Veronica Fuentes-Santamaria

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. J. Comp. Neurol. 483:458–475, 2005.

Alterations in calretinin immunostaining in the ferret superior olivary complex after cochlear ablation

In this study, we used image analysis to assess changes in calretinin immunoreactivity in the lateral (LSO) and medial (MSO) superior olivary nuclei in ferrets 2 months after unilateral cochlear ablations at 30–40 days of age, soon after hearing onset. These two nuclei are the first significant sites of binaural convergence in the ascending auditory system, and both receive direct projections from the deafferented cochlear nucleus. Cochlear ablation results in a decrease in the overall level of calretinin immunostaining within the LSO ipsilaterally compared with the contralateral side and with control animals and within the MSO bilaterally compared with control ferrets. In addition, the level of calretinin immunostaining ipsilaterally within neurons in the LSO was significantly less in cochlear ablated than control animals. In contrast, there was no effect of cochlear ablation on the level of calretinin immunostaining within neurons either in the contralateral LSO or in the MSO. These results are consistent with a downregulation in calretinin within the neuropil of MSO bilaterally and LSO ipsilaterally, as well as a downregulation in calretinin within somata in the ipsilateral LSO as a result of unilateral cochlear ablation soon after hearing onset. Thus, cochlear‐driven activity appears to affect calcium binding protein levels in both neuropil and neurons within the superior olivary complex. J. Comp. Neurol. 470:63–79, 2004.

Cochlear ablation in adult ferrets results in changes in insulin-like growth factor-1 and synaptophysin immunostaining in the cochlear nucleus

Afferent activity modulates synaptic plasticity as well as the levels of activity-dependent molecules such as growth factors. Disruption of this activity due to deafferentation has been shown to result in an altered trophic support and consequently in changes in neuronal excitability and synaptic transmission. In the present study, to test whether lack of cochlear integrity results in changes in insulin-growth factor-1 (IGF-1) and synaptophysin immunostaining in the cochlear nucleus, the first relay structure in the auditory pathway, unilateral cochlear ablations were performed in adult ferrets. Changes in IGF-1 and synaptophysin immunostaining were assessed in the anteroventral (AVCN), posteroventral (PVCN) and dorsal cochlear nucleus (DCN) at 1, 20 and 90 days after deafferentation. An increase in IGF-1 immunostaining within AVCN, PVCN and DCN was observed ipsilaterally at all survival times after cochlear ablation when compared with the contralateral side and unoperated animals. This increase was accompanied by a significant ipsilateral increase in the mean gray level of synaptophysin immunostaining as well as a decrease in the area of synaptophysin immunostaining at 1 and 20 days after the ablation in AVCN, PVCN and DCN compared with the contralateral side and control animals. These changes in synaptophysin immunostaining were no longer present 90 days after cochlear ablation. The present results provide evidence of a persistent upregulation in IGF-1 and a transitory upregulation in synaptophysin levels in the cochlear nucleus that may reflect neuroprotective mechanisms following the loss of trophic support from spiral ganglion neurons.

Upregulation of calretinin immunostaining in the ferret inferior colliculus after cochlear ablation

In many systems, including ascending auditory pathways, calcium‐binding proteins are markers of specific neuronal circuits. Previous studies suggest that calretinin immunostaining may be a specific marker for circuits in the inferior colliculus (IC) that code timing information. We undertook experiments to determine the changes in calretinin immunostaining in the IC that take place in response to cochlear ablation. Cochlear ablation was performed unilaterally in ferrets just after hearing onset. Animals survived for 2–3 months after ablation and brains were then processed for calretinin immunocytochemistry. The mean optical density and stained area of the calretinin immunopositive plexus in the IC were determined for five coronal sections through the right and left IC. In controls (n = 3), measurements of these parameters in the central nucleus of the IC showed symmetry between the two sides. In experimental animals (n = 8) the calretinin immunopositive plexus contralateral to the cochlear ablation was denser and larger than that in either the ipsilateral IC or in the IC of control animals. The calretinin plexus in the ipsilateral IC was slightly less dense and smaller than in controls but the differences did not reach statistical significance. IC volume measurements and synaptophysin immunostaining analysis in the central nucleus of the IC revealed no statistical differences between ablated and control animals or between the two sides in ablated animals. The significant increase in both mean optical density and immunostained area of the calretinin plexus in the IC contralateral to the cochlear ablation may reflect an upregulation in calretinin expression in the nuclei that contribute to this plexus. J. Comp. Neurol. 460:585–596, 2003.

Axon Morphologies and Convergence Patterns of Projections from Different Sensory-Specific Cortices of the Anterior Ectosylvian Sulcus onto Multisensory Neurons in the Cat Superior Colliculus

Corticofugal projections to the thalamus reveal 2 axonal morphologies, each associated with specific physiological attributes. These determine the functional characteristics of thalamic neurons. It is not clear, however, whether such features characterize the corticofugal projections that mediate multisensory integration in superior colliculus (SC) neurons. The cortico-collicular projections from cat anterior ectosylvian sulcus (AES) are derived from its visual, auditory, and somatosensory representations and are critical for multisensory integration. Following tracer injections into each subdivision, 2 types of cortico-collicular axons were observed. Most were categorized as type I and consisted of small-caliber axons traversing long distances without branching, bearing mainly small boutons. The less frequent type II had thicker axons, more complex branching patterns, larger boutons, and more complex terminal boutons. Following combinatorial injections of 2 different fluorescent tracers into defined AES subdivisions, fibers from each were seen converging onto individual SC neurons and indicