Publications using TDT

The neural substrate for the perception of vocalizations is relatively well described, but how their timing and specificity are tightly coupled with accompanying physiological changes and context-appropriate behaviors remains unresolved. We hypothesized that temporally integrated vocal and emotive responses, especially the expression of fear, vigilance and aggression, originate within the amygdala. To test this hypothesis, we performed electrical microstimulation at 461 highly restricted loci within the basal and central amygdala in awake mustached bats. At a subset of these sites, high frequency stimulation with weak constant current pulses presented at near-threshold levels triggered vocalization of either echolocation pulses or social calls. At the vast majority of locations, microstimulation produced a constellation of changes in autonomic and somatomotor outputs. These changes included widespread co-activation of significant tachycardia and hyperventilation and/or rhythmic ear pinna movements (PMs). In a few locations, responses were constrained to vocalization and/or PMs despite increases in the intensity of stimulation. The probability of eliciting echolocation pulses vs. social calls decreased in a medial-posterior to anterolateral direction within the centrobasal amygdala. Microinjections of kainic acid (KA) at stimulation sites confirmed the contribution of cellular activity rather than fibers-of-passage in the control of multimodal outputs. The results suggest that localized clusters of neurons may simultaneously modulate the activity of multiple central pattern generators (CPGs) present within the brainstem.

Gentamicin is an aminoglycoside antibiotic used to treat gram-negative bacterial infections. Treatment with this antibiotic carries the potential for adverse side effects, including ototoxicity and nephrotoxicity. Ototoxic effects are at least in part a consequence of oxidative stress, and various antioxidants have been used to attenuate gentamicin-induced hair cell death and hearing loss. Here, a combination of nutrients previously shown to reduce oxidative stress in the hair cells and attenuate hearing loss after other insults was evaluated for potential protection against gentamicin-induced ototoxicity. Guinea pigs were maintained on a nutritionally complete standard laboratory animal diet or a diet supplemented with β-carotene, vitamins C and E, and magnesium. Three diets with iterative increases in nutrient levels were screened; the final diet selected for study use was one that produced statistically reliable increases in plasma levels of vitamins C and E and magnesium. In two separate studies, significant decreases in gentamicin-induced hearing loss at frequencies including 12 kHz and below were observed, with less benefit at the higher frequencies. Consistent with the functional protection, robust protection of both the inner and outer hair cell populations was observed, with protection largely in the upper half of the cochlea. Protection was independently assessed in two different laboratories, using two different strains of guinea pigs. Additional in vitro tests did not reveal any decrease in antimicrobial activity with nutrient additives. Currently, there are no FDA-approved treatments for the prevention of gentamicin-induced ototoxicity. The current data provide a rationale for continued investigations regarding translation to human patients.

Purpose The use of bilateral stimulation is becoming common for cochlear implant (CI) recipients with either (a) a CI in one ear and a hearing aid (HA) in the nonimplanted ear (CI&HA—bimodal) or (b) CIs in both ears (CI&CI—bilateral). The objective of this study was to evaluate 4 individuals who transitioned from bimodal to bilateral stimulation. Method Participants had completed a larger study of bimodal hearing and subsequently received a second CI. Test procedures from the bimodal study, including roaming speech recognition, localization, and a questionnaire (the Speech, Spatial, and Qualities of Hearing Scale; Gatehouse & Noble, 2004) were repeated after 6–7 months of bilateral CI experience. Results Speech recognition and localization were not significantly different between bimodal and unilateral CI. In contrast, performance was significantly better with CI&CI compared with unilateral CI. Speech recognition with CI&CI was significantly better than with CI&HA for 2 of 4 participants. Localization was significantly better for all participants with CI&CI compared with CI&HA. CI&CI performance was rated as significantly better on the Speech, Spatial, and Qualities of Hearing Scale compared with CI&HA. Conclusions There was a strong preference for CI&CI for all participants. The variability in speech recognition and localization, however, suggests that performance under these stimulus conditions is individualized. Differences in hearing and/or HA history may explain performance differences.

