Electrophysiological responses of cochlear root neurons.

Cochlear root neurons (CRNs) are second-order neurons interspersed among the fibers of the cochlear nerve in certain rodents. They project, among other nuclei, mainly to the pontine reticular nucleus, and participate in the acoustic startle response (ASR), a short-latency motor reflex initiated by sudden intense sounds. The sound-evoked activity of CRNs has not previously been described. Here we describe extracellular responses of CRNs located in the infranuclear portion of the cochlear nerve root. CRNs exhibited secure responses to tone bursts, with first-spike latencies of approximately 2.2 ms. The characteristic frequencies of the recorded CRNs were about 30 kHz, and the best-characterized CRN had a threshold of 10 dB sound pressure level and sharpness of tuning similar to that of cochlear nerve fibers. The peristimulus time histograms were primary-like with notch. The observed response properties were consistent with the suggestion that CRNs provide the short-latency acoustic input to the reticular formation that leads to an ASR.

Postnatal development of efferent synapses in the rat cochlea.

The development of olivocochlear efferent axons and their contacts in the postnatal cochlea was studied after DiI applications to the olivocochlear bundle in the ipsilateral brainstem of rats from 0 to 10 days of age (P0-10). Light microscopic analyses showed that labeled axons reached the vicinity of inner hair cells by P0 and outer hair cells by P2. Electron microscopic analyses demonstrated that labeled immature efferent axons are present among supporting cells of the greater epithelial ridge as well as inner hair cells at P0. The first efferent contacts that contacted inner hair cells contained a few irregularly sized vesicles and, occasionally, mitochondria. Postsynaptic specializations within inner hair cells apposed to labeled efferent axons included subsynaptic cisterns, irregularly sized vesicles, and synaptic bodies. Similar features were present in unlabeled profiles, presumed to be afferents, indicating that immature efferent axons could not be reliably distinguished from afferents without positive labeling. Efferent axons synapsed with outer hair cells by P4 and had synapse-like contacts at the bases of Deiters' cells at P4 and P6. Contacts between afferents and efferents were observed frequently in the inner spiral bundle from P6. As they matured, efferent axon terminals contacting hair cells contained increasing numbers of synaptic vesicles and were typically apposed by well-defined postsynaptic cisterns, thus acquiring distinctive profiles.

Projections of cochlear root neurons, sentinels of the rat auditory pathway.

In certain rodents, the root of the cochlear nerve contains a population of large neurons, known as cochlear root neurons (CRNs), an essential element of the primary acoustic startle pathway. To characterize the projections of the CRNs, we made stereotaxically guided, iontophoretic injections of biotinylated tracers into the cochlear nerve root of albino rats. CRN axons, which are remarkably thick, enter the trapezoid body, cross the midline, and ascend in the rostral aspect of the lateral lemniscus to reach the upper levels of the midbrain. As a group, CRN axons produce a characteristic pattern of profusely ramified collaterals that innervate specific brainstem regions. The main target of CRN axons is the contralateral pontine reticular formation, where collaterals terminate in the caudal pontine reticular nucleus (PnC) and, to a lesser degree, in the ventrolateral tegmental area, the oral pontine reticular nucleus, and the rostral and medial paralemniscal regions. Other targets of CRN axons include the lateral paragigantocellular nucleus of both sides, the ipsilateral facial motor nucleus and PnC, and the contralateral intercollicular tegmentum and superior colliculus. Notably, CRNs apparently do not innervate any of the nuclei of the auditory brainstem, as usually defined, even though their axons pass through or in close proximity to them. The fact that CRNs innervate several reticular and tectal structures that mediate auditory alerting and escape behaviors suggests that they are "early warning neurons," i.e., true sentinels of the auditory pathway.

Olivocochlear neurons in the chinchilla: a retrograde fluorescent labelling study.

