Pharmacological specialization of learned auditory responses in the inferior colliculus of the barn owl

Neural tuning for interaural time difference (ITD) in the optic tectum of the owl is calibrated by experience-dependent plasticity occurring in the external nucleus of the inferior colliculus (ICX). When juvenile owls are subjected to a sustained lateral displacement of the visual field by wearing prismatic spectacles, the ITD tuning of ICX neurons becomes systematically altered; ICX neurons acquire novel auditory responses, termed "learned responses," to ITD values outside their normal, pre-existing tuning range. In this study, we compared the glutamatergic pharmacology of learned responses with that of normal responses expressed by the same ICX neurons. Measurements were made in the ICX using iontophoretic application of glutamate receptor antagonists. We found that in early stages of ITD tuning adjustment, soon after learned responses had been induced by experience-dependent processes, the NMDA receptor antagonist D, L-2-amino-5-phosphonopentanoic acid (AP-5) preferentially blocked the expression of learned responses of many ICX neurons compared with that of normal responses of the same neurons. In contrast, the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) blocked learned and normal responses equally. After long periods of prism experience, preferential blockade of learned responses by AP-5 was no longer observed. These results indicate that NMDA receptors play a preferential role in the expression of learned responses soon after these responses have been induced by experience-dependent processes, whereas later in development or with additional prism experience (we cannot distinguish which), the differential NMDA receptor-mediated component of these responses disappears. This pharmacological progression resembles the changes that occur during maturation of glutamatergic synaptic currents during early development.     

Regional distribution of neurotrophin receptors in the developing auditory brainstem

Neuron survival and axonal regeneration become severely limited during early postnatal development. In conjunction with our recent organotypic analysis of regeneration in the auditory midbrain, we wished to determine whether neurotrophins could serve as a trophic substance during the postnatal period. Therefore, the current study examines the development of three neurotrophin receptor tyrosine kinases (TrkA, TrkB, and TrkC) in the gerbil auditory brainstem. Immunoreactivity to TrkA, the nerve growth-factor receptor, was observed in nonneuronal cells during the first two postnatal weeks. In the cochlear nucleus of mature animals, however, there was a TrkA-positive neuronal subpopulation. In contrast, immunoreactivity to TrkB and TrkC (the receptors for brain-derived neurotrophic factor and neurotrophin-3, respectively) displayed a widespread distribution in the auditory brainstem. At postnatal day 0, TrkB and TrkC staining was virtually absent from auditory nuclei, although immunopositive neurons were present in the mesencephalic trigeminal nucleus. By postnatal day 7, TrkB- and TrkC-positive neurons were present in most brainstem auditory nuclei. At postnatal day 15, TrkB immunoreactivity was observed throughout the inferior colliculus (IC), the cochlear nucleus, the medial and lateral nuclei of the trapezoid body, and the lateral superior olive, whereas TrkC labeled only a subpopulation of neurons within the central nucleus of the IC. The TrkB immunoreactivity was present on both neuronal somata and dendrites, whereas TrkC was generally restricted to cell bodies. At postnatal day 30, TrkB immunostaining was observed on most neurons of the IC. The medial and lateral nuclei of the trapezoid body displayed extremely strong TrkB staining, followed by the cochlear nucleus. In contrast, the TrkC immunostaining was decreased dramatically by postnatal day 21. Observations at the ultrastructural level confirmed a neuronal localization of TrkB and TrkC. Immunostaining for both receptors was restricted largely to the postsynaptic density of synaptic profiles in both dendrites and somata. In summary, this study illustrates a differential pattern of immunoreactivity between three neurotrophin receptors during development. The general increase of TrkB expression is well correlated with the onset of sound-evoked activity in this system, and its synaptic localization suggests that it may be involved in the modulation or maintenance of postsynaptic physiology.     