Although many studies have examined the precedence effect (PE), few have tested whether it shows a buildup and breakdown in nonhuman animals comparable to that seen in humans. These processes are thought to reflect the ability of the auditory system to adjust to a listener's acoustic environment, and their mechanisms are still poorly understood. In this study, ferrets were trained on a two-alternative forced-choice task to discriminate the azimuthal direction of brief sounds. In one experiment, pairs of noise bursts were presented from two loudspeakers at different interstimulus delays (ISDs). Results showed that localization performance changed as a function of ISD in a manner consistent with the PE being operative. A second experiment investigated buildup and breakdown of the PE by measuring the ability of ferrets to discriminate the direction of a click pair following presentation of a conditioning train. Human listeners were also tested using this paradigm. In both species, performance was better when the test clicks and conditioning train had the same ISD but deteriorated following a switch in the direction of the leading and lagging sounds between the conditioning train and test clicks. These results suggest that ferrets, like humans, experience a buildup and breakdown of the PE.

Purpose In this study, the authors examined the effects of aging and residual hearing on the identification of acoustically similar and dissimilar vowels in adults with postlingual deafness who use hearing aids (HAs) and/or cochlear implants (CIs). Method The authors used two groups of acoustically similar and dissimilar vowels to assess vowel identification. Also, the Consonant–Nucleus–Consonant Word Recognition Test (Peterson & Lehiste, 1962) and sentences from the Hearing in Noise Test (Nilsson, Soli, & Sullivan, 1994) were administered. Forty CI recipients with postlingual deafness (ages 31–81 years) participated in the study. Results Acoustically similar vowels were more difficult to identify than acoustically dissimilar vowels. With increasing age, performance deteriorated when identifying acoustically similar vowels. Vowel identification was also affected by the use of a contralateral HA and the degree of residual hearing prior to implantation. Moderate correlations were found between speech perception and vowel identification performance. Conclusions Identification performance was affected by the acoustic similarity of the vowels. Older adults experienced more difficulty identifying acoustically similar confusable vowels than did younger adults. The findings might lend support to the ease of language understanding model (Ronnberg, Rudner, Foo, & Lunner, 2008), which proposes that the quality and perceptual robustness of acoustic input affects speech perception. Supplemental Material https://doi.org/10.23641/asha.6170174

Mutations in the connexin 26 gene (GJB2) are the most common genetic cause of deafness, leading to congenital bilateral non-syndromic sensorineural hearing loss. Here we report the generation of a mouse model for a connexin 26 (Cx26) mutation, in which cre-Sox10 drives excision of the Cx26 gene from non-sensory cells flanking the auditory epithelium. We determined that these conditional knockout mice, designated Gjb2-CKO, have a severe hearing loss. Immunocytochemistry of the auditory epithelium confirmed absence of Cx26 in the non-sensory cells. Histology of the organ of Corti and the spiral ganglion neurons (SGNs) performed at ages 1, 3, or 6 months revealed that in Gjb2-CKO mice, the organ of Corti began to degenerate in the basal cochlear turn at an early stage, and the degeneration rapidly spread to the apex. In addition, the density of SGNs in Rosenthal's canal decreased rapidly along a gradient from the base of the cochlea to the apex, where some SGNs survived until at least 6 months of age. Surviving neurons often clustered together and formed clumps of cells in the canal. We then assessed the influence of brain derived neurotrophic factor (BDNF) gene therapy on the SGNs of Gjb2-CKO mice by inoculating Adenovirus with the BDNF gene insert (Ad.BDNF) into the base of the cochlea via the scala tympani or scala media. We determined that over-expression of BDNF beginning around 1 month of age resulted in a significant rescue of neurons in Rosenthal's canal of the cochlear basal turn but not in the middle or apical portions. This data may be used to design therapies for enhancing the SGN physiological status in all GJB2 patients and especially in a sub-group of GJB2 patients where the hearing loss progresses due to ongoing degeneration of the auditory nerve, thereby improving the outcome of cochlear implant therapy in these ears.

Cochlear implant electrical stimulation of the auditory system to rehabilitate deafness has been remarkably successful. Its deployment requires both an intact auditory nerve and a suitably patent cochlear lumen. When disease renders prerequisite conditions impassable, such as in neurofibromatosis type II and cochlear obliterans, alternative treatment targets are considered. Electrical stimulation of the cochlear nucleus and midbrain in humans has delivered encouraging clinical outcomes, buttressing the promise of central auditory prostheses to mitigate deafness in those who are not candidates for cochlear implantation. In this study we explored another possible implant target: the auditory thalamus. In anesthetized cats, we first presented pure tones to determine frequency preferences of thalamic and cortical sites. We then electrically stimulated tonotopically organized thalamic sites while recording from primary auditory cortical sites using a multichannel recording probe. Cathode-leading biphasic thalamic stimulation thresholds that evoked cortical responses were much lower than published accounts of cochlear and midbrain stimulation. Cortical activation dynamic ranges were similar to those reported for cochlear stimulation, but they were narrower than those found through midbrain stimulation. Our results imply that thalamic stimulation can activate auditory cortex at low electrical current levels and suggest an auditory thalamic implant may be a viable central auditory prosthesis.

