Patterns of cell death in mouse anteroventral cochlear nucleus neurons after unilateral cochlea removal

Developmental changes that influence the results of removal of afferent input on the survival of neurons of the anteroventral cochlear nucleus (AVCN) of mice were examined with the hope of providing a suitable model for understanding the cellular and molecular basis for these developmental changes in susceptibility. We performed unilateral cochlear ablation on wild-type mice at a variety of ages around the time of hearing onset to determine developmental changes in the sensitivity of AVCN neurons to afferent deprivation. In postnatal day 5 (P5) mice, cochlea removal resulted in 61% neuronal loss in the AVCN. By age P14, fewer than 1% of AVCN neurons were lost after this manipulation. This reveals a rather abrupt change in the sensitivity to disruption of afferent input, a critical period. We next investigated the temporal events associated with neuron loss after cochlea removal in susceptible animals. We demonstrate that significant cell loss occurs within 48 hours of cochlea removal in P7 animals. Furthermore, evidence of apoptosis was observed within 12 hours of cochlea removal, suggesting that the molecular events leading to cell loss after afferent deprivation begin to occur within hours of cochlea removal. Finally, we began to examine the role of the bcl-2 gene family in regulating afferent deprivation-induced cell death in the mouse AVCN. AVCN neurons in mature bcl-2 knockout mice demonstrate susceptibility to removal of afferent input comparable to neonatal sensitivity of wild-type controls. These data suggest that bcl-2 is one effector of cell survival as these cells switch from afferent-dependent to -independent survival mechanisms.     

Effects of unilateral cochlea removal on anteroventral cochlear nucleus neurons in developing gerbils

Afferent regulation of neurons in the cochlear nucleus as a function of age was investigated at the light microscope level. Unilateral cochlea removal was performed on Mongolian gerbils of three age groups: 1, 8, and 20 weeks postnatal. Animals survived for either 2 days or 2 weeks. An additional group of neonatally operated animals had a prolonged survival of 9 weeks. The number of neurons in anteroventral cochlear nucleus (AVCN) was counted, and cross-sectional area measurements of large spherical cells in AVCN were made. In animals 1 week old at the time of surgery, there was a 35% reduction in neuron number in AVCN after 2 days, a 58% reduction after 2 weeks, and a 59% reduction 9 weeks after inner ear destruction. However, in animals 20 weeks old at the time of surgery, there was no cell loss in AVCN either 2 days or 2 weeks after surgery. Animals in each age group showed a reduction in cross-sectional area of large spherical cells in AVCN after cochlea ablation. The gerbils that underwent cochlea removal at 8 and 20 weeks showed an average decrease of 14-18%. This effect was seen as early as 2 days after cochlea removal. Animals that underwent cochlea removal at 1 week exhibited the greatest change; a 25% decrease at 2 days progressed to 38% at 2 weeks following cochlea removal. No appreciable further changes were seen at 9 weeks after neonatal cochlea removal. The results indicate greater susceptibility of 1-week-old gerbil cochlear nucleus neurons to peripheral loss than found in older animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative changes in calretinin immunostaining in the cochlear nuclei after unilateral cochlear removal in young ferrets