Frequency tuning and spontaneous activity in the auditory nerve and cochlear nucleus magnocellularis of the barn owl Tyto alba

Single-unit recordings were obtained from the brain stem of the barn owl at the level of entrance of the auditory nerve. Auditory nerve and nucleus magnocellularis units were distinguished by physiological criteria, with the use of the response latency to clicks, the spontaneous discharge rate, and the pattern of characteristic frequencies encountered along an electrode track. The response latency to click stimulation decreased in a logarithmic fashion with increasing characteristic frequency for both auditory nerve and nucleus magnocellularis units. The average difference between these populations was 0.4-0.55 ms. The average most sensitive thresholds were approximately 0 dB SPL and varied little between 0.5 and 9 kHz. Frequency-threshold curves showed the simple V shape that is typical for birds, with no indication of a low-frequency tail. Frequency selectivity increased in a gradual, power-law fashion with increasing characteristic frequency. There was no reflection of the unusual and greatly expanded mapping of higher frequencies on the basilar papilla of the owl. This observation is contrary to the equal-distance hypothesis that relates frequency selectivity to the spatial representation in the cochlea. On the basis of spontaneous rates and/or sensitivity there was no evidence for distinct subpopulations of auditory nerve fibers, such as the well-known type I afferent response classes in mammals. On the whole, barn owl auditory nerve physiology conformed entirely to the typical patterns seen in other bird species. The only exception was a remarkably small spread of thresholds at any one frequency, this being only 10-15 dB in individual owls. Average spontaneous rate was 72.2 spikes/s in the auditory nerve and 219.4 spikes/s for nucleus magnocellularis. This large difference, together with the known properties of endbulb-of-Held synapses, suggests a convergence of approximately 2-4 auditory nerve fibers onto one nucleus magnocellularis neuron. Some auditory nerve fibers as well as nucleus magnocellularis units showed a quasiperiodic spontaneous discharge with preferred intervals in the time-interval histogram. This phenomenon was observed at frequencies as high as 4.7 kHz.     

Glutamate-induced Co2+ uptake in rat auditory brainstem neurons reveals developmental changes in Ca2+ permeability of glutamate receptors

Ca2+ influx through glutamate receptors (GluRs) is thought to play a crucial part in developmental processes and neuronal plasticity. Here we have examined the spatiotemporal distribution of Ca2+-permeable GluRs in auditory brainstem neurons of the rat from birth to adulthood, using the cobalt-staining technique of Pruss and collaborators. In slices of young adult rats, 1 mM glutamate evoked intense cobalt uptake in subsets of neurons in the ventral cochlear nuclei, the medial nucleus of the trapezoid body, the lateral and the medial superior olive, and the lateral lemniscal nuclei. Neurones in the central nucleus of the inferior colliculus, and thalamic auditory nuclei appear to express few, if any, Ca2+-permeable GluRs. Thus, in adults, Ca2+-permeable GluRs are present in neurons of almost all main relay stations of the auditory brainstem. During development, cobalt-stained cells first appeared at about hearing onset (at postnatal day 12 [P12]). At P16, staining levels were highest and the pattern of distribution was already adult-like. The staining intensity slightly declined during the fourth postnatal week. In contrast, Ca2+-permeable receptors were detected in the external cortex of the inferior colliculus as early as P4. Our results show that auditory neurons, characterized by a high temporal precision in neuronal activity, display Ca2+-permeable GluRs. Because Ca2+ permeability appears at about the onset of hearing and is highest during the following 2 weeks, Ca2+ influx through GluRs is likely to be implicated in remodelling processes occurring during this ontogenetic period.     

Simulation of ILD sensitive neurons in the inferior colliculus of the barn owl

The barn owl (Tyto alba) uses interaural level difference (ILD) as a cue for the localization of sound. The first site of binaural convergence in the pathway that processes ILD is the ventral lateral lemniscus pars posterior (VLVp). Neurons in VLVp receive excitatory input from the contralateral nucleus angularis, and inhibitory input from the contralateral VLVp. Within the lateral shell of the inferior colliculus are ILD sensitive neurons that show maximum spike rate at a specific ILD value, with response falling off sharply on each side. Adolphs has developed a model of such lateral shell neurons based on anatomic and physiological data. In his model, lateral shell neurons receive inhibitory input from VLVp on both sides, and this inhibition, applied against a constant excitatory input, produces the observed two-sided response curves. We simulated, in Matlab 4, Adolphs' model, and obtained supporting results. Our simulation suggests that VLVp provides a repository of simple ILD filters from which higher centers construct more complex filters, including the single-peaked curves observed by Adolphs. The VLVp filters are organized along the inhibitory gradient, with broad filters ventral, sharp filters dorsal.     