Age-related hearing loss - presbycusis - is the number one neurodegenerative disorder and top communication deficit of our aged population. Like many aging disorders of the nervous system, damage from free radicals linked to production of reactive oxygen and/or nitrogen species (ROS and RNS, respectively) may play key roles in disease progression. The efficacy of the antioxidant systems, e.g., glutathione and thioredoxin, is an important factor in pathophysiology of the aging nervous system. In this investigation, relations between the expression of antioxidant-related genes in the auditory portion of the inner ear - cochlea, and age-related hearing loss was explored for CBA/CaJ mice. Forty mice were classified into four groups according to age and degree of hearing loss. Cochlear mRNA samples were collected and cDNA generated. Using Affymetrix® GeneChip, the expressions of 56 antioxidant-related gene probes were analyzed to estimate the differences in gene expression between the four subject groups. The expression of Glutathione peroxidase 6, Gpx6; Thioredoxin reductase 1, Txnrd1; Isocitrate dehydrogenase 1, Idh1; and Heat shock protein 1, Hspb1; were significantly different, or showed large fold-change differences between subject groups. The Gpx6, Txnrd1 and Hspb1 gene expression changes were validated using qPCR. The Gpx6 gene was upregulated while the Txnrd1 gene was downregulated with age/hearing loss. The Hspb1 gene was found to be downregulated in middle-aged animals as well as those with mild presbycusis, whereas it was upregulated in those with severe presbycusis. These results facilitate development of future interventions to predict, prevent or slow down the progression of presbycusis.

Improving performance in motor skill acquisition is proposed to be supported by tuning of neural networks. To address this issue we investigated changes of phase-amplitude cross-frequency coupling (paCFC) in neuronal networks during motor performance improvement. We recorded intracranially from subdural electrodes (electrocorticogram; ECoG) from 6 patients who learned 3 distinct motor tasks requiring coordination of finger movements with an external cue (serial response task, auditory motor coordination task, go/no-go). Performance improved in all subjects and all tasks during the first block and plateaued in subsequent blocks. Performance improvement was paralled by increasing neural changes in the trial-to-trial paCFC between theta ([Formula: see text]; 4-8 Hz) phase and high gamma (HG; 80-180 Hz) amplitude. Electrodes showing this covariation pattern (Pearson's r ranging up to .45) were located contralateral to the limb performing the task and were observed predominantly in motor brain regions. We observed stable paCFC when task performance asymptoted. Our results indicate that motor performance improvement is accompanied by adjustments in the dynamics and topology of neuronal network interactions in the [Formula: see text] and HG range. The location of the involved electrodes suggests that oscillatory dynamics in motor cortices support performance improvement with practice.

Stimulus-specific adaptation (SSA) is the reduction in response to a common stimulus that does not generalize, or only partially generalizes, to rare stimuli. SSA is strong and widespread in primary auditory cortex (A1) of rats, but is weak or absent in the main input station to A1, the ventral division of the medial geniculate body. To study SSA in A1, we recorded neural activity in A1 intracellularly using sharp electrodes. We studied the responses to tone pips of the same frequency in different contexts: as Standard and Deviants in Oddball sequences; in equiprobable sequences; in sequences consisting of rare tone presentations; and in sequences composed of many different frequencies, each of which was rare. SSA was found both in subthreshold membrane potential fluctuations and in spiking responses of A1 neurons. SSA for changes in frequency was large at a frequency difference of 44% between Standard and Deviant, and clearly present with tones separated by as little as 4%, near the behavioral frequency difference limen in rats. When using equivalent measures, SSA in spiking responses was generally larger than the SSA at the level of the membrane potential. This effect can be traced to the nonlinearity of the transformation between membrane potential to spikes. Using the responses to the same tone in different contexts made it possible to demonstrate that cortical SSA could not be fully explained by adaptation in narrow frequency channels, even at the level of the membrane potential. We conclude that local processing significantly contributes to the generation of cortical SSA.

