Biomedical subjects

D K Ryugo

Publications and source records attributed to D K Ryugo.

At least 37 records · Page 2Linked to original sources

PubMed1996

Pyramidal cells in primary auditory cortex project to cochlear nucleus in rat.

Recent work has demonstrated that the auditory cortex in rat sends direct projections to the auditory nuclei of the brainstem, including the cochlear nucleus and superior olive. To determine the cortical origin of the projections to cochlear nucleus, Fast Blue, a retrograde fluorescent tracer, was injected into the cochlear nucleus. Labeled cells in the forebrain were then studied with light microscopy and mapped. The projection was found to originate from large pyramidal neurons in layer V of primary auditory cortex. The projection was predominantly ipsilateral, and no labeled neurons were found in other cortical areas. These data imply that primary auditory cortex exerts influence over ascending auditory information at the earliest stages of the central auditory system.

Amidines↗

PubMed1996

Immunocytochemical localization of glycine in a subset of cartwheel cells of the dorsal cochlear nucleus in rats.

Glycine is an inhibitory neurotransmitter and a glutamate cofactor for N-methyl-D-aspartate (NMDA) receptors in the central nervous system. The distribution of glycine in the auditory system will therefore provide clues as to synaptic mechanisms underlying auditory signal processing. Previous studies have reported the immunocytochemical presence of glycine in the dorsal cochlear nucleus of a variety of mammals, but the specificity with respect to particular cell types has proven elusive at the light microscopic level. We sought to identify cell types in the superficial regions of the dorsal cochlear nucleus that were immunoreactive to glycine using light and electron microscopy in the rat. At the light microscopic level, glycine immunoreactivity was present in some but not all medium-sized cells in layers I and II. The somata of pyramidal and granule cells were not stained. At the electron microscopic level, using previously published ultrastructural criteria, we examined the glycine-labeled cells and determined that many but not all cartwheel cells were labeled. We also observed unlabeled unipolar brush cells, Golgi cells, and stellate cells. As some of the labeled cells could not be identified, we could not determine whether unipolar brush cells, Golgi cells or stellate cells had both labeled and unlabeled subpopulations. Our observations indicate that within the population of cartwheel cells, only a subset are glycine-immunoreactive.

Animals↗

PubMed1996

Peripheral course of genioglossal motor axons within the hypoglossal nerve of the rat.

The spatial compartmentalization of motor axons in cranial nerves has not been previously demonstrated. In the present study, motor axons in the medial lingual ramus of the hypoglossal nerve of rats were labeled using horseradish peroxidase and diaminobenzidine staining methods. Neuronal axons were segregated into fascicles within the main nerve trunk (average total length: 10.4 mm). Light microscopic examination of stained nerve sections revealed reaction product within individual axons and showed that the grouping of labeled nerve fibers in the medial ramus was maintained for at least the peripheral half of the nerve before the stain faded. The authors propose that the fascicular anatomy of the rat hypoglossal cranial nerve resembles that of peripheral spinal nerves in humans. This functional and structural compartmentalization may have a clinical impact on the repair and treatment of cranial nerve pathologies.

Animals↗

PubMed1995

Inositol 1,4,5-trisphosphate receptors: immunocytochemical localization in the dorsal cochlear nucleus.

In the cochlear nucleus of mammals, the relatively homogeneous responses of auditory nerve fibers are transformed into a variety of different response patterns by the different classes of resident neurons. The spectrum of these responses is hypothesized to depend on the types and distribution of receptors, ion channels, G proteins, and second messengers that form the signaling capabilities in each cell class. In the present study, we examined the immunocytochemical distribution of the inositol 1,4,5-triphosphate (IP3) receptor in the dorsal cochlear nucleus to better understand how this second messenger might be involved in shaping the neural signals evoked by sound. Affinity-purified polyclonal antibodies directed against the IP3 receptor labeled a homogeneous population of neurons in the dorsal cochlear nucleus of rats, guinea pigs, mustache bats, cats, New World owl monkeys, rhesus monkeys, and humans. These cells were all darkly immunostained except in the human where the labeling was less intense. Immunoblots of dorsal cochlear nucleus tissue from the rat revealed a single band of protein of molecular weight approximately 260 kD, which is the same size as the purified receptor, indicating that our antibodies reacted specifically with the IP3 receptor. These immunolabeled neurons were identified as cartwheel cells on the basis of shared characteristics across species, including cell body size and distribution, the presence of a highly invaginated nucleus, and a well-developed system of cisternae. Reaction product was localized along the membranes of rough and smooth endoplasmic reticulum, subsurface cisternae, and the nuclear envelope. This label was distributed throughout the cartwheel cell body and dendritic shafts but not within dendritic spines, axons, or axons terminals. The regular pattern of immunolabeling across mammals suggests that IP3 and cartwheel cells are conserved in evolution and that both play an important but as yet unknown role in hearing.