How the owl resolves auditory coding ambiguity

The barn owl (Tyto alba) uses interaural time difference (ITD) cues to localize sounds in the horizontal plane. Low-order binaural auditory neurons with sharp frequency tuning act as narrow-band coincidence detectors; such neurons respond equally well to sounds with a particular ITD and its phase equivalents and are said to be phase ambiguous. Higher-order neurons with broad frequency tuning are unambiguously selective for single ITDs in response to broad-band sounds and show little or no response to phase equivalents. Selectivity for single ITDs is thought to arise from the convergence of parallel, narrow-band frequency channels that originate in the cochlea. ITD tuning to variable bandwidth stimuli was measured in higher-order neurons of the owl's inferior colliculus to examine the rules that govern the relationship between frequency channel convergence and the resolution of phase ambiguity. Ambiguity decreased as stimulus bandwidth increased, reaching a minimum at 2-3 kHz. Two independent mechanisms appear to contribute to the elimination of ambiguity: one suppressive and one facilitative. The integration of information carried by parallel, distributed processing channels is a common theme of sensory processing that spans both modality and species boundaries. The principles underlying the resolution of phase ambiguity and frequency channel convergence in the owl may have implications for other sensory systems, such as electrolocation in electric fish and the computation of binocular disparity in the avian and mammalian visual systems.     

Head-related transfer functions of the barn owl: measurement and neural responses

Sounds arriving at the eardrum are filtered by the external ear and associated structures in a frequency and direction specific manner. When convolved with the appropriate filters and presented to human listeners through headphones, broadband noises can be precisely localized to the corresponding position outside of the head (reviewed in Blauert, 1997). Such a 'virtual auditory space' can be a potentially powerful tool for neurophysiological and behavioral work in other species as well. We are developing a virtual auditory space for the barn owl, Tyto alba, a highly successful auditory predator that has become a well-established model for hearing research. We recorded catalogues of head-related transfer functions (HRTFs) from the frontal hemisphere of 12 barn owls and compared virtual and free sound fields acoustically and by their evoked neuronal responses. The inner ca. 1 cm of the ear canal was found to contribute little to the directionality of the HRTFs. HRTFs were recorded by inserting probetube microphones to within about 1 or 2 mm of the eardrum. We recorded HRTFs at frequencies between 2 and 11 kHz, which includes the frequencies most useful to the owl for sound localization (3-9 kHz; Konishi, 1973). Spectra of virtual sounds were within +/- 1 dB of amplitude and +/- 10 degrees of phase of the spectra of free field sounds measured near to the eardrum. The spatial pattern of responses obtained from neurons in the inferior colliculus were almost indistinguishable in response to virtual and to free field stimulation.     

Arginine-481 mutation abolishes ligand-binding of the AMPA-selective glutamate receptor channel alpha1-subunit

Arginine-481 is located in the putative agonist-binding region preceding the putative transmembrane segment M1 of the alpha1-subunit of the AMPA-selective glutamate receptor (GluR) channel. This amino acid is completely conserved among GluR proteins. A site-directed mutagenesis study using a baculovirus expression system showed that substitution of glutamate, glutamine and lysine for arginine-481 of the recombinant alpha1-subunit protein abolishes binding to [3H]AMPA completely. The present study provides the first direct experimental evidence that the conserved charged arginine-481 residue is essential, directly or indirectly, for the acquisition of ligand-binding activity by the receptor protein.     