Objective: Prevention of temporary threshold shift (TTS) after laboratory-based exposure to pure-tones, broadband noise, and narrowband noise signals has been achieved, but prevention of TTS under these experimental conditions may not accurately reflect protection against hearing loss following impulse noise. This study used a controlled laboratory-based TTS paradigm that incorporated impulsive stimuli into the exposure protocol; development of this model could provide a novel platform for assessing proposed therapeutics. Design: Participants played a video game that delivered gunfire-like sound through headphones as part of a target practice game. Effects were measured using audiometric threshold evaluations and distortion product otoacoustic emissions (DPOAEs). The sound level and number of impulses presented were sequentially increased throughout the study. Study sample: Participants were normal-hearing students at the University of Florida who provided written informed consent prior to participation. Results: TTS was not reliably induced by any of the exposure conditions assessed here. However, there was significant individual variability, and a subset of subjects showed TTS under some exposure conditions. Conclusions: A subset of participants demonstrated reliable threshold shifts under some conditions. Additional experiments are needed to better understand and optimize stimulus parameters that influence TTS after simulated impulse noise.

Auditory Scene Analysis provides a useful framework for understanding atypical auditory perception in autism. Specifically, a failure to segregate the incoming acoustic energy into distinct auditory objects might explain the aversive reaction autistic individuals have to certain auditory stimuli or environments. Previous research with non-autistic participants has demonstrated the presence of an Object Related Negativity (ORN) in the auditory event related potential that indexes pre-attentive processes associated with auditory scene analysis. Also evident is a later P400 component that is attention dependent and thought to be related to decision-making about auditory objects. We sought to determine whether there are differences between individuals with and without autism in the levels of processing indexed by these components. Electroencephalography (EEG) was used to measure brain responses from a group of 16 autistic adults, and 16 age- and verbal-IQ-matched typically-developing adults. Auditory responses were elicited using lateralized dichotic pitch stimuli in which inter-aural timing differences create the illusory perception of a pitch that is spatially separated from a carrier noise stimulus. As in previous studies, control participants produced an ORN in response to the pitch stimuli. However, this component was significantly reduced in the participants with autism. In contrast, processing differences were not observed between the groups at the attention-dependent level (P400). These findings suggest that autistic individuals have difficulty segregating auditory stimuli into distinct auditory objects, and that this difficulty arises at an early pre-attentive level of processing.

Besides the rapid fluctuations in pressure that constitute the “fine structure” of a sound stimulus, slower fluctuations in the sound's envelope represent an important temporal feature. At various stages in the auditory system, neurons exhibit tuning to envelope frequency and have been described as modulation filters. We examine such tuning in the ventral nucleus of the lateral lemniscus (VNLL) of the pentobarbital-anesthetized cat. The VNLL is a large but poorly accessible auditory structure that provides a massive inhibitory input to the inferior colliculus. We test whether envelope filtering effectively applies to the envelope spectrum when multiple envelope components are simultaneously present. We find two broad classes of response with often complementary properties. The firing rate of onset neurons is tuned to a band of modulation frequencies, over which they also synchronize strongly to the envelope waveform. Although most sustained neurons show little firing rate dependence on modulation frequency, some of them are weakly tuned. The latter neurons are usually band-pass or low-pass tuned in synchronization, and a reverse-correlation approach demonstrates that their modulation tuning is preserved to nonperiodic, noisy envelope modulations of a tonal carrier. Modulation tuning to this type of stimulus is weaker for onset neurons. In response to broadband noise, sustained and onset neurons tend to filter out envelope components over a frequency range consistent with their modulation tuning to periodically modulated tones. The results support a role for VNLL in providing temporal reference signals to the auditory midbrain.