Although the chinchilla is widely used as a model for auditory research, little is known about the distribution and morphology of its olivocochlear neurons. Here, we report on the olivocochlear neurons projecting to one cochlea, as determined by single and double retrograde fluorescent tracer techniques. 10 adult chinchillas were anesthetized and given either unilateral or bilateral injections of a fluorescent tracer (either Fluoro-Gold or Fast Blue) into scala tympani or as a control, a unilateral injection into the middle ear cavity. The results indicate that there are similarities as well as significant differences between the chinchilla and other species of rodents in the distributions of their olivocochlear neurons. Based on three well-labelled cases, there was a mean total of 1168 olivocochlear neurons in the chinchilla. Of these, the majority (mean 787) were small, lateral olivocochlear neurons found almost exclusively within the ipsilateral lateral superior olivary nucleus. The next largest group consisted of a mean of 280 medial olivocochlear neurons virtually all of which were located in the dorsomedial peri-olivary nucleus. Chinchilla medial olivocochlear neurons were more predominantly crossed in their projections (4:1) than in any known species. The smallest group of olivocochlear neurons (mean 101) consisted of larger lateral olivocochlear neurons (shell neurons) which were located on the margins of the superior olivary nucleus and which projected mainly (2.2:1) ipsilaterally. Double retrograde labelling was observed only in medial olivocochlear neurons and occurred in only 1-2% of these cells. The results confirm previous findings which indicated a relative paucity of fibers belonging to the uncrossed as compared to the crossed olivocochlear bundle. This, together with the strong apical bias of the uncrossed projection reported previously, offers possible explanations for the apparent absence of efferent-mediated suppressive effects of contralateral acoustic stimulation in this species. Regarding the lateral olivocochlear system, the chinchilla is shown to possess both intrinsic and shell neurons, as in the rat.

Efferent innervation of the inner hair cell region: origins and terminations of two lateral olivocochlear systems.

The projections of lateral olivocochlear neurons (LOC), which terminate beneath inner hair cells (IHCs), were investigated by injecting biotinylated dextran amine into the lateral superior olivary nucleus (LSO) and the surrounding region in the rat. This region has been definitively shown to contain two types of olivocochlear neurons: small cells within the LSO (intrinsic neurons) and large cells (shell neurons) surrounding it (Vetter, D.E., Mugnaini, E., 1992. Distribution and dendritic features of three groups of rat olivocochlear neurons. Anat. Embryol. 185, 1-16). Labeled efferent axons were studied by light microscopy in whole mounts and radial sections of the organ of Corti (OC). It was found that injections confined to the LSO, which presumably affected mainly intrinsic neurons, labeled a cluster of axons in the osseous spiral lamina that entered the inner spiral bundle (ISB) and terminated in one or more dense patches that, in total basal-apical extent, spanned no more than 10-20% (1-2 mm) of the total length of the OC (10 mm). In contrast, injections affecting shell neurons produced labeled axons that entered the OC over a span of more than 50% of its length and which, as a group, coursed in the ISB for at least 80%, and sometimes more than 95% of total cochlear length. Study of individual axons in the OC revealed that intrinsic axons did not bifurcate upon entering the OC and traveled less than 1 mm before terminating in a discrete, dense arbor. In contrast, shell axons typically bifurcated into basal and apical branches that, in toto, traveled between 1 and 2 mm beneath the IHCs, forming numerous en passant swellings and a few terminal branches en route. The fact that localized injections of intrinsic neurons produced focal peaks of labeling in the cochlea, whereas similar injections of shell neurons produced a diffuse, non-focal projection that could extend for nearly the entire length of the cochlea, suggests that significant differences exist between these two populations in their capacity to influence localized, frequency-specific regions of the OC, and thus in their probable functional roles. The present findings in the rat not only confirm a previous study in the guinea pig which found a similar dual efferent innervation beneath the IHCs (Brown, M.C., 1987. Morphology of labeled efferent fibers in the guinea pig cochlea. J. Comp. Neurol. 260, 605-618), but extend those observations by linking two axonal types beneath the IHCs to their respective cell bodies of origin in the lateral zone of the superior olivary complex.

Regional distribution of a creatine transporter in rat auditory brainstem: an in-situ hybridization study.