Effects of acute brainstem compression on auditory brainstem response in the guinea pig

BACKGROUND: The purpose of this study was to establish the norm for parameters of auditory brainstem response (ABR) in the guinea pig and to investigate if acute brainstem compression results in significant changes to these parameters. METHODS: Thirty-six guinea pigs with positive Preyer's reflex were anesthetized. A craniectomy was performed to remove the right occipital bone and the dura mater was opened to expose the brain, cerebellum and cerebellopontine angle (CPA). A small inflatable balloon was placed into the CPA precisely and slowly. ABR was recorded before incision of the skin as a baseline value, after placement and after inflation of the balloon with water at 0.1-ml intervals. RESULTS: Five stable peaks were recorded in 27 experimental animals. When the balloon was inflated with 0.1 ml water, the absolute latency (AL) of peaks IV and V and the interpeak latency (IPL) of peaks III and IV, and IV and V were prolonged. The amplitude ratios (AR) of peaks II, III, IV and V to peak I decreased. Inflation of the balloon with 0.2 ml of water caused further elongation of ALs of peaks IV and V and decreases in each AR. When the balloon volume increased to 0.3 ml, peak V became unrecognizable and peaks III and IV showed significant elongation of AL; peaks I and II did not show significant change in ALs. Further increase of the balloon volume to 0.4 ml resulted in disappearance of peaks III, IV and V; AL of peak II was also elongated. However, the amplitude and AL of peak I remained unchanged. Similar changes were observed in IPLs. CONCLUSIONS: This study establishes the norm of parameters of ABR in guinea pigs and demonstrates that acute brainstem compression causes elongation of ALs and IPLs of peaks II, III, IV and V. This suggests that peaks II, III, IV and V come from the brainstem and that peak I is not generated from the brainstem in the guinea pig.     

Development of an expert system for pediatric auditory brainstem response interpretation

Expert systems are computer programs which incorporate artificial intelligence technology and are created to emulate the decision-making abilities of human experts. The advantage of such systems lies in their ability to capture and model expert problem solving knowledge in a domain and make it available to an unlimited number of consumers in an economic and efficient way. The purpose of this project was to develop an expert system to interpret infant auditory brainstem response data as entered by the user. The resulting system provides diagnostic conclusions regarding hearing status, type of hearing loss, and brainstem function at an accuracy level equal to that of a human expert.     

Neural coding of relational invariance in speech: Human language analogs to the barn owl.

The ability to form perceptual equivalence classes from variable input stimuli is common in both animals and humans. Neural circuitry that can disambiguate ambiguous stimuli to arrive at perceptual constancy has been documented in the barn owl's inferior colliculus where sound-source azimuth is signaled by interaural phase differences spanning the frequency spectrum of the sound wave. Extrapolating from the sound-localization system of the barn owl to human speech, 2 hypothetical models are offered to conceptualize the neural realization of relative invariance in (a) categorization of stop consonants /b, d, g/ across varying vowel contexts and (b) vowel identity across speakers. 2 computational algorithms employing real speech data were used to establish acoustic commonalities to form neural mappings representing phonemic equivalence classes in the form of functional arrays similar to those seen in the barn owl. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Early monaural occlusion alters the neural map of interaural level differences in the inferior colliculus of the barn owl

Monaural occlusion during early life causes adaptive changes in the tuning of units in the owl's optic tectum to interaural level differences (ILD) that tend to align the auditory with the visual map of space. We investigated whether these changes could be due to experience-dependent plasticity occurring in the auditory pathway prior to the optic tectum. Units were recorded in the external nucleus of the inferior colliculus (ICx), which is a major source of auditory input to the optic tectum. The tuning of ICx units to ILD was measured in normal barn owls and in barn owls raised with one ear occluded. ILD tuning at each recording site was measured with dichotic noise bursts, presented at a constant average binaural level, 20 dB above threshold. The best ILD at each site was defined as the midpoint of the range of ILD values which elicited more than 50% of the maximum response. A physiological map of ILD was found in the ICx of normal owls: best ILDs changed systematically from right-ear-greater to left-ear-greater as the electrode progressed from dorsal to ventral. Best ILDs ranged from 13 dB right-ear-greater to 15 dB left-ear-greater and progressed at an average rate of 12 dB/mm. The representations of ILD were similar on both sides of the brain. In the ICx of owls raised with one ear occluded, the map of ILD was shifted in the adaptive direction: ILD tuning was shifted towards values favoring the non-occluded ear (the direction that would restore a normal space map). The average magnitude of the shift was on the order of 8-10 dB in each of 4 owls. In one owl, the mean shift in ILD tuning was almost identical on both sides of the brain. In another owl, the mean shift was much larger on the side ipsilateral to the occlusion than on the contralateral side. In both cases, the mean shifts measured in each ICx were comparable to the mean shifts measured in the optic tectum on the same sides of the brain. Thus, the adjustments in ILD tuning that have been observed in the optic tectum in response to monaural occlusion are almost entirely due to adaptive mechanisms that operate at or before the level of the ICx.     

Development and differentiation of the anuran auditory brainstem during metamorphosis: an acetylcholinesterase histochemical study

The time course of cell differentiation and the presence of histochemically defined areas in brainstem auditory nuclei were examined in developing bullfrogs, Rana catesbeiana, using cresyl violet staining and acetylcholinesterase (AChE) histochemistry. In the medulla, the dorsolateral nucleus (DLN) can be seen as a distinct structure in its adult location only at Gosner stage 40 and beyond. The majority of cells in the DLN are not fully differentiated until late metamorphic climax (stages 45-46) and early postmetamorphosis. The more ventral vestibular nucleus differentiates earlier (stage 37) than the DLN. Adult-like organization of auditory nuclei in the torus semicircularis (TS) of the midbrain cannot be reliably discerned until metamorphic climax stages. Cellular masses in the brainstem reveal AChE from the earliest stage examined (stage 27) but the intensity of staining differs among cell groups. Staining intensity in the DLN is at a peak in recently metamorphosed froglets. The time course of cell differentiation in the DLN precedes slightly or is coincident with the increased, transient presence of AChE. Staining of the superior olive stabilizes at a moderate level in early postmetamorphic stages. Ventral regions of the principal nucleus in the TS stain more intensely than dorsal regions beginning at stage 40. This dorsal-ventral gradient in staining persists in adult stages. There is a transient decline in staining of the laminar nucleus in metamorphic climax stages. Staining intensity in the magnocellular nucleus peaks during stages 40-46 and in early postmetamorphic froglets and then declines in adults, paralleling the pattern seen in the DLN. These data suggest that metamorphic climax and early froglet periods are an important developmental window for major differentiation and maturational events in the auditory brainstem.     

Use of contralateral masking in the measurement of the auditory brainstem response

In the first portion of this study, the effects of two levels of contralateral masking on the auditory brainstem response (ABR) were investigated in 10 normal-hearing subjects. No significant changes were observed in the mean latency-intensity functions or the mean amplitude-intensity functions of this group of subjects when noise of various levels was added to the nontest ear. In the second portion of this study, ABRs were also recorded from the poorer ear of four subjects with a profound unilateral sensorineural hearing loss. Results from the latter group revealed a crossed-over wave V in all cases when the stimulus was delivered to the poorer ear and the nontest (better) ear was not masked. Contralateral masking obliterated this "crossed ABR" in all four unilaterally impaired subjects. These results provide support for the use of contralateral masking when recording from the poorer ear of subjects having asymmetrical hearing loss.     

Capacity for plasticity in the adult owl auditory system expanded by juvenile experience

In the process of creating a multimodal map of space, auditory-visual neurons in the optic tectum establish associations between particular values of auditory spatial cues and locations in the visual field. In the barn owl, tectal neurons reveal these associations in the match between their tuning for interaural time differences (ITDs) and the locations of their visual receptive fields (VRFs). In young owls ITD-VRF associations can be adjusted by experience over a wide range, but the range of adjustment normally becomes quite restricted in adults. This normal range of adjustment in adults was greatly expanded in owls that had previously learned abnormal ITD-VRF associations as juveniles. Thus, the act of learning abnormal associations early in life leaves an enduring trace in this pathway that enables unusual functional connections to be reestablished, as needed, in adulthood, even when the associations represented by these connections have not been used for an extended period of time.     