Neurons of the cochlear nuclei receive axosomatic endings from primary afferent fibers from the cochlea and have projections that diverge to form parallel ascending auditory pathways. These cells are characterized by neurochemical phenotypes such as levels of calretinin. To test whether or not early deafferentation results in changes in calretinin immunostaining in the cochlear nucleus, unilateral cochlear ablations were performed in ferrets soon after hearing onset (postnatal day [P]30-P40). Two months later, changes in calretinin immunostaining as well as cell size, volume, and synaptophysin immunostaining were assessed in the anteroventral (AVCN), posteroventral (PVCN), and dorsal cochlear nucleus (DCN). A decrease in calretinin immunostaining was evident ipsilaterally within the AVCN and PVCN but not in the DCN. Further analysis revealed a decrease both in the calretinin-immunostained neuropil and in the calretinin-immunostained area within AVCN and PVCN neurons. These declines were accompanied by significant ipsilateral decreases in volume as well as neuron area in the AVCN and PVCN compared with the contralateral cochlear nucleus and unoperated animals, but not compared with the DCN. In addition, there was a significant contralateral increase in calretinin-immunostained area within AVCN and PVCN neurons compared with control animals. Finally, a decrease in area of synaptophysin immunostaining in both the ipsilateral AVCN and PVCN without changes in the number of boutons was found. The present data demonstrate that unilateral cochlear ablation leads to 1) decreased immunostaining of the neuropil in the AVCN and PVCN ipsilaterally, 2) decreased calretinin immunostaining within AVCN and PVCN neurons ipsilaterally, 3) synaptogenesis in the AVCN and PVCN ipsilaterally, and 4) increased calretinin immunostaining within AVCN and PVCN neurons contralaterally.     

Afferent reorganisation within the superior olivary complex of the gerbil: Development and induction by neonatal,unilateral cochlear removal

Cochlear removal in young animals has been shown to produce a variety of degenerative and generative effects within the auditory brainstem. A primary target for axons of neurons in the anteroventral cochlear nucleus (AVCN) is the superior olivary complex (SOC). Following unilateral cochlear removal in neonatal gerbils, AVCN neurons on the side of the removal die, and axons deriving from the AVCN on the unlesioned side produce new endings that innervate previously inappropriate target zones within the ipsilateral medial nucleus of the trapezoid body, both medial superior olives, and the contralateral lateral superior olive. In this study, we have used the anterograde transport of Dil and HRP from the AVCN to relate the formation of these endings to the time course of normal development in the gerbil brainstem. We have also examined the effects of cochlear removal at different ages, and survival to various ages after the removal, to define the time course for these generative phenomena. The results show that, while the major projection pathways from the AVCN to the SOC are in place at the time of birth, further and subtle development of AVCN terminal arbors occurs during the first postnatal week. This overlaps with the time during which cochlear removal produces the formation of exuberant afferents to the SOC from the intact AVCN. The exuberant afferents form through axon sprouting rather than through a suppression of normal, developmental regression. They appear to innervate tonotopically appropriate target regions within the SOC. The formation of the novel afferents begins within 3 days of the removal and appears to be complete within a further 5–7 days. By postnatal day (P) 10, both the normal development of the AVCN to SOC projection and the potential for alteration of that projection by removal of the contralateral. Cochlea appear to be over. These results suggest that the potential for forming novel projections in the gerbil auditory brainstem is lost before the onset of functional hearing (at P12) and is, therefore, unlikely to result from changes in auditory experience. © 1995 Wiley-Liss, Inc.     

Neonatal deafness results in degraded topographic specificity of auditory nerve projections to the cochlear nucleus in cats

We previously examined the early postnatal maturation of the primary afferent auditory nerve projections from the cat cochlear spiral ganglion (SG) to the cochlear nucleus (CN). In normal kittens these projections exhibit clear cochleotopic organization before birth, but quantitative data showed that their topographic specificity is less precise in perinatal kittens than in adults. Normalized for CN size, projections to the anteroventral (AVCN), posteroventral (PVCN), and dorsal (DCN) subdivisions are all significantly broader in neonates than in adults. By 6-7 postnatal days, projections are proportionate to those of adults, suggesting that significant refinement occurs during the early postnatal period. The present study examined SG projections to the CN in adult cats deafened as neonates by ototoxic drug administration. The fundamental organization of the SG-to-CN projections into frequency band laminae is clearly evident despite severe auditory deprivation from birth. However, when normalized for the smaller CN size in deafened animals, projections are disproportionately broader than in controls; AVCN, PVCN, and DCN projections are 39, 26, and 48% broader, respectively, than predicted if they were precisely proportionate to projections in normal hearing animals. These findings suggest that normal auditory experience and neural activity are essential for the early postnatal development (or subsequent maintenance) of the topographic precision of SG-to-CN projections. After early deafness, the basic cochleotopic organization of the CN is established and maintained into adulthood, but the CN is severely reduced in size and the topographic specificity of primary afferent projections that underlies frequency resolution in the normal central auditory system is significantly degraded.     