Aged↗

PubMed1995

Neuronal organization of the cochlear nuclei in alligator lizards: a light and electron microscopic investigation.

The organization of neurons and fibers in the cochlear nuclei of the alligator lizard (Gerrhonotus multicarinatus) was examined with light and electron microscopy. In this species, much is known about the anatomy and physiology of the inner ear including the cochlear nerve, but little is known about the synaptic connections of cochlear fibers on second-order neurons. These data will help to develop general principles addressing the cellular organization of the vertebrate auditory system. Subdivisions of the cochlear nuclei were defined on the basis of their histologic appearance and neuronal composition. Neuron classes were proposed from their light microscopic and ultrastructural features. Nucleus magnocellularis medialis consists of a homogeneous population of neurons called "lesser ovoid" cells. Nucleus magnocellularis lateralis consists of "greater ovoid" and "small" cells. Nucleus angularis lateralis consists of "spindle" cells. Lastly, nucleus angularis medialis contains a population of large neurons called "duckhead" and "multipolar" cells, and a population of smaller neurons called "bulb" and "agranular" cells. These neuron populations are differentially innervated by tectorial and free-standing cochlear fibers that are associated with separate frequency ranges. All neuronal populations except agranular cells were observed to receive synaptic input from cochlear nerve fibers. In nucleus magnocellularis medialis and nucleus angularis medialis, primary afferents form both chemical and electrical synapses with resident neurons. These observations imply that acoustic information is synaptically processed in fundamentally distinct ways in the cochlear nuclei of alligator lizards and distributed along separate neural circuits. Thus, the characteristic structural and functional dichotomy of the alligator lizard inner ear is extended to central auditory pathways by way of cochlear nerve projections.

Animals↗

PubMed1994

Physiology and morphology of complex spiking neurons in the guinea pig dorsal cochlear nucleus.

Intracellular recordings from the dorsal cochlear nucleus have identified cells with both simple and complex action potential waveforms. We investigated the hypothesis that cartwheel cells are a specific cell type that generates complex action potentials, based on their analogous anatomical, developmental, and biochemical similarities to cerebellar Purkinje cells, which are known to discharge complex action potentials. Intracellular recordings were made from a brain slice preparation of the guinea pig dorsal cochlear nucleus. A subpopulation of cells discharged a series of two or three action potentials riding on a slow depolarization as an all-or-none event; this discharge pattern is called a complex spike or burst. These cells also exhibited anodal break bursts, anomalous rectification, subthreshold inward rectification, and frequent inhibitory postsynaptic potentials (IPSPs). Seven complex-spiking cells were stained with intracellular dyes and subsequently identified as cartwheel neurons. In contrast, six identified simple-spiking cells recorded in concurrent experiments were pyramidal cells. The cartwheel cell bodies reside in the lower part of layer 1 and the upper part of layer 2 of the nucleus. The cells are characterized by spiny dendrites penetrating the molecular layer, a lack of basal dendritic processes, and an axonal plexus invading layers 2 and 3, and the inner regions of layer 1. The cartwheel cell axons made putative synaptic contacts at the light microscopic level with pyramidal cells and small cells, including stellate cells, granule cells, and other cartwheel cells in layers 1 and 2. The axonal plexus of individual cartwheel cells suggests that they can inhibit cells receiving input from either the same or adjacent parallel fibers and that this inhibition is distributed along the isofrequency contours of the nucleus.

Action Potentials↗

PubMed1994

Central trajectories of type II (thin) fibers of the auditory nerve in cats.

This paper describes the central projections of thin fibers of the auditory nerve in cats. Both thin (type II) and thick (type I) fibers are labeled by extracellular injections of horseradish peroxidase (HRP) into the auditory nerve. Type I and almost all type II fibers bifurcate upon reaching the auditory nerve root of the cochlear nucleus. For a given bundle of auditory nerve fibers labeled by a discrete injection of HRP, bifurcations of type II and type I fibers are restricted to a narrow region of the nerve root. After the bifurcation, the pathways of type II branches within the anteroventral cochlear nucleus (AVCN) and posteroventral cochlear nucleus (PVCN) are similar to those of type I branches. This similarity in bifurcation and course of type I and type II fibers was observed in the ventral as well as dorsal parts of the ventral cochlear nucleus. The complete axonal course of most type II fibers could not be reconstructed, however, due to fading of the reaction product. Type II fibers produce very few collaterals in the cochlear nucleus (CN), but possess many 'en passant' swellings along their main processes and collaterals. Compared with type II fibers previously studied in mice (Berglund and Brown, 1989; 1994; Brown and Ledwith, 1990), cat type II fibers are similar in their general projections within the main body of the nucleus and in the frequency of 'en passant' swellings per length of fiber, but cat fibers have a higher percentage of 'complex' or pedunculated 'en passant' swellings.

Animals↗

PubMed1993

The projections of intracellularly labeled auditory nerve fibers to the dorsal cochlear nucleus of cats.

The cochlear nucleus receives incoming auditory nerve discharges, preserves or transforms the signals, and distributes outgoing activity to higher centers. The organization of auditory nerve input to the cochlear nucleus will heavily influence the mechanisms by which acoustic information is processed. In order to study structure-function relationships between auditory nerve and cochlear nucleus, the axonal arborizations of type I spiral ganglion cells were labeled with intracellular injections of horseradish peroxidase after first being electrophysiologically characterized by recording with a micropipette inserted into the axon. For each auditory nerve fiber, spontaneous discharge rate (SR) and a frequency tuning curve were determined. The tuning curve yielded the characteristic frequency (CF, that frequency to which the fiber is most sensitive) and CF threshold in dB SPL. Individual axonal arborizations including all terminal swellings were reconstructed through serial sections with the aid of a light microscope and drawing tube. On average, 13.4 +/- 8.1% of the terminal swellings were found in the dorsal cochlear nucleus (DCN) and the remaining terminal swellings were located in the ventral cochlear nucleus. In the DCN, the terminal fields of auditory nerve fibers were restricted to layer III, contributed to cytoarchitectonic striations, and exhibited a systematic relationship between fiber CF and position along the strial (or long) axis of the nucleus. Computer-aided rotations revealed that the terminal fields were anisotropic, being flattened within the trans-strial axis. The maximal width of the terminal fields along the strial axis ranged from 31-321 microns and was inversely related to fiber CF and SR. Variation in the number of terminals or depth of the terminal field within layer III was not related to SR grouping or CF of the fiber.

Animals↗

PubMed1993

Frequency organization of the dorsal cochlear nucleus in cats.

Sensory epithelia are often spatially reiterated throughout their representation in the central nervous system. Differential expression of this representation can reveal specializations of the organism's behavioral repertoire. For example, the nature of the central representation of sound frequency in the auditory system has provided important clues in understanding ecological pressures for acoustic processing. In this context, we used electrophysiological techniques to map the frequency organization of the dorsal cochlear nucleus in nine cats. Frequency responses were sampled in increments of 100-200 microns along electrode tracks that entered the dorsomedial border of the nucleus and exited at the ventrolateral border. Electrode tracks were oriented parallel to the long (or strial) axis of the nucleus so that each penetration sampled neural responses for most of the cat's audible frequencies and remained in or near the pyramidal cell layer for several millimeters. Nearly identical distance versus frequency relationships were obtained for different rostral-caudal locations within the same cat as well as for different cats. Frequency responses systematically decreased from above 50 kHz at the most dorsomedial locations in the nucleus to below 1 kHz in the most ventrolateral regions. The rate of frequency change was roughly three times greater in high frequency regions than in low frequency regions. In addition, the highest pyramidal cell density and longest rostral-caudal axis was observed for the middle third of the dorsal-ventral axis of the nucleus. As a result, roughly half of all pyramidal cells responded to frequencies between 8-30 kHz. The representation of neural tissue for these frequencies may be related to the importance of spectral cues in sound locations.

Animals↗

PubMed1993

Neuronal inositol 1,4,5-trisphosphate receptor localized to the plasma membrane of olfactory cilia.

Both the cyclic adenosine 3',5'-monophosphate and the phosphoinositide second messenger systems are involved in olfactory signal transduction. The inositol 1,4,5-trisphosphate receptor is one of the principal intracellular calcium channels responsible for mobilizing stored calcium. The precise location of the 1,4,5-trisphosphate receptor (endoplasmic reticulum vs surface) and its role in the events of olfactory signal transduction need to be defined. By light microscopic and confocal immunohistochemistry we show expression of the olfactory-enriched G-protein, G(olf), associated with cyclic AMP responses, and of the inositol 1,4,5-trisphosphate receptor in the dendritic projections and cilia of essentially all olfactory receptor neurons, indicating that individual neurons express components of both second messenger systems. By immunoelectron microscopy, we demonstrate that the inositol 1,4,5-trisphosphate receptor is localized to the surface membrane of rat olfactory cilia. This is the first morphological demonstration of the surface membrane localization of inositol 1,4,5-trisphosphate receptor in olfactory cilia. Our findings, taken in conjunction with electrophysiological data from other workers, are supportive of the inositol 1,4,5-trisphosphate receptor playing a novel role in regulating calcium flux at the ciliary surface membrane.

Animals↗

PubMed1991

Unmyelinated axons of the auditory nerve in cats.

This paper describes some central terminations of type II spiral ganglion neurons as labeled by extracellular injections of horseradish peroxidase (HRP) into the auditory nerve of cats. After histological processing with diaminobenzidine, both thick (2-4 microns) and thin (0.5 microns) fibers of the auditory nerve were stained. Whenever traced, thick fibers always originated from type I spiral ganglion neurons and thin fibers always from type II ganglion neurons. Because the labeling of type II axons faded as fibers projected into the cochlear nucleus, this report is limited to regions of the ventral cochlear nucleus near the auditory nerve root. The central axons of type II neurons are unmyelinated, have simple yet variable branching patterns in the cochlear nucleus, and form both en passant and terminal swellings. Under the light microscope, most swellings are located in the neuropil but they are also found in the vicinity of cell bodies, nodes of Ranvier of type I axons, and blood vessels. Eighteen en passant swellings in the neuropil were located by light microscopy and resectioned for electron microscopy; two of these swellings exhibited ultrastructural features characteristic of chemical synapses. The data indicate that inputs from outer hair cells might be able to influence auditory processing in the cochlear nucleus through type II primary neurons.

Animals↗

PubMed1991

Neurofilament antibodies and spiral ganglion neurons of the mammalian cochlea.

The spiral ganglia of the cat, gerbil, mouse, rat, and human were immunohistochemically stained with various monoclonal neurofilament antibodies. Three antibodies to the 200-kD neurofilament protein (R-3, Dräger et al., '84; ICN anti-200, clone NE14, Debus et al., '83; RT-97, Wood and Anderton, '81) labeled the somata of type II spiral ganglion neurons but not those of type I ganglion neurons. In the extreme base of the cochlea of cats, mice and rats, there was intense labeling of a few (less than 0.5% of the total ganglion population) large neurons resembling type I ganglion neurons. Several other neurofilament antibodies (Amersham anti-68, Amersham and ICN anti-160, and SMI-32) did not specifically label type II ganglion neurons but instead labeled all neurons of the spiral ganglion. These two patterns of labeling prompted us to investigate the cause for this difference. Because antibodies against the 200-kD neurofilament protein preferentially labeled type II neurons and because 200-kD neurofilament is highly phosphorylated, we treated cochlear tissue with alkaline phosphatase in order to remove phosphate groups. This treatment eliminated the intense labeling of type II neurons with R-3, ICN anti-200, and RT-97, but had no effect on the intense labeling of ganglion cell bodies observed with the other neurofilament antibodies tested. This evidence suggests that labeling occurs because of the cytoplasmic presence of phosphorylated 200-kD neurofilament protein in type II ganglion neurons. Populations of neurons may thus differ in their neurofilament epitopes and monoclonal antibodies can be used to mark such differences.

Alkaline Phosphatase↗

PubMed1991

Synaptic connections of the auditory nerve in cats: relationship between endbulbs of held and spherical bushy cells.

This report focuses on a class of large synaptic endings, the endbulbs of Held. These endings are located in the anteroventral cochlear nucleus and arise from the axons of type I spiral ganglion neurons. Axons were stained with horseradish peroxidase (HRP) using intracellular injections of single fibers or extracellular injections into the auditory nerve. Individual endbulbs or pairs of endbulbs that converged onto the same spherical bushy cell were examined with the aid of a light microscope and subjected to morphometric analyses. Endbulbs of fibers having low spontaneous discharge rates (SR, less than or equal to 18 spikes/sec) have a more complex shape than those of high SR fibers (greater than 18 s/s), a feature represented by systematic differences in endbulb silhouette perimeter without differences in endbulb silhouette area. Consequently, the ratio, silhouette area divided by silhouette perimeter, yields a "form factor" separating endbulbs of high SR from those of low SR. High SR fibers had ratios greater than 0.52 (mean = 0.63 +/- 0.09), whereas low SR fibers had ratios less than 0.52 (mean = 0.45 +/- 0.06). Pairs of endbulbs with unknown physiological properties had similar form factor values, despite the wide range of values observed in the endbulb population. These data imply that endbulbs converging upon the cell body of a spherical bushy cell arise from fibers of the same SR group. Electron microscopic examination was conducted on the endbulb of one physiologically characterized and intracellularly stained auditory nerve fiber (CF = 1.4 kHz; SR = 55 s/s) and its unstained endbulb mate with the aid of serial ultrathin sections. In addition to the well-known axosomatic synapses, these endbulbs formed axodendritic synapses: 11.7% for the HRP-labeled endbulb and 13.3% for the unlabeled endbulb. The axodendritic synapses appear to occur on dendrites of nearby spherical bushy cells and may represent a mechanism whereby single endbulbs can disperse activity to multiple neurons in the cochlear nucleus. We propose that axosomatic synapses preserve fiber SR groupings, whereas axodendritic synapses may not.

Animals↗

PubMed1990

Central projections of cochlear nerve fibers in the alligator lizard.

The auditory (cochlear) ganglion cells of the alligator lizard (Gerrhonotus multicarinatus) give rise to two types of peripheral fibers: tectorial fibers, which contact hair cells covered by a tectorial membrane, and free-standing fibers, which contact hair cells without a tectorial membrane. To determine the central projections of these fibers, we applied intracellular and extracellular injections of horseradish peroxidase (HRP) to the peripheral component of the cochlear nerve. After histological processing with diaminobenzidine, individual cochlear nerve fibers could be traced through serial sections with the aid of a light microscope and drawing tube. The projection patterns formed two morphologically distinct groups. Neurons whose peripheral processes contacted tectorial hair cells in the cochlea projected to three divisions of the cochlear nucleus: nucleus magnocellularis lateralis (NML), nucleus magnocellularis medialis (NMM), and nucleus angularis lateralis (NAL). Neurons whose peripheral processes contacted free-standing hair cells projected primarily to the nucleus angularis medialis (NAM), although some also sent a single, thin branch to the NML; these neurons never projected to NAL or NMM. Morphometric comparisons of tectorial and free-standing fibers demonstrate that tectorial fibers have a larger axonal diameter, form a greater number of terminal swellings, and make proportionally more somatic contacts. By correlating the morphologically defined groups with previously reported physiologically defined groups, we conclude that different divisions of the cochlear nucleus are associated with separate frequency ranges and that stimuli in the different frequency ranges may be processed separately in the brain.

Acoustic Stimulation↗

PubMed1989

Endbulbs of held and spherical bushy cells in cats: morphological correlates with physiological properties.

Single auditory nerve fibers of type I spiral ganglion cells in cats were electrophysiologically characterized by recording with micropipettes inserted into the axon and then labeled by intracellular injections of horseradish peroxidase (HRP) through the same pipettes. This method for staining and studying single neurons allowed us to describe structure-function relationships for labeled endbulbs of Held and the somata of their postsynaptic spherical bushy cells. The silhouette areas of terminal endbulbs and the corresponding somata of spherical bushy cells were determined by planimetry from drawings made with a light microscope and drawing tube. On the presynaptic side, endbulb area is related to fiber characteristic frequency (CF, the frequency to which a fiber is most sensitive) such that the largest endbulbs arise from fibers having CFs between 1 and 4 kHz; smaller endbulbs can arise from fibers of any CF. Endbulb area is not correlated with fiber spontaneous discharge rate (SR). Dividing the endbulb's silhouette area by its silhouette perimeter, however, yields a "form factor" that is a reliable indicator of fiber SR: Endbulbs from fibers of low-medium SR (less than or equal to 18 spikes/second) have form factor values less than 0.52, whereas endbulbs of high SR fibers (greater than 18 spikes/second) have values greater than 0.52. This form factor should therefore be predictive of SR groupings in auditory fibers for which physiological data are not available. On the postsynaptic side, the somata of spherical bushy cells receiving endbulbs from low-medium SR fibers are on average smaller than those receiving endbulbs from high SR fibers. In contrast, the nuclei of the spherical bushy cells are the same size regardless of presynaptic fiber SR. Some of the effects of low-medium SR fibers on their postsynaptic targets, when compared to those of high SR fibers, appear to be mimicked by effects of experimentally induced deprivation.

Acoustic Stimulation↗

PubMed1988

Central trajectories of type II spiral ganglion neurons.

Previous attempts to trace the central pathways of the thin axons from type II spiral ganglion neurons have been hampered by technical difficulties such as fading of the reaction product as distance increases from the injection site (Ryugo et al.: Soc. Neurosci. Abstr. 12:779, '86; Brown: J. Comp. Neurol. 260:591-604, '87). By using small rodents (gerbils and mice), which have short auditory nerves, we have succeeded in filling the entire central axon and terminals of type II neurons after peripheral injections of horseradish peroxidase. The general course of the type II fibers within the auditory nerve and cochlear nucleus is similar to that of type I fibers except that terminals from type II neurons are often found in regions of the cochlear nucleus that have high densities of granule cells.

Animals↗

PubMed1988

Brainstem branches from olivocochlear axons in cats and rodents.

Horseradish peroxidase was used to label axons of olivocochlear (OC) neurons by intracellular injections in cats and extracellular injections in rodents. These axons arise from cell bodies in the superior olivary complex and project to the cochlea. En route to the cochlea, the thick axons (greater than 0.7 micron diam.) of medial olivocochlear (MOC) neurons formed collaterals that terminated in the ventral cochlear nucleus, the interstitial nucleus of the vestibular nerve (in cats), and the inferior vestibular nucleus (in rodents). The thin axons (less than 0.7 micron diam.), presumed to arise from lateral olivocochlear (LOC) neurons, did not branch near the CN. Within the CN, the MOC collaterals tended to ramify in and near regions with high densities of granule cells, regions also associated with the terminals of type II afferent axons (Brown et al.: J. Comp. Neurol. 278:581-590, '88). These results suggest that those fibers associated peripherally with outer hair cells (MOC efferents and type II afferents) are associated centrally with regions containing granule cells, whereas those fibers associated with inner hair cells peripherally (LOC efferents and type I afferents) are not.

Afferent Pathways↗

PubMed1988

Central projections of intracellularly labeled auditory nerve fibers in cats: morphometric correlations with physiological properties.

The central arborizations and endings of type I spiral ganglion neurons were labeled with intracellular injections of horseradish peroxidase (HRP) after their characteristic frequency (CF) and spontaneous discharge rate (SR) were physiologically determined. A fiber-by-fiber analysis was conducted and the morphological data compared with the fiber's response properties. The total number of branch points was correlated with total fiber length, a relationship that remained relatively constant when analyzing the ascending and descending branches together or separately. On the other hand, the ascending branches of four out of five fibers having CFs below 0.5 kHz bifurcated and gave rise to a pair of terminal endbulbs of Held. Low- and medium-SR fibers gave rise to more endings than did high-SR fibers, especially on the ascending branch. This difference was accounted for by small endings, a category composed of terminal boutons, string endings, and small complex endings. The categories of modified endbulbs, and endbulbs of Held did not vary in number with respect to fiber SR. The mean area of each ending type within the small ending category was statistically smaller for low- and medium-SR fibers than for high-SR fibers, whereas the mean area of modified endbulbs and endbulbs of Held was not correlated with fiber SR. Total ending area per fiber appeared independent of either CF or SR. These results are discussed in relation to issues of conservation of axon arborizations and terminals, and convergence of input from the different SR groups.

Acoustic Stimulation↗