Congenital Deaf Induces CaBPs Change in Auditory Brainstem Miyako Hatano, MD Makoto Ito, MD, PhD Department of Otolaryngology-Head and Neck Surgery, Division of Neuroscience, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan Abstract We investigated the neurobiological changes that occur after unilateral cochlear ablation before hearing onset by examining calcium-binding proteins (CaBPs), which are sequentially expressed in the auditory brainstem nuclei during definite developmental stages. CaBPs may play a specific role during neuronal development of the central auditory system. In the present study, we examined the changes in the distribution of the CaBPs calbindin-D28k (CB) and parvalbumin (PV) in the superior olivary complex (SOC) and the cochlear nucleus (CN) following unilateral cochlear ablation in neonatal rats. Upon reaching adulthood, a marked decrease in CB and PV immunoreactive neurons was observed in the contralateral SOC, particularly in the medial nucleus of the trapezoid body (MNTB), but no neuronal cell death was observed. A volume reduction in the ipsilateral CN was observed, and immunostaining for PV was also decreased. Immunostaining for CB showed bilateral symmetry in the CN. These results suggest that unilateral congenital deafness with a volume reduction in CN induces changes in the CaBPs in the contralateral SOC in rat. The loss of neurons and the volume reduction in the CN may decrease the input to the contralateral SOC, resulting in downregulation of CaBPs in these nuclei. This study provides an anatomical basis for a better understanding of the changes in the auditory brainstem of deaf children. CN volume ipsilateral to cochlear ablation was reduced compared with the contralateral CN (Figs. 1A, B). The volume of the AVCN, PVCN and DCN was reduced by 59.5%, 62.1% and 21.5%, respectively, compared with each contralateral subdivision of the CN (Fig. 2). CB immunoreactivity was not significantly different between the ipsilateral and contralateral cochlear nucleus (Figs. 1C, D) We observed a substantial decrease in the number of fibers and neurons that were immunoreactive for PV in the VCN on the ablated side compared with the contralateral VCN (Figs. 1E, F), whereas no differences in PV immunoreactivity were noted between the ipsilateral and contralateral DCN. Following neonatal unilateral cochlear ablation, a decrease in synaptic density was shown in the contralateral LSO (lateral superior olive) and SPN (superior periolivary nucleus) (Figs. 3A, B). Neonatal cochlear ablation did not result in any numerical loss of neurons in the MNTB (Figs. 4A, B) as determined by cresyl violet staining. In contrast to the cresyl violet cell count result, neonatal cochlear ablation was found to produce a substantial reduction in the number of CB immunopositive cells in the MNTB on the side contra to ablation (Figs. 4C, D). PV immunostaining suggested a reduction in cell numbers in the contralateral MNTB after neonatal cochlear ablation. However, it was difficult to count the PV-positive cells due to the heavy background staining in the SOC area. 1. Andressen, C. et al. 1993. Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 271, 181-208. 2. Friauf, E. 1994. Distribution of calcium- binding protein calbindin-D28k in the auditory system of adult and developing rats. J Comp Neurol 349, 193-211. 3. Celio, M.R. 1990. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375-475. A total of 10 adult male or female Sprague–Dawley rats were used. P0-P1 rat pups in the experimental group (n=7) were removed the stapes, a syringe needle was inserted through the oval window and the cochlea contents were aspirated. Age- matched rats without cochlear ablation were assessed as the non-surgical controls (n=3). Auditory function was assessed by recording auditory brainstem response (ABR). The measurements of ABR thresholds were performed at frequencies of 4, 8 and 16 kHz after a post-surgical survival time of 5 months. Coronal frozen sections (40 μm) of brains were cut using a sliding microtome and processed in three alternating series. One series of sections was stained with cresyl violet, while the other two were analyzed by immunocytochemistry. The primary antibody was either a mouse monoclonal antibody against CB (Sigma, Saint Louis, Missouri USA; clone CB-955; dilution of 1:12000) or a mouse monoclonal antibody against PV (Sigma, Saint Louis, Missouri USA; clone PA-235; dilution of 1:30000). The secondary antibody was a polymer HRP-labeled anti-mouse antibody (EnVision+ System). These results suggest that 1) The loss of neurons and the volume reduction in the cochlear nucleus may decrease the input to contralateral MNTB 2) CB and PV were downregurated in the contralateral MNTB, LSO and SPN after neonatal cochlear ablation 3) The