William R. Lippe

The effect of unilateral basilar papilla removal upon nuclei laminaris and magnocellularis of the chick examined with [3H]2-deoxy-d-glucose autoradiography

The effect of unilateral basilar papilla removal on glucose uptake in the 2nd and 3rd order auditory nuclei in the chicks brain stem, nucleus magnocellularis and nucleus laminaris, respectively, was examined with [3H]2-deoxy-D-glucose (2-DG) autoradiography. The tissue was processed according to a thaw-mount technique, and the number of grains in the resulting autoradiographs was counted to assess changes in glucose uptake. It was observed that there is a greater density of grains over the neuropil regions of nucleus laminaris which receive input from the normal ear than over the corresponding regions which receive input from the operated ear. Similarly, differences in grain density are found between the normally innervated and deafferented magnocellular nuclei although these differences are not as great as those in nucleus laminaris. Differences in grain density were also apparent between the glial/fiber regions which bound the neuropil areas of nucleus laminaris; there is a greater density of grains overlying those glial/fiber regions through which fibers receiving input from the normal ear course than over those regions through which fibers which normally carry input from the operated ear travel. It is likely that this difference mainly reflects glucose uptake in the fibers although a possible contribution of glial tissue cannot be excluded. All these effects of basilar papilla removal are seen with survival times as short as 70 min and thus likely reflect the reduction of neural activity rather than the degeneration of pre- or postsynaptic elements. Finally, the same pattern of results as described above was found when using the more common [14C]2-DG procedure or when using [3H]2-DG but processing the tissue using the freeze-dried technique. The present results thus show the neuropil regions of nucleus laminaris and the adjacent glial/fiber areas to be areas of high glucose utilization. Unilateral basilar papilla removal results in the removal of an excitatory input to these regions, and this results in a reduction of glucose utilization that is specific to those neuropil regions and glial/fiber areas that receive input from the operated ear. These findings are contrasted with another study in which removal of a major excitatory input to the dentate gyrus of the rat results in a reduced glucose utilization which is not specific to the deafferented region and which largely reflects post- rather than presynatic events.

Development of the Place Principle

Two experiments using embryonic and hatchling chickens examined how the representation of frequency along the basilar membrane changed during hearing development. In experiment 1, chicks were exposed to high intensity pure tones (500, 1,500, or 3,000 Hz) at one of three different ages. Analysis of hair cell degeneration indicated a discrete region of damage which systematically changed as a function of exposure frequency and age. With maturation, each frequency produced damage at progressively more apical locations. In experiment 2, the representation of frequency in the brain stem auditory nuclei was compared in embryonic, hatchling, and adult chickens. Microelectrode recordings indicated a systematic shift in the frequency representation. Neurons, which are activated by high frequencies in the adult, initially respond to only low frequencies. These experiments indicate how the mature pattern of frequency representation along the basilar membrane gradually emerges during the stages of hearing development.

The Development of Cochlear Function

The cochlea is the window through which the central auditory system views its acoustic environment. The transduction of air borne sound by the hair cells and the neural encoding at the periphery place constraints on the acoustic features that are available for further processing by auditory neurons in the brain. At birth, the cochlea in most altricial mammals is still very immature. It is effectively unresponsive to sound and generates little sustained (spontaneous) activity. During the first month after the onset of hearing, significant changes occur in cochlear functioning, changes that are reflected in both the overall level and spatiotemporal pattern of nerve impulses that are transmitted centrally over the auditory nerve. The task of determining the extent to which maturational changes in auditory perception, spontaneous activity, and central responses to sound originate within the cochlea and to what degree these changes reflect the development of central synaptic processes remains a formidable challenge.

Relationship between frequency of spontaneous bursting and tonotopic position in the developing avian auditory system.

Neural activity in the developing brainstem auditory pathway of the chick embryo is dominated by a rhythmic pattern of spontaneous discharge. Neurons in nucleus magnocellularis (NM) and nucleus laminaris (NL), second and third order auditory nuclei, discharge spontaneously in synchronous bursts at periodic intervals. Rhythmic bursting is present as early as embryonic day 14 (E14), shortly after the onset of functional synaptogenesis, and gives way to an adult-like, steady level of firing on E19, two days prior to hatching. In the present experiment, multiple-unit recording techniques were used in E17 and E18 embryos to examine the relationship between rate of rhythmic bursting and tonotopic position in NM and NL. The mean rate of rhythmic bursting ranged from 0.21-0.71 Hz. Bursting rate varied systematically as a function of position, being faster at progressively higher frequency regions of the nuclei at both E17 (r = 0.75) and E18 (r = 0.86). In addition, the rate of bursting at a given location in the nuclei increased during development. The presence of a systematic relationship between the rate of rhythmic bursting and tonotopic location suggests that the spatio-temporal pattern of spontaneous discharges could provide developmental cues for the spatial ordering of auditory projections.

Cochlear microphonic measurements of interaural time differences in the chick

The major cues for the sound localization are the interaural differences in the timing and intensity of acoustic information. This poses a difficult coding problem for animals with relatively small heads, such as birds, because interaural time differences (ITDs) would have a small range and magnitude and interaural intensity differences (IIDs) would be significant for only high frequency sounds. It has been suggested that this coding problem is mitigated in birds by an enhancement of ITDs and IIDs resulting from the acoustic coupling of the two middle ear cavities through an interaural canal. In this report, the functional ITDs for sounds at different azimuthal locations were recorded in young chicks, and the contribution of middle ear acoustic coupling was evaluated. ITDs were calculated from simultaneous cochlear microphonic (CM) recordings evoked by pure tone stimuli. These effective ITDs were larger than predicted by the physical separation of the two ears, and this enhancement was more pronounced at low (0.8 and 1 kHz) than at high (2 and 4 kHz) frequencies, reaching maximum values of approximately 180 and 100 microseconds, respectively. The amplitude of the CM also varied as a function of sound source location. This variation was as much as +/- 30%, even for the low frequency tones. This suggests that IID cues are also available to the chick. To determine the contribution of middle ear acoustic coupling to the timing and amplitude of the CM response, the CM in one ear was measured prior to, and following occlusion of the contralateral external auditory canal. The cochlear microphonic from the ear distal to the sound source advanced in time and increased in amplitude when the ear proximal to the sound source was sealed. These effects were more pronounced for low frequency sounds. These findings confirm that acoustic coupling of the middle ear cavities plays a role in enhancing sound localization cues in the chick.

REDUCTION AND RECOVERY OF NEURONAL SIZE IN THE COCHLEAR NUCLEUS OF THE CHICKEN FOLLOWING AMINOGLYCOSIDE INTOXICATION

The effect of aminoglycoside intoxication on the cross-sectional area of neurons in nucleus magnocellularis (NM) was studied in neonatal chickens. Birds received daily injections of 100 mg/kg body weight of gentamicin for 10 consecutive days. Cell area was measured at five different tonotopic regions along the posterior-to-anterior dimension of NM (low-to-high frequency) after post-treatment survival times of 8, 23 and 40 days. Gentamicin caused a reversible reduction of cell area that varied as a function of location and survival time. Significant decreases of cell area occurred only in the rostral half of the nucleus. Cell area was reduced at 8 and 23 days survival and recovered to near control values by 40 days post-treatment. Body weight, brain weight and the cross-sectional area of cerebellar Purkinje neurons were also reduced but did not recover. The present results show that aminoglycoside toxicity can affect auditory neurons in the brain. It is suggested that two factors contributed to the changes in NM neuron size: (1) Processes specifically related to the loss and regeneration of cochlear hair cells, most likely changes in afferent activity. (2) A general retardation in growth.

Glutamatergic and GABAergic agonists increase [Ca2+]i in avian cochlear nucleus neurons

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca2+ indicator dye Fura-2 was used to measure Ca2+ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca2+ responses were evoked by kainic acid (KA), alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D-aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca2+]i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca2+ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca2+]i were carried by Ca2(+)-permeable receptor channels. Significantly smaller changes in [Ca2+]i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca2+ free-EGTA--buffered media, including L-cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclo-pentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca2+]i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca2+]i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca2+ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca2+]i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca2+]i to increase in NM neurons. Presumably, each route represents a means by which Ca2+ can alter cellular processes.

Effects of middle ear pressure on frequency representation in the central auditory system.

Changes in middle ear pressure (MEP) are known to produce an attenuation of sound transmission through the outer and middle ear, but the effects on frequency representation in the auditory system have not previously been studied. This issue is of particular interest because of changes in MEP occurring during episodes of otitis media. We have investigated the effect of changes in MEP on the tuning of neurons in the inferior colliculus (IC) of the gerbil to calibrated tone stimulation of the contralateral, pressurized ear. Both negative and positive non-atmospheric MEP produced an elevation of neural thresholds that was inversely related to IC neuron best frequency (BF). A robust, linear relationship was found between BF at atmospheric MEP (control) and BF at -20 daPa MEP. Higher resolution analysis was performed on a sub-sample of neurons that had particularly stable BFs with repeated, control MEP. For the majority of these neurons, alternation of MEP between control and -20 daPa had no effect on BF. However, a few neurons showed small (up to 5%), significant shifts in BF with -20 daPa MEP. These results are consistent with previous reports of the effects of MEP on spontaneous otoacoustic emissions. We conclude that non-atmospheric MEP acts as a high-pass filter on the input to the cochlea, but does not change the frequency organization of the auditory system to any marked extent.

Development of the place principle

Two experiments using embryonic and hatchling chickens examined how the representation of frequency along the basilar membrane changed during hearing development. In experiment 1, chicks were exposed to high intensity pure tones (500, 1,500, or 3,000 Hz) at one of three different ages. Analysis of hair cell degeneration indicated a discrete region of damage which systematically changed as a function of exposure frequency and age. With maturation, each frequency produced damage at progressively more apical locations. In experiment 2, the representation of frequency in the brain stem auditory nuclei was compared in embryonic, hatchling, and adult chickens. Microelectrode recordings indicated a systematic shift in the frequency representation. Neurons, which are activated by high frequencies in the adult, initially respond to only low frequencies. These experiments indicate how the mature pattern of frequency representation along the basilar membrane gradually emerges during the stages of hearing development.