Auditory prostheses can partially restore speech comprehension when hearing fails. Sound coding with current prostheses is based on electrical stimulation of auditory neurons and has limited frequency resolution due to broad current spread within the cochlea. In contrast, optical stimulation can be spatially confined, which may improve frequency resolution. Here, we used animal models to characterize optogenetic stimulation, which is the optical stimulation of neurons genetically engineered to express the light-gated ion channel channelrhodopsin-2 (ChR2). Optogenetic stimulation of spiral ganglion neurons (SGNs) activated the auditory pathway, as demonstrated by recordings of single neuron and neuronal population responses. Furthermore, optogenetic stimulation of SGNs restored auditory activity in deaf mice. Approximation of the spatial spread of cochlear excitation by recording local field potentials (LFPs) in the inferior colliculus in response to suprathreshold optical, acoustic, and electrical stimuli indicated that optogenetic stimulation achieves better frequency resolution than monopolar electrical stimulation. Virus-mediated expression of a ChR2 variant with greater light sensitivity in SGNs reduced the amount of light required for responses and allowed neuronal spiking following stimulation up to 60 Hz. Our study demonstrates a strategy for optogenetic stimulation of the auditory pathway in rodents and lays the groundwork for future applications of cochlear optogenetics in auditory research and prosthetics.

Anuran ears function as pressure difference receivers, and the amplitude and phase of tympanum vibrations are inherently directional, varying with sound incident angle. We quantified the nature of this directionality for Cope's gray treefrog, Hyla chrysoscelis. We presented subjects with pure tones, advertisement calls, and frequency-modulated sweeps to examine the influence of frequency, signal level, lung inflation, and sex on ear directionality. Interaural differences in the amplitude of tympanum vibrations were 1-4 dB greater than sound pressure differences adjacent to the two tympana, while interaural differences in the phase of tympanum vibration were similar to or smaller than those in sound phase. Directionality in the amplitude and phase of tympanum vibration were highly dependent on sound frequency, and directionality in amplitude varied slightly with signal level. Directionality in the amplitude and phase of tone- and call-evoked responses did not differ between sexes. Lung inflation strongly affected tympanum directionality over a narrow frequency range that, in females, included call frequencies. This study provides a foundation for further work on the biomechanics and neural mechanisms of spatial hearing in H. chrysoscelis, and lends valuable perspective to behavioral studies on the use of spatial information by this species and other frogs.

Manipulations of the sensory environment typically induce greater changes to the developing nervous system than they do in adulthood. The relevance of these neural changes can be evaluated by examining the age-dependent effects of sensory experience on quantitative measures of perception. Here, we measured frequency modulation (FM) detection thresholds in adult gerbils and investigated whether diminished auditory experience during development or in adulthood influenced perceptual performance. Bilateral conductive hearing loss (CHL) of ≈30 dB was induced either at postnatal day 10 or after sexual maturation. All animals were then trained as adults to detect a 5 Hz FM embedded in a continuous 4 kHz tone. FM detection thresholds were defined as the minimum deviation from the carrier frequency that the animal could reliably detect. Normal-hearing animals displayed FM thresholds of 25 Hz. Inducing CHL, either in juvenile or adult animals, led to a deficit in FM detection. However, this deficit was greater for juvenile onset hearing loss (89 Hz) relative to adult onset hearing loss (64 Hz). The effects could not be attributed to sensation level, nor were they correlated with proxies for attention. The thresholds displayed by CHL animals were correlated with shallower psychometric function slopes, suggesting that hearing loss was associated with greater variance of the decision variable, consistent with increased internal noise. The results show that decreased auditory experience has a greater impact on perceptual skills when initiated at an early age and raises the possibility that altered development of CNS synapses may play a causative role.

Insulin-like growth factor-I (IGF-I) deficiency causes growth delay, and IGF-I has been shown to partially mediate bone anabolism by parathyroid hormone (PTH). PTH-related protein (PTHrP) is abundant in bone, and has osteogenic features by poorly defined mechanisms. We here examined the capacity of PTHrP (1-36) and PTHrP (107-111) (osteostatin) to reverse the skeletal alterations associated with IGF-I deficiency. Igf1-null mice and their wild type littermates were treated with each PTHrP peptide (80 µg/Kg/every other day/2 weeks; 2 males and 4 females for each genotype) or saline vehicle (3 males and 3 females for each genotype). We found that treatment with either PTHrP peptide ameliorated trabecular structure in the femur in both genotypes. However, these peptides were ineffective in normalizing the altered cortical structure at this bone site in Igf1-null mice. An aberrant gene expression of factors associated with osteoblast differentiation and function, namely runx2, osteoprotegerin/receptor activator of NF-κB ligand ratio, Wnt3a , cyclin D1, connexin 43, catalase and Gadd45, as well as in osteocyte sclerostin, was found in the long bones of Igf1-null mice. These mice also displayed a lower amount of trabecular osteoblasts and osteoclasts in the tibial metaphysis than those in wild type mice. These alterations in Igf1-null mice were only partially corrected by each PTHrP peptide treatment. The skeletal expression of Igf2, Igf1 receptor and Irs2 was increased in Igf1-null mice, and this compensatory profile was further improved by treatment with each PTHrP peptide related to ERK1/2 and FoxM1 activation. In vitro, PTHrP (1-36) and osteostatin were effective in promoting bone marrow stromal cell mineralization in normal mice but not in IGF-I-deficient mice. Collectively, these findings indicate that PTHrP (1-36) and osteostatin can exert several osteogenic actions even in the absence of IGF-I in the mouse bone.

Hypoxia-ischemia (HI) occurs when blood and/or oxygen delivery to the brain is compromised. HI injuries can occur in infants born prematurely (<37 weeks gestational age) or at very low birth weight (<1500 g), as well as in term infants with birth complications. In both preterm and term HI populations, brain injury is associated with subsequent behavioral deficits. Neonatal HI injury can be modeled in rodents (e.g., the Rice-Vannucci method, via cautery of right carotid followed by hypoxia). When this injury is induced early in life (between postnatal day (P)1-5), neuropathologies typical of human preterm HI are modeled. When injury is induced later (P7-12), neuropathologies typical of those seen in HI term infants are modeled. The current study sought to characterize the similarities/differences between outcomes following early (P3) and late (P7) HI injury in rats. Male rats with HI injury on P3 or P7, as well as sham controls, were tested on a variety of behavioral tasks in both juvenile and adult periods. Results showed that P7 HI rats displayed deficits on motor learning, rapid auditory processing (RAP), and other learning/memory tasks, as well as a reduction in volume in various neuroanatomical structures. P3 HI animals showed only transient deficits on RAP tasks in the juvenile period (but not in adulthood), yet robust deficits on a visual attention task in adulthood. P3 HI animals did not show any significant reductions in brain volume that we could detect. These data suggest that: (1) behavioral deficits following neonatal HI are task-specific depending on timing of injury; (2) P3 HI rats showed transient deficits on RAP tasks; (3) the more pervasive behavioral deficits seen following P7 HI injury were associated with substantial global tissue loss; and (4) persistent deficits in attention in P3 HI subjects might be linked to neural connectivity disturbances rather than a global loss of brain volume, given that no such pathology was found. These combined findings can be applied to our understanding of differing long-term outcomes following neonatal HI injury in premature versus term infants.

High frequency deep brain stimulation (DBS) of the subthalamic nucleus (STN) is a widely used treatment for Parkinson's disease, but its effects on neural activity in basal ganglia circuits are not fully understood. DBS increases the excitation of STN efferents yet decouples STN spiking patterns from the spiking patterns of STN synaptic targets. We propose that this apparent paradox is resolved by recent studies showing an increased rate of axonal and synaptic failures in STN projections during DBS. To investigate this hypothesis, we combine in vitro and in vivo recordings to derive a computational model of axonal and synaptic failure during DBS. Our model shows that these failures induce a short term depression that suppresses the synaptic transfer of firing rate oscillations, synchrony and rate-coded information from STN to its synaptic targets. In particular, our computational model reproduces the widely reported suppression of parkinsonian β oscillations and synchrony during DBS. Our results support the idea that short term depression is a therapeutic mechanism of STN DBS that works as a functional lesion by decoupling the somatic spiking patterns of STN neurons from spiking activity in basal ganglia output nuclei.

Mosquito flight produces a tone as a side effect of wing movement; this tone is also a communication signal that is frequency-modulated during courtship. Recordings of tones produced by tethered flying male and female Aedes aegypti were undertaken using pairs of pressure-gradient microphones above and below, ahead and behind, and to the left and right over a range of distances. Fundamental frequencies were close to those previously reported, although amplitudes were lower. The male fundamental frequency was higher than that of the female and males modulated it over a wider range. Analysis of harmonics shows that the first six partials were nearly always within 1 Hz of integer multiples of the fundamental, even when the fundamental was being modulated. Along the front-back axis, amplitude attenuated as a function of distance raised to the power 2.3. Front and back recordings were out of phase, as were above and below, while left and right were in phase. Recordings from ahead and behind showed quadratic phase coupling, while others did not. Finally, two methods are presented for separating simultaneous flight tones in a single recording and enhancing their frequency resolution. Implications for mosquito behavior are discussed.