reduction of CB and PV in the contralateral MNTB can be directly affected neuronal cell populations in the CN. The number of indications of cochlear implantation for deaf children has been increasing in recent years. Brain plasticity is a complex property with massive developmental changes after birth. In general, input deprivation in the nervous system causes functional and structural changes through expression of neural plasticity. Despite the many morphological studies that have investigated the changes in the central auditory system caused by deafness before hearing onset, the neurobiological plasticity of congenital deafness remains unknown. Calbindin D-28k (CB) and parvalbumin (PV) are widely expressed in the brainstem auditory nuclei. CB and PV are calcium- binding proteins (CaBPs) that occur in distinct subpopulations of neurons in the central nervous system. CaBPs are involved in the release of neurotransmitters, the functioning of ion channels in neuronal membranes, and the regulation of enzyme activities at pre- and postsynaptic sites and synaptic short-time plasticity. In the present study, we investigated the changes in CaBPs expression within the auditory brainstem in an animal model with congenital deafness. Unilateral cochlear ablations before hearing onset have been used as models of congenital deafness. Introduction Methods and Materials Results Conclusions Bibliography Figure 1. Low magnification images illustrating the pattern of cresyl violet staining (A, B), CB immunoreactivity (C, D) and PV immunoreactivity (E, F) in coronal sections of the AVCN in ablated rats. Scale bar = 200 μm in B Figure 2. Bar graphs showing the mean volume reduction percentage of AVCN, PVCN and DCN compared with the contralateral lesion side. Whiskers on bars are standard errors of the mean. Figure 3. Low magnification images illustrating the pattern of CB immunoreactivity (A, B) and PV immunoreactivity (C, D) in coronal sections of the LSO and the SPN in ablated rats. Scale bar = 200 μm in B Figure 4. High magnification images illustrating the pattern of cresyl violet stain (A, B), CB immunoreactivity (C, D) and PV immunoreactivity (E, F) in coronal sections of the MNTB in ablated rats. Scale bar = 100 μm in B
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Congenital Deaf Induces CaBPs Change in Auditory Brainstem
Miyako Hatano, MD Makoto Ito, MD, PhDDepartment of Otolaryngology-Head and Neck Surgery, Division of Neuroscience,
Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
Abstract
We investigated the neurobiological changes that occur after unilateral cochlear ablation before hearing onset by examining calcium-binding proteins (CaBPs), which are sequentially expressed in the auditory brainstem nuclei during definite developmental stages. CaBPs may play a specific role during neuronal development of the central auditory system. In the present study, we examined the changes in the distribution of the CaBPs calbindin-D28k (CB) and parvalbumin (PV) in the superior olivary complex (SOC) and the cochlear nucleus (CN) following unilateral cochlear ablation in neonatal rats. Upon reaching adulthood, a marked decrease in CB and PV immunoreactive neurons was observed in the contralateral SOC, particularly in the medial nucleus of the trapezoid body (MNTB), but no neuronal cell death was observed. A volume reduction in the ipsilateral CN was observed, and immunostaining for PV was also decreased. Immunostaining for CB showed bilateral symmetry in the CN. These results suggest that unilateral congenital deafness with a volume reduction in CN induces changes in the CaBPs in the contralateral SOC in rat. The loss of neurons and the volume reduction in the CN may decrease the input to the contralateral SOC, resulting in downregulation of CaBPs in these nuclei. This study provides an anatomical basis for a better understanding of the changes in the auditory brainstem of deaf children.
CN volume ipsilateral to cochlear ablation was reduced compared with the contralateralCN (Figs. 1A, B). The volume of the AVCN, PVCN and DCN was reduced by 59.5%, 62.1% and 21.5%, respectively, compared with each contralateral subdivision of the CN (Fig. 2). CB immunoreactivity was not significantly different between the ipsilateraland contralateral cochlear nucleus (Figs. 1C, D) We observed a substantial decrease in the number of fibers and neurons that were immunoreactive for PV in the VCN on the ablated side compared with the contralateralVCN (Figs. 1E, F), whereas no differences in PV immunoreactivity were noted between the ipsilateral and contralateral DCN.
Following neonatal unilateral cochlear ablation, a decrease in synaptic density was shown in the contralateral LSO (lateral superior olive) and SPN (superior periolivarynucleus) (Figs. 3A, B).