The expression of an mRNA encoding a creatine transporter (CRT1) was investigated in the rat auditory system under ambient sound conditions, using radiolabeled and non-radiolabeled oligonucleotide in-situ hybridization. The results indicated that CRT1 mRNA is widely distributed in auditory nuclei, including the fusiform and deep layers of the dorsal cochlear nucleus, the ventral cochlear nucleus, the superior olivary complex, the nuclei of the lateral lemniscus and the inferior colliculus. The molecular layer of the dorsal cochlear nucleus and the medial geniculate have low levels of label. Creatine provides cells with a reservoir of high-energy phosphate. Neurons do not synthesize creatine but accumulate it by a transport mechanism, which is probably the limiting step in the regulation of intracellular creatine. Therefore, the quantity of transporter expressed may reflect the utilization of creatine and could serve as an in-vitro indicator of endogenous high-energy metabolism in some cells. Although most auditory nuclei express CRT1 mRNA, the quantity of CRT1 mRNA varies among auditory nuclei, indicating that many auditory nuclei have high and fluctuating energy requirements. The level of CRT1 transcript or protein may be regulated by chronic metabolic changes in the auditory system that may occur, for example, with damage to the acoustic organ or the aging process.

Multiple projections from the ventral nucleus of the trapezoid body in the rat.

An analysis of the central projections of the ventral nucleus of the trapezoid body (VNTB) in the rat, a region of the superior olivary complex known for its neuronal heterogeneity, was made using two anterograde axonal tracers, [3H]leucine and biotinylated dextran amine (BDA). A mixture of these tracers was injected iontophoretically into the VNTB and the results analyzed by first assessing magnitudes of autoradiographic signal in nuclei receiving projections and then identifying the axons and terminals responsible for this signal in parallel sets of sections processed for BDA. Our analysis showed that in addition to its projections to each cochlea via the olivocochlear bundle, the VNTB has 3 major central sites of axonal terminations: (1) the cochlear nucleus, particularly the molecular layer of the contralateral dorsal cochlear nucleus, (2) the contralateral lateral superior olive, and (3) the ipsilateral inferior colliculus. Other sites receiving projections from the VNTB included the VNTB itself and the nuclei of the lateral lemniscus. Significantly, the relative magnitudes of labeling within the nuclei receiving inputs from the VNTB varied consistently as a function of the dorsoventral location of the injection site, confirming previous work showing that there is a partial segregation within this nucleus of neurons according to their projections. Our data also revealed an orderly topographic pattern of projections to the cochlear nuclei, lateral superior olive and the inferior colliculus which is consistent with the known tonotopic organization both of the VNTB and these projection targets. Methodologically, the co-injection of two tracers was advantageous in that patterns of silver grains in autoradiographs could be used to confirm whether axons and terminals labeled with BDA had originated from labeled somata at the injection site or were the result of uptake of BDA by fibers of passage.

Changes in spontaneous otoacoustic emissions produced by acoustic stimulation of the contralateral ear.

Spontaneous otoacoustic emissions (SOAEs) were measured in human ear canals before, during and after presentation of tonal stimuli to the contralateral ear. Stimuli were presented in 1/8 octave steps from 2 octaves below to 1 octave above the SOAE frequency at sound levels below the observed contralateral acoustic reflex threshold. For certain conditions there was an abrupt upward frequency shift at stimulus onset. For a fixed level the effect was frequency selective; the maximum frequency shift was obtained with tones approximately 1/2 octave below the SOAE. SOAE amplitude usually decreased but in some cases increased or remained unchanged. When amplitude changes were observed, the maximum shifts were observed for tones at or near the SOAE frequency. Changes in SOAEs were not observed for stimulus levels below 60 dB SPL. The effect is believed to be mediated by medial efferent neurons of the uncrossed olivocochlear bundle which arise in the medial region of the superior olivary complex and terminate on outer hair cells (OHCs). These results support those models which attribute SOAE generation to OHCs, and are indicative of an efferent influence on cochlear mechanics. A simple model is presented that proposes that efferent activity alters the tuning of the emission generator by causing changes in OHC membrane conductance.

Descending projections from the superior olivary complex to the cochlear nucleus of the cat.

Subdivisions of the cochlear nuclear complex give rise to a number of discrete projections to certain cell groups of the superior olivary complex and also received substantial descending projections from the periolivary nuclei. In the present study, we sought to determine by means of retrograde transport of horseradish peroxidase (HRP), and anterograde transport of radiolabeled protein, if the periolivary nuclei give rise to discrete projections to the various subdivisions of the cochlear nuclear complex. Following medium to large injections of HRP into the cochlear nucleus, irrespective of location, labeled cells were found in all periolivary nuclei bilaterally. In every case more than 40% of the labeled cells were found in the lateral nucleus of the trapezoid body on the same side and the ventral nucleus of the trapezoid body of both sides. Other periolivary nuclei contributing more than 5% of the total number of cells in individual cases were the contralateral lateral nucleus of the trapezoid body and the ipsilateral anterolateral and dorsal periolivary nuclei. Injections of tritiated leucine into periolivary nuclei gave rise to axonal labeling to the trapezoid body and the dorsal acoustic stria, usually bilaterally, and to terminal labeling that was widely distributed within the cochlear nuclear complex. In several cases with small injections, particularly in the lateral nucleus of the trapezoid body, the projections from the periolivary nuclei to the anteroventral and dorsal cochlear nuclei connected areas described as having similar best-frequency representation. The autoradiographic data corroborated the main results from the HRP experiments and provided additional information permitting these conclusions: the projections from the periolivary nuclei to the cochlear nuclear complex are organized tonotopically, at least in part; each periolivary nucleus (and perhaps individual cells), projects widely throughout the cochlear nuclear complex; the pattern of termination of projections from different periolivary nuclei to a given region of the cochlear nuclear complex are similar, as seen in autoradiograms, and the lateral and dorsal periolivary nuclei project mainly ipsilaterally, while the medial periolivary nuclei project bilaterally with a contralateral bias. The magnitude of these projections and their widespread distribution within the cochlear nuclear complex would suggest an important role for the descending projections in the normal functioning of the cochlear nucleus.

The projections of principal cells of the medial nucleus of the trapezoid body in the cat.

Previous studies suggest that the principal cells of the medial nucleus of the trapezoid body (MNTB) give rise to the projection from MNTB to the lateral superior olivary nucleus (LSO) of the same side, where they mediate rapid inhibitory effects of contralateral sound stimulation. In the present study, we explored certain morphological features of this connection as well as several other projections of the MNTB by using anterograde and retrograde axonal tracing methods. Following injections of tritiated leucine into MNTB, labeled axons reached LSO by passing ventral to, dorsal to, and through the medial superior olivary nucleus, and gave rise to labeling around the somata and proximal dendrites of LSO fusiform cells. As measured in autoradiograms of 2 micron plastic sections, these axons had a modal diameter of 5-6 micron. Terminal labeling, tentatively attributed to principal cell axons, was also seen in the ventral nucleus of the lateral lemniscus (VNLL) and the dorsomedial and ventromedial periolivary nuclei. HRP injections into the LSO and the VNLL showed that the principal cell projected to both of these nuclei and revealed a topographic arrangement of the projection to the LSO which is consistent with tonotopic maps determined electrophysiologically. Control HRP injections demonstrated that other minor projections of the MNTB arose from minor cell populations in this nucleus. The findings provide a morphological correlate of certain physiological findings and suggest a wider role for the MNTB in the ascending auditory system than previously has been supposed.

Topographic organization of the olivocochlear projections from the lateral and medial zones of the superior olivary complex.

By anterograde tracing using autoradiography, we have found topographic organizations in the projections of both medial and lateral olivocochlear (OC) systems in the cat. Lateral-zone injections show an ipsilateral cochleotopic projection pattern with more medial injections projecting more basally in the cochlea. In the contralateral cochlea, in contrast, the projections from all of the lateral-zone injections were predominantly to the apex. However, detailed analysis suggests the possibility that the contralateral lateral-zone projections may have the same cochleotopic organization as the ipsilateral projections but with a heavy bias toward the apex. Medial-zone injections show a pattern in which more dorsal regions project more basally in both cochleas. The ipsilateral projections of lateral OC neurons appear to connect regions with similar best frequencies but the projections of medial OC neurons do not. Summation of data from all of the injections in each zone indicates that lateral OC projections are relatively evenly distributed throughout the ipsilateral cochlea but are predominantly to the apex in the contralateral cochlea. Medial OC projections are predominantly to the middle and basal parts of the cochlea on both sides with contralateral projections somewhat more basal than ipsilateral projections.

Differential olivocochlear projections from lateral versus medial zones of the superior olivary complex.