Long-term interaction between microglial cells and cochlear nucleus neurons after bilateral cochlear ablation

The removal of afferent activity has been reported to modify neuronal activity in the cochlear nucleus of adult rats. After cell damage, microglial cells are rapidly activated, initiating a series of cellular responses that influences neuronal function and survival. To investigate how this glial response occurs and how it might influence injured neurons, bilateral cochlear ablations were performed on adult rats to examine the short-term (16 and 24 hours and 4 and 7 days) and long-term (15, 30, and 100 days) changes in the distribution and morphology of microglial cells (immunostained with the ionized calcium-binding adaptor molecule 1; Iba-1) and the interaction of microglial cells with deafferented neurons in the ventral cochlear nucleus. A significant increase in the mean cross-sectional area and Iba-1 immunostaining of microglial cells in the cochlear nucleus was observed at all survival times after the ablation compared with control animals. These increases were concomitant with an increase in the area of Iba-1 immunostaining at 24 hours and 4, 7, and 15 days postablation. Additionally, microglial cells were frequently seen apposing the cell bodies and dendrites of auditory neurons at 7, 15, and 30 days postablation. In summary, these results provide evidence for persistent glial activation in the ventral cochlear nucleus and suggest that long-term interaction occurs between microglial cells and deafferented cochlear nucleus neurons following bilateral cochlear ablation, which could facilitate the remodeling of the affected neuronal circuits.     

Auditory brainstem of the ferret: early cessation of developmental sensitivity of neurons in the cochlear nucleus to removal of the cochlea

The role of primary afferent innervation in the maintenance of neurons in the mammalian auditory system was assessed by performing unilateral removals of the cochlea in neonatal and mature ferrets of known birth dates. Removals were performed under steroid anesthesia and resulted in the complete destruction of the organ of Corti and the loss of at least 80% of type I spiral ganglion neurons. Four main age groups [postnatal days (P)5, P24, P90, and P180] were used. Additional animals received no surgery, partial removals, or complete removals at older ages. Three months after the cochlear removal the animals were reanesthetized and perfused. The brainstem and the temporal bones were wax-embedded, frontally sectioned, and Nissl-stained. Sections of the right and left cochlear nuclei were compared quantitatively. Removal of the cochlea at P5 resulted in the loss of more than 50% of large (nongranular) neurons throughout the ipsilateral cochlear nucleus. Lesions at older ages did not produce any neuron loss. The size of the remaining neurons was reduced by 10-15% in all age groups. Partial lesions at P5 produced a graded response in the cochlear nucleus that was related to the extent of the lesion. The developmental sensitive period for the effects of cochlear removal on the ferret cochlear nucleus is therefore over before the age (P28-P30) at which the animal begins to hear. The present result differs markedly from the chicken, in which the sensitive period for removal of the cochlea persists for at least 2 months after the onset of hearing.     

Relationships between neuronal birthdates and tonotopic positions in the mouse cochlear nucleus

Tonotopy is a key anatomical feature of the vertebrate auditory system, but little is known about the mechanisms underlying its development. Since date of birth of a neuron correlates with tonotopic position in the cochlea, we investigated if it also correlates with tonotopic position in the cochlear nucleus (CN). In the cochlea, spiral ganglion neurons are organized in a basal to apical progression along the length of the cochlea based on birthdates, with neurons in the base (responding to high-frequency sounds) born early around mouse embryonic day (E) 9.5–10.5, and those in the apex (responding to low-frequency sounds) born late around E12.5‑13.5. Using a low-dose thymidine analog incorporation assay, we examine whether CN neurons are arranged in a spatial gradient according to their birthdates. Most CN neurons are born between E10.5 ānd E13.5, with a peak at E12.5. A second wave of neuron birth was observed in the dorsal cochlear nucleus (DCN) beginning on E14.5 and lasts until E18.5. Large excitatory neurons were born in the first wave, and small local circuit neurons were born in the second. No spatial gradient of cell birth was observed in the DCN. In contrast, neurons in the anteroventral cochlear nucleus (AVCN) were found to be arranged in a dorsal to ventral progression according to their birthdates, which are aligned with the tonotopic axis. Most of these AVCN neurons are endbulb-innervated bushy cells. The correlation between birthdate and tonotopic position suggests testable mechanisms for specification of tonotopic position.     

TrkB levels in the cochlear nucleus after unilateral cochlear ablation: correlations with post-lesion plasticity

Tyrosine kinase B (TrkB) levels in the adult guinea pig cochlear nucleus (CN) were determined from Western blots for up to 60 days after unilateral cochlear ablation (UCA). Compared to TrkB levels on the intact side, those on the lesioned side were elevated in the anteroventral CN (AVCN) at 7 and 60 days and in the posteroventral CN (PVCN) at 30 days. TrkB levels were depressed in the AVCN and the dorsal CN (DCN) at 30 days. Elevations in the AVCN on the lesioned side at 7 days coincided with a period of synaptogenesis. Other changes were temporally related to up- or downregulations of transmitter release and synaptic receptor activities that were evident after UCA. The findings suggest that changes in signaling through TrkB may contribute to the plasticities in the CN that were evident after UCA.     

Influence of neonatal cochlear removal on the development of mouse cochlear nucleus: I. Number,size, and density of its neurons

Right cochleae were aspirated from 6-day-old mice to determine the influence of cochlear integrity on the development of cochlear nucleus (CN). At 45 days of age, cochlear destruction was confirmed histologically, and the CN of unilaterally deafferented and control animals were analyzed morphometrically. The molecular, fusiform, and polymorphic layers of deafferented dorsal CN were reduced in volume, and the polymorphic layer neurons were fewer, smaller, and less dense. The octopus and multipolar cell regions in deafferented ventral CN (VCN) were smaller, and their neurons were fewer, smaller, and more densely packed. The VCN globular and small spherical cell regions were also smaller with fewer, denser, but normal-sized neurons. There were fewer VCN large spherical cells, but no change was measured in their size. The granule cell regions throughout CN were also reduced in volume. Overall, CN was reduced to 46% of its normal size and 34% of its normal neuronal numbers. These results in the mouse show that deafferentation before the onset of hearing causes more severe CN changes than those reported after adult deafferentation in other mammals and support the theory of a critical period in development when presynaptic integrity is much more important for neuronal maturation than it is for maintenance after the neuron is mature. This suggests that any congenital pathology that compromises the sensorineural structures of the cochlea may cause severe structural and functional abnormalities in the maturing central auditory nuclei.     

Selective vulnerability of adult cochlear nucleus neurons to de-afferentation by mechanical compression

It is well established that the cochlear nucleus (CN) of developing species is susceptible to loss of synaptic connections from the auditory periphery. Less information is known about how de-afferentation affects the adult auditory system. We investigated the effects of de-afferentation to the adult CN by mechanical compression. This experimental model is quantifiable and highly reproducible. Five weeks after mechanical compression to the axons of the auditory neurons, the total number of neurons in the CN was evaluated using un-biased stereological methods. A region-specific degeneration of neurons in the dorsal cochlear nucleus (DCN) and posteroventral cochlear nucleus (PVCN) by 50% was found. Degeneration of neurons in the anteroventral cochlear nucleus (AVCN) was not found. An imbalance between excitatory and inhibitory synaptic transmission after de-afferentation may have played a crucial role in the development of neuronal cell demise in the CN. The occurrence of a region-specific loss of adult CN neurons illustrates the importance of evaluating all regions of the CN to investigate the effects of de-afferentation. Thus, this experimental model may be promising to obtain not only the basic knowledge on auditory nerve/CN degeneration but also the information relevant to the application of cochlear or auditory brainstem implants.     