Neonatal cochlear ablation did not result in any numerical loss of neurons in the MNTB (Figs. 4A, B) as determined by cresyl violet staining. In contrast to the cresyl violet cell count result, neonatal cochlear ablation was found to produce a substantial reduction in the number of CB immunopositive cells in the MNTB on the side contra to ablation (Figs. 4C, D). PV immunostaining suggested a reduction in cell numbers in the contralateral MNTB after neonatal cochlear ablation. However, it was difficult to count the PV-positive cells due to the heavy background staining in the SOC area.
1. Andressen, C. et al. 1993. Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 271, 181-208.
2. Friauf, E. 1994. Distribution of calcium-binding protein calbindin-D28k in the auditory system of adult and developing rats. J Comp Neurol 349, 193-211.
3. Celio, M.R. 1990. Calbindin D-28k and parvalbumin in the rat nervous system. Neuroscience 35, 375-475.
A total of 10 adult male or female Sprague–Dawley rats were used. P0-P1 rat pups in the experimental group (n=7) were removed the stapes, a syringe needle was inserted through the oval window and the cochlea contents were aspirated. Age-matched rats without cochlear ablation were assessed as the non-surgical controls (n=3).
Auditory function was assessed by recording auditory brainstem response (ABR). The measurements of ABR thresholds were performed at frequencies of 4, 8 and 16 kHz after a post-surgical survival time of 5 months.
Coronal frozen sections (40 μm) of brainswere cut using a sliding microtome and processed in three alternating series. One series of sections was stained with cresylviolet, while the other two were analyzed by immunocytochemistry.
The primary antibody was either a mouse monoclonal antibody against CB (Sigma, Saint Louis, Missouri USA; clone CB-955; dilution of 1:12000) or a mouse monoclonal antibody against PV (Sigma, Saint Louis, Missouri USA; clone PA-235; dilution of 1:30000). The secondary antibody was a polymer HRP-labeled anti-mouse antibody(EnVision+ System).
These results suggest that 1) The loss of neurons and the volume reduction in the cochlear nucleus may decrease the input to contralateral MNTB 2) CB and PV were downregurated in the contralateral MNTB, LSO and SPN after neonatal cochlear ablation 3) The reduction of CB and PV in the contralateral MNTB can be directly affected neuronal cell populations in the CN.
The number of indications of cochlear implantation for deaf children has been increasing in recent years. Brain plasticity is a complex property with massive developmental changes after birth. In general, input deprivation in the nervous system causes functional and structural changes through expression of neural plasticity. Despite the many morphological studies that have investigated the changes in the central auditory system caused by deafness before hearing onset, the neurobiological plasticity of congenital deafness remains unknown.
Calbindin D-28k (CB) and parvalbumin(PV) are widely expressed in the brainstem auditory nuclei. CB and PV are calcium-binding proteins (CaBPs) that occur in distinct subpopulations of neurons in the central nervous system. CaBPs are involved in the release of neurotransmitters, the functioning of ion channels in neuronal membranes, and the regulation of enzyme activities at pre- and postsynaptic sites and synaptic short-time plasticity.
In the present study, we investigated the changes in CaBPs expression within the auditory brainstem in an animal model with congenital deafness. Unilateral cochlear ablations before hearing onset have been used as models of congenital deafness.
Introduction
Methods and Materials
Results
Conclusions
Bibliography
Figure 1. Low magnification images illustrating the pattern of cresyl violet staining (A, B), CB immunoreactivity (C, D) and PV immunoreactivity (E, F) in coronal sections of the AVCN in ablated rats. Scale bar = 200 μm in B
Figure 2. Bar graphs showing the mean volume reduction percentage of AVCN, PVCN and DCN compared with the contralateral lesion side. Whiskers on bars are standard errors of the mean.
Figure 3. Low magnification images illustrating the pattern of CB immunoreactivity (A, B) and PV immunoreactivity (C, D) in coronal sections of the LSO and the SPN in ablated rats. Scale bar = 200 μm in B
Figure 4. High magnification images illustrating the pattern of cresyl violet stain (A, B), CB immunoreactivity (C, D) and PV immunoreactivity (E, F) in coronal sections of the MNTB in ablated rats. Scale bar = 100 μm in B
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