An anterograde tracer (35S-methionine) was injected unilaterally in the superior olivary complex (SOC) at regions previously demonstrated by retrograde labeling to contain olivocochlear (OC) cell bodies. Quantitative analysis of cochlear autoradiographs from these cats demonstrates that there are two OC systems. The lateral OC system has cell bodies lateral to the medial superior olivary nucleus (MSO) and projects to the inner hair cell (IHC) region bilaterally (mostly ipsilaterally). The medial OC system has cell bodies medial, ventral, and anterior to the MSO and projects to the outer hair cell (OHC) region bilaterally (mostly contralaterally). A single medial OC neuron innervates many small patches of OHCs with substantial gaps between the patches. Medial OC neurons also appear to project to the IHC region to a small extent. A review of the literature with the medial-lateral division of OC efferents in mind reveals many differences between these two systems. In particular, lateral OC axons are unmyelinated and innervate the dendrites of radial afferent fibers under IHCs, whereas medial OC axons are myelinated and directly innervate OHCs. Although both systems appear to be cholinergic, the lateral OC system also shows met-enkephalin-like immunoreactivity. The synapses of the medial OC system are formed in development before those of the lateral OC system and they degenerate more slowly after the OC axons are cut. The many differences between these two OC systems suggest that they are functionally separate systems.

The dual origins of the olivocochlear bundle in the albino rat.

Recent studies of the origins and terminations of the olivocochlear bundle (OCB) in the cat provide evidence that separate efferent systems differentially innervate the two types of hair cells in the organ of Corti. To begin to test the generality of these separate olivocochlear systems, the cells of origin of the OCB were determined in the albino rat by using axonal transport of horseradish peroxidase. Our findings are that, as in the cat, two classes of olivocochlear (OC) neurons project to the cochlea. These neurons could be dichotomized according to their location in the superior olivary complex (lateral or medial), their size (small or large), and their preferred side of projection (ipsilateral or contralateral). All labeled OC neurons also exhibited a positive reaction for acetylcholinesterase. In the rat, however, lateral and medial OC neurons are each restricted to a single nucleus, and, furthermore, the lateral OC neurons project only ipsilaterally. The rat also has significantly fewer mean totals of both lateral (240 vs. 710) and medial (240 vs. 520) OC neurons than the cat. The present methods also demonstrated the course of axons of the OCB and axon collaterals entering the cochlear nuclear complex. Vestibular efferent neurons were also labeled bilaterally medial and lateral to the facial genu. Our results suggest that the general organizational plan of OC neurons in the rat may offer advantages over the situation in the cat for studies of connectional neuroanatomy, neurophysiology, and behavioral functions of the two olivocochlear systems.

Efferent components of the auditory system.

The origins and terminations of the olivocochlear bundle, which provides an efferent innervation to the organ of Corti, are described on the basis of experiments using axonal transport of tracer substances and light microscopy in the cat. The cells of origin were labeled by the retrograde tracer horseradish peroxidase which was injected unilaterally into the cochlea. Labeled cells in the superior olivary complex could be dichotomized according to their location (lateral or medial), their size (small or large), and their preferred side of projection (uncrossed or crossed). All labeled olivocochlear neurons exhibited a positive reaction for acetylcholinesterase. To determine the cochlear projections of the neurons, injections of a radioactive amino acid were made into either the lateral or medial olivocochlear cell group. After allowing time for synthesis and axonal transport of radio-labeled protein to reach synaptic endings in the cochleas, the tissue sections of these specimens were processed for autoradiography. The results indicate that lateral olivocochlear neurons project to the regional beneath the inner hair cells of both sides, whereas medial olivocochlear neurons project to the region beneath the outer hair cells of both sides. These findings are in substantial accord with previous experimental work but suggest that the organ of Corti receives a dual efferent innervation which is organized according to the location and morphology of its cells of origin. Accordingly, it is proposed that the two efferent components of the cochlear innervation described here be referred to as the lateral and medial olivocochlear systems, replacing the current designations of crossed and uncrossed olivocochlear bundles, the latter which are demonstrably heterogeneous in their origins and terminations and, probably, also in their functions.

Distribution and light microscopic features of granule cells in the cochlear nuclei of cat, rat, and mouse.