Degeneration of axons in the brainstem of the chinchilla after auditory overstimulation

The patterns of axonal degeneration following acoustic overstimulation of the cochlea were traced in the brainstem of adult chinchillas. The Nauta-Rasmussen method for axonal degeneration was used following survivals of 1-32 days after a 105 min exposure to an octave-band noise with a center frequency of 4 kHz and a sound pressure level of 108 dB. Hair-cell and myelinated nerve-fiber loss were assessed in the cochlea. The cochleotopic pattern of terminal degeneration in the ventral cochlear nucleus correlated with the sites of myelinated fiber and inner-hair-cell loss: this correlation was less rigorous with outer-hair-cell loss, especially in the dorsal cochlear nucleus. These results are consistent with a dystrophic process with a slow time course depending on hair-cell loss and/or direct cochlear nerve-fiber damage. However, in a number of cases with no damage in the apical cochlea, fine fiber degeneration occurred with a faster course in low-frequency regions in the dorsal cochlear nucleus and, transynaptically, in a non-cochleotopic pattern in the superior olive and inferior colliculus. These findings suggest that neuronal hyperactivity plays a role in the central degeneration following acoustic overstimulation, possibly by an excitotoxic process.     

General characteristics and suppression tuning properties of the distortion-product otoacoustic emission 2f1-f2 in the barn owl

The distortion-product otoacoustic emission (DPOAE) 2f1-f2 was measured in the ear canal of the barn owl. DPOAE were elicited by primary tones in 11 frequency regions from 1 to 9 kHz. The highest DPOAE output levels and best thresholds were found for f1 frequencies of 4 to 7 kHz and additionally at the lowest f1 frequency investigated. In some cases, the DPOAE sound pressures were only 37 dB below the primary-tone levels (PTL). The optimal primary-tone frequency ratios ranged from 1.05 to 1.45 and varied strongly among the different frequency regions investigated. The largest optimal ratios were measured in the middle frequency range for f1. At lower and higher f1, the optimal ratios decreased. DPOAE levels could be suppressed in a frequency-selective way by adding a third tone. As in other non-mammals, the best suppressive frequencies were near f1, suggesting DPOAE generation near the frequency place of this primary tone. This is in contrast to what is known for mammalian species, where the DPOAE is thought to be generated near f2. To obtain 6 dB of suppression of the DPOAE level, suppressor-tone levels ranging from 13 dB below to 4 dB above the primary-tone level were necessary. The Q10dB-values of suppression tuning curves increased as a function of frequency up to a value of 15.8. This tendency resembled the increase in frequency selectivity of auditory nerve fibers in this species.     

Isochronic mapping of the auditory brainstem response: normative results

Isochronic mapping involves recording multi-channel evoked potentials from scalp electrodes and plotting contours of peak latencies. In this study, auditory brainstem responses were recorded from 20 electrode sites for left, right and binaural stimulation of each ear of 10 male and 10 female, normally hearing, young adults. Analysis of the data showed that the stimulus parameters of intensity, polarity and rate had no significant effect on the maps. On monaural stimulation, wave V was recorded first at the contralateral mastoid and ipsilaterally frontally and last at the ipsilateral mastoid some 350, mu s later. Binaural stimulation gave a symmetrical map, with wave V recorded first frontally and last at the occiput. In contrast, wave III was recorded first ipsilaterally and frontally and last at the contralateral mastoid. Wave II was recorded first at the rear of the contralateral mastoid and last forward of the ipsilateral mastoid. Comparisons between these results and human physiological studies are in agreement for waves V and III but do not support the concept of the VIIth nerve alone as the generator for wave II. These results suggest that this technique is a potentially useful diagnostic tool and it is intended to evaluate it by testing patients with a range of sensory, peripheral-neural and central-neural pathologies.     

Methods of sedation for auditory brainstem response testing

The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue alpha-aminoisobutyrate. alpha-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of alpha-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated alpha-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for alpha-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated alpha-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mM. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells.