. Taken in the context of the large body of literature that has used the behavioural test as a demonstration of the presence of tinnitus, these results are suggestive of objective neural correlates of the presence of noise-induced tinnitus and hyperacusis.

Communication calls produced by electrical stimulation of four structures in the guinea pig brain.

One of the main central processes affecting the cortical representation of conspecific vocalizations is the collateral output from the extended motor system for call generation. Before starting to study this interaction we sought to compare the characteristics of calls produced by stimulating four different parts of the brain in guinea pigs (Cavia porcellus). By using anaesthetised animals we were able to reposition electrodes without distressing the animals. Trains of 100 electrical pulses were used to stimulate the midbrain periaqueductal grey (PAG), hypothalamus, amygdala, and anterior cingulate cortex (ACC). Each structure produced a similar range of calls, but in significantly different proportions. Two of the spontaneous calls (chirrup and purr) were never produced by electrical stimulation and although we identified versions of chutter, durr and tooth chatter, they differed significantly from our natural call templates. However, we were routinely able to elicit seven other identifiable calls. All seven calls were produced both during the 1.6 s period of stimulation and subsequently in a period which could last for more than a minute. A single stimulation site could produce four or five different calls, but the amygdala was much less likely to produce a scream, whistle or rising whistle than any of the other structures. These three high-frequency calls were more likely to be produced by females than males. There were also differences in the timing of the call production with the amygdala primarily producing calls during the electrical stimulation and the hypothalamus mainly producing calls after the electrical stimulation. For all four structures a significantly higher stimulation current was required in males than females. We conclude that all four structures can be stimulated to produce fictive vocalizations that should be useful in studying the relationship between the vocal motor system and cortical sensory representation.

Effects of the cannabinoid CB1 agonist ACEA on salicylate ototoxicity, hyperacusis and tinnitus in guinea pigs.

Cannabinoids have been suggested as a therapeutic target for a variety of brain disorders. Despite the presence of their receptors throughout the auditory system, little is known about how cannabinoids affect auditory function. We sought to determine whether administration of arachidonyl-2'-chloroethylamide (ACEA), a highly-selective CB1 agonist, could attenuate a variety of auditory effects caused by prior administration of salicylate, and potentially treat tinnitus. We recorded cortical resting-state activity, auditory-evoked cortical activity and auditory brainstem responses (ABRs), from chronically-implanted awake guinea pigs, before and after salicylate + ACEA. Salicylate-induced reductions in click-evoked ABR amplitudes were smaller in the presence of ACEA, suggesting that the ototoxic effects of salicylate were less severe. ACEA also abolished salicylate-induced changes in cortical alpha band (6-10 Hz) oscillatory activity. However, salicylate-induced increases in cortical evoked activity (suggestive of the presence of hyperacusis) were still present with salicylate + ACEA. ACEA administered alone did not induce significant changes in either ABR amplitudes or oscillatory activity, but did increase cortical evoked potentials. Furthermore, in two separate groups of non-implanted animals, we found no evidence that ACEA could reverse behavioural identification of salicylate- or noise-induced tinnitus. Together, these data suggest that while ACEA may be potentially otoprotective, selective CB1 agonists are not effective in diminishing the presence of tinnitus or hyperacusis.

Histological Basis of Laminar MRI Patterns in High Resolution Images of Fixed Human Auditory Cortex.

Functional magnetic resonance imaging (fMRI) studies of the auditory region of the temporal lobe would benefit from the availability of image contrast that allowed direct identification of the primary auditory cortex, as this region cannot be accurately located using gyral landmarks alone. Previous work has suggested that the primary area can be identified in magnetic resonance (MR) images because of its relatively high myelin content. However, MR images are also affected by the iron content of the tissue and in this study we sought to confirm that different MR image contrasts did correlate with the myelin content in the gray matter and were not primarily affected by iron content as is the case in the primary visual and somatosensory areas. By imaging blocks of fixed post-mortem cortex in a 7 T scanner and then sectioning them for histological staining we sought to assess the relative contribution of myelin and iron to the gray matter contrast in the auditory region. Evaluating the image contrast in [Formula: see text]-weighted images and quantitative [Formula: see text] maps showed a reasonably high correlation between the myelin density of the gray matter and the intensity of the MR images. The correlation with T1-weighted phase sensitive inversion recovery (PSIR) images was better than with the previous two image types, and there were clearly differentiated borders between adjacent cortical areas in these images. A significant amount of iron was present in the auditory region, but did not seem to contribute to the laminar pattern of the cortical gray matter in MR images. Similar levels of iron were present in the gray and white matter and although iron was present in fibers within the gray matter, these fibers were fairly uniformly distributed across the cortex. Thus, we conclude that T1- and [Formula: see text]-weighted imaging sequences do demonstrate the relatively high myelin levels that are characteristic of the deep layers in primary auditory cortex and allow it and some of the surrounding areas to be reliably distinguished.

Responses to the purr call in three areas of the guinea pig auditory cortex.

Single electrodes were used to record from anaesthetized animals stimulated with a closed sound system. Neural responses to the purr call were very different in the dorsocaudal core field and in two long-latency belt areas, the ventrorostral belt and the dorsocaudal belt. Responses in the dorsocaudal core field were accurately timed to the start of the nine rhythmic pulses within the purr while the ventrorostral belt responses were more sustained and less temporally precise and most dorsocaudal belt units did not respond. These results are consistent with the separate processing of narrow-band tonal stimuli such as the purr by a ventrorostral pathway involving the primary auditory area and the ventrorostral belt but not by a dorsocaudal pathway from the dorsocaudal core field to the dorsocaudal belt area.

Representation of the purr call in the guinea pig primary auditory cortex.

Guinea pigs produce the low-frequency purr or rumble call as an alerting signal. A digitised example of the call was presented to anaesthetised guinea pigs via a closed sound system while recording from the primary auditory cortex. The exemplar used in this study had 9 regular phrases each spaced with their centres about 80 ms apart. Low-frequency (1.1 kHz) units responded best to the call but within this population there were four separate groups: (1) cells that responded vigorously to many or all of the 9 phrases; (2) cells that gave an onset response; (3) cells that only responded to a click embedded in the call; (4) cells that did not respond. Particular response types were often grouped together. Thus when orthogonal electrode tracks were used most units gave a similar response. There was no correlation between the type of response and the cortical depth. A similar range of response types was also found in the thalamus and there was no evidence of a distinct response in the cortex that was due to intracortical processing. Cells in the cortex were able to represent the temporal structure of the purr with the same fidelity as cells in the thalamus.

Onset neurones in the anteroventral cochlear nucleus project to the dorsal cochlear nucleus.

Considerable circumstantial evidence suggests that cells in the ventral cochlear nucleus, that respond predominantly to the onset of pure tone bursts, have a stellate morphology and project, among other places, to the dorsal cochlear nucleus. The characteristics of such cells make them leading candidates for providing the so-called "wideband inhibitory input" which is an essential part of the processing machinery of the dorsal cochlear nucleus. Here we use juxtacellular labeling with biocytin to demonstrate directly that large stellate cells, with onset responses, terminate profusely in the dorsal cochlear nucleus. They also provide widespread local innervation of the anteroventral cochlear nucleus and a small innervation of the posteroventral cochlear nucleus. In addition, some onset cells project to the contralateral dorsal cochlear nucleus.

Phase-locked responses to pure tones in the primary auditory cortex.

At the level of the brainstem, precise temporal information is essential for some aspects of binaural processing, while at the level of the cortex, rate and place mechanisms for neural coding seem to predominate. However, we now show that precise timing of steady-state responses to pure tones occurs in the primary auditory cortex (AI). Recordings were made from 163 multi-units in guinea pig AI. All units increased their firing rate in response to pure tones at 100 Hz and 46 (28%) gave sustained responses which were synchronised with the stimulus waveform (phase-locking). The phase-locking units were clustered together in columns. Phase-locking was generally strongest in layers III and IV but was also recorded in layers I, II and V. Good phase-locking was observed over a range of 60-250 Hz: some units (30%) were narrow band while others (37%) were low-pass (33% were not determined). Phase-locking strength was also influenced by sound level: some units showed monotonic increases in strength with level and others were non-monotonic. Ten of the units provided a good temporal representation of the fundamental frequency (270 Hz) of a guinea pig vocalisation (rumble) and may be involved in analysing communication calls.

Changes in the response properties of inferior colliculus neurons relating to tinnitus.

Tinnitus is often identified in animal models by using the gap prepulse inhibition of acoustic startle. Impaired gap detection following acoustic over-exposure (AOE) is thought to be caused by tinnitus "filling in" the gap, thus, reducing its salience. This presumably involves altered perception, and could conceivably be caused by changes at the level of the neocortex, i.e., cortical reorganization. Alternatively, reduced gap detection ability might reflect poorer temporal processing in the brainstem, caused by AOE; in which case, impaired gap detection would not be a reliable indicator of tinnitus. We tested the latter hypothesis by examining gap detection in inferior colliculus (IC) neurons following AOE. Seven of nine unilaterally noise-exposed guinea pigs exhibited behavioral evidence of tinnitus. In these tinnitus animals, neural gap detection thresholds (GDTs) in the IC significantly increased in response to broadband noise stimuli, but not to pure tones or narrow-band noise. In addition, when IC neurons were sub-divided according to temporal response profile (onset vs. sustained firing patterns), we found a significant increase in the proportion of onset-type responses after AOE. Importantly, however, GDTs were still considerably shorter than gap durations commonly used in objective behavioral tests for tinnitus. These data indicate that the neural changes observed in the IC are insufficient to explain deficits in behavioral gap detection that are commonly attributed to tinnitus. The subtle changes in IC neuron response profiles following AOE warrant further investigation.

A novel behavioural approach to detecting tinnitus in the guinea pig.

Tinnitus, the perception of sound in the absence of an external stimulus, is a particularly challenging condition to demonstrate in animals. In any animal model, objective confirmation of tinnitus is essential before we can study the neural changes that produce it. A gap detection method, based on prepulse inhibition of the whole-body startle reflex, is often used as a behavioural test for tinnitus in rodents. However, in the guinea pig the whole-body startle reflex is subject to rapid habituation and hence is not an ideal behavioural measure. By contrast, in this species the Preyer or pinna reflex is a very reliable indicator of the startle response and is much less subject to habituation. We have developed a novel adaptation of the gap detection paradigm, which uses the Preyer reflex to measure the startle response, rather than whole-body movement. Using this method, we have demonstrated changes in gap detection, in guinea pigs where tinnitus had been induced by the administration of a high dose of salicylate. Our data indicate that the Preyer reflex gap detection method is a reliable test for tinnitus in guinea pigs.