Rapid deafferentation-induced upregulation of bcl-2 mRNA in the chick cochlear nucleus

Neuronal survival in developing animals is often dependent on afferent activity. In the posthatch chick, approximately 30% of the neurons in the avian cochlear nucleus, nucleus magnocellularis (NM) die following elimination of VIIIth nerve activity. The factors that influence death or survival of an individual NM neuron are largely unknown. Previous studies indicate that both cell death and cell survival mechanisms compete to determine cell fate. One factor that has been shown to suppress cell death cascades in a variety of systems is bcl-2. If this gene product plays a role in regulating cell survival following deafferentation, then one might expect the expression of this gene to be influenced by removal of afferent input. In the present study, in situ hybridization revealed a rapid and transient increase in bcl-2 mRNA in NM neurons following deafferentation. Enhanced bcl-2 mRNA expression was observed at 6 and 12 h following deafferentation, but not at 3 or 24 h. Surprisingly, the upregulation of bcl-2 mRNA was limited to a subpopulation (20-30%) of deafferented neurons corresponding to the number of NM neurons that eventually die following cochlea removal. The robust and rapid upregulation of this gene suggests that cell death cascades regulated by bcl-2 may be initiated following deafferentation.     

Influence of neonatal cochlear removal on the development of mouse cochlear nucleus: II. Dendritic morphometry of its neurons

Right cochleae were aspirated from 6-day-old mice to determine the influence of cochlear integrity on the dendritic development of neurons within cochlear nucleus (CN). At 45 days of age, cochlear destruction was confirmed histologically and the brains were stained by the Golgi-Cox method to permit dendritic morphometry in CN ipsilateral (deafferented) and contralateral (normal) to the neonatally lesioned cochleae. The dendritic field cross-sectional area of ventral CN bushy cells was reduced on the deafferented side, as was the total dendritic length of stellate cells throughout ventral and dorsal CN. The neonatal deafferentation had no statistically significant effect on the total dendritic length of those dorsal CN fusiform cells that developed. These dendritic changes are interpreted as lack of development due to the loss of auditory nerve afferents during a critical period of development and indicate that any congenital pathology that compromises the cochlear sensorineural structures may lead to central auditory abnormalities as well.     

Small neurons in the vestibular nerve root project to the marginal shell of the anteroventral cochlear nucleus in the cat

We injected biotinylated dextran amine (BDA) into marginal shell regions of the anteroventral cochlear nucleus (AVCN) of the cat. These injections led to retrograde labeling of cells including small cells (median soma area = 111 μm², equivalent diameter = 11.9 μm) in the vestibular nerve root (VNR), just ventral to an anterior part of the AVCN. This is an unexpected new finding. The cells were scattered among BDA-labeled fibers and were oriented parallel to the course of the VNR fibers. We suggest that the small neurons of the VNR might serve as second-order vestibular neurons conveying information from vestibular end organs to the cochlear nucleus (CN) and/or act as interneurons between the olivocochlear fibers in the VNR and the CN.     

Naturally occurring neuron death during postnatal development of the gerbil ventral cochlear nucleus begins at the onset of hearing

Postnatal development of the gerbil ventral cochlear nucleus (VCN) was studied quantitatively under the light microscope in Nissl-stained serial sections at postnatal day 0 (P0), P5, P7, P10, P12, P15, and P140. VCN boundaries were unambiguous at all ages, and nucleus volume was calculated planimetrically for all groups. Measurements of neuron soma cross-sectional area and number were made in all groups except P0. Both VCN volume and soma area doubled between P5 and P10. Although somatic growth did not continue beyond P10, VCN volume increased a further 57% between P15 and P140. Neuron number did not change significantly between P5 and P10, averaging approximately 36,000 neurons. Between P10 and P12, neuron number decreased significantly by 22%, with no further change thereafter. Our data show that, following significant postnatal growth in the gerbil VCN, a brief period of naturally occurring neuron death begins at the onset of hearing. J. Comp. Neurol. 387:421–429, 1997. © 1997 Wiley-Liss, Inc.     

Auditory brainstem of the ferret: Long survival following cochlear removal progressively changes projections from the cochlear nucleus to the inferior colliculus

Some effects on auditory brainstem connections of long (1–2.3 years) survival following unilateral cochlear removal in infant and adolescent ferrets were examined by making multiple injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) in either the left or the right inferior colliculus (IC). Previous studies have shown that, in normal adult ferrets, about 50 times as many cochlear nucleus (CN) neurons project to the contralateral as to the ipsilateral IC. Right cochlear removal at P25 increased, within 30 days, the number of retrogradely labeled left CN neurons projecting to the left ipsilateral IC by 17% (from n = 235 to n = 275), relative to normals. In this study, longer survival (3 months to 1 year) after right cochlear removal at P25 resulted in larger increases (38–47%; n ≈ 100) in the number of neurons labeled in the left CN after injections of WGA-HRP in the left IC. No change occurred in the number of neurons labeled in the right CN. Taken together, the results of these experiments show that the ratio of the number of labeled neurons in the left CN to that in the right CN increases progressively with survival time out to the maximum time tested (1 year). In contrast to these results, we have previously reported that right cochlear removal at P90 did not change the number of neurons projecting from the left CN to the left IC after 90 days of survival. However, in this study, very long survival (2–3 years) following right cochlear removal at P90 resulted in an increased (51%, from n = 235 to n = 355) number of left CN neurons labeled by WGA-HRP injections into the left IC, relative to normals. The increased number of labeled neurons included neurons throughout each division of the CN and all of the principal morphological types. In a separate series of experiments involving long survival (1-2 years), right cochlear removal at P25 or P40 did not significantly change the number of neurons in either CN retrogradely labeled by injections of WGA-HRP in the right IC, or the ratio between the number of neurons labeled in each CN. Long survival following cochlear removal at P25–P90 did not result in any loss of neurons in the ipsilateral CN or in any shrinkage of CN neurons further than the 10–20% seen at a shorter survival time (90 days). These results show that at least one pathway within the auditory brainstem continues to be changed in later life following either early or late cochlear removal. They provide a possible anatomical basis for long-term changes seen in sensory systems following lesions of the sense organs of mature animals. © 1994 Wiley-Liss, Inc.     

Trauma-specific insults to the cochlear nucleus in the rat

The effect of acoustic overstimulation on the neuronal number of the cochlear nucleus (CN) was investigated by using unbiased stereological methods in rats. We found that, after 9 weeks of recovery, neurons in the anteroventral cochlear nucleus (AVCN) degenerated, whereas those in the posteroventral and dorsal cochlear nuclei (PVCN and DCN) were preserved. The noise trauma induced near complete loss of the outer hair cells throughout the cochlea, and the inner hair cells were preserved only in the more apical regions. This pattern of selective loss of AVCN neurons in this study was different from trauma induced by auditory deafferentation by mechanical compression of auditory neurons. In contrast to noise trauma, mechanical compression caused loss of neurons in the PVCN and DCN. After 5 weeks of recovery from mechanical compression, there was no loss of inner or outer hair cells. These findings indicate that auditory deprivation, induced by different experimental manipulations, can have strikingly different consequences for the central auditory system. We hypothesized that AVCN neuronal death was induced by excitotoxic mechanisms via AMPA‐type glutamate receptors and that excitatory neuronal circuits developed after acoustic overstimulation protected the PVCN and DCN against neuronal death. The results of the present study demonstrate that hearing loss from different etiologies will cause different patterns of neuronal degeneration in the CN. These findings are important for enhancing the performance of cochlear implants and auditory brainstem implants, because diverse types of hearing loss can selectively affect neuronal degeneration of the CN. © 2012 Wiley Periodicals, Inc.