In the present study the cytology and the topography of the cochlear granule cell domain (a comprehensive term introduced here for all granule cell-containing regions of the cochlear nuclear complex) have been studied light microscopically in Nissl, Bielschowsky, and Golgi-Del Rio-Hortega material of cats, rats, and mice; in Golig rapid material of 0-14-day-old kittens; and in sections of 6-week-old kittens following HRP injections in the superficial dorsal cochlear nucleus (DCN). The domain has been parcellated in seven subdivisions which, in spite of some species' differences, are easily identifiable in all of the included animals. The cochlear granule cells are considered as a particular class of neuron, which is slightly different from, but nevertheless principally similar to the cerebellar granule cells in both shape and mode of neuronal connections. The digitiform terminals of the cochlear granule cells differentiate after the first two weeks of extrauterine life. In several respects these cells show larger variation among species than do the cerebellar granules, the similarity between the two classes of granule cells being most conspicuous in the rodent. The silver, Golgi rapid, and HRP material suggest that all, or at least the majority, of the granule cell axons project to the molecular layer of the DCN, forming parallel fibers similar to those of the cerebellar cortex. Also, the cochlear parallel fibers traverse the spiny apical dendrites of principal neurons (the pyramidal cells) and the smoother dendrites of molecular layer stellate cells.

Origins of axons in the cat's acoustic striae determined by injection of horseradish peroxidase into severed tracts.

Origins and terminations of fibers of the dorsal and intermediate acoustic striae were studied by surgically severing these tracts and injecting HRP into the incision. This procedure results in filling the severed axons with HRP. Filled axons were traced to cell groups of origin and to some terminations of the acoustic striae. HRP-labeled terminals were found in the cochlear nuclei as well as in periolivary cell groups. Filling of cells with HRP RANged from being complete, resulting in Golgi-like images, to being barely detectable. Labeled cells were abundant in the dorsal and posteroventral cochlear nucleus adjacent to the injection as well as scattered throughout the periolivary cell groups of both sides, being highest in concentration around the ipsilateral lateral superior olive. On the side contralateral to the injection, labeled cells were found along the medial border of the dorsal cochlear nucleus, in the interstitial nucleus of the stria of Held, and sparsely throughout the ventral cochlear nucleus. The distribution of labeled cells was similar following HRP injections of the dorsal cochlear nucleus, except that these injections revealed additional descending projections from the inferior colliculi and from the ventral nucleus of the trapezoid body of both sides. These additional projections were interpreted as entering the CN by a ventral route. Findings of this study are in accord with physiological recordings made from fibers of the acoustic striae.

Olivocochlear and vestibular efferent neurons of the feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry.

Anterograde degeneration studies have shown that the cochlear and vestibular receptor organs receive an efferent innervation from neurons in the brain stem. This pathway may provide a mechanism by which the CNS could modulate its own afferent input. The neurons which provide this innervation have so far escaped positive identification with methods which depend on retrograde cell changes after axotomy. In the present study, horseradish peroxidase (HRP) was injected into the labryinths of kittens and after allowing 24 hours for the retrograde axonal transport of this tracer, its presence in neurons of the brain stem was demonstrated histochemically. Because there is evidence that the efferent innervation of the labyrinth is cholinergic, acetylcholinesterase (AChE) was also demonstrated histochemically in the same or in adjacent tissue sections. Neurons labelled with HRP were found bilaterally in most periolivary cell groups of the superior olivary complex (cochlear efferents) and in the parvocellular reticular nucleus lateral to the abducens nucleus (vestibular efferents). Counts of labelled neurons yielded estimated totals of 1,700-1,800 cochlear and 400-500 vestibular efferent neurons. Approximately 60% of the neurons in each total were located on the side ipsilateral to the injection. The distribution of HRP-labelled neurons was virtually identical to that of AChE-positive neurons found in adjacent sections, and in those regions with predominantly ipsilateral or contralateral projections, there was an approximate correspondence in number of HRP- and AChE-positive neurons. In tissue sections processed successively for demonstration of HRP and AChE, virtually all HRP-labelled neurons were found to be AChE-positive. These findings suggest that a number of current conceptions regarding labyrinthine efferent systems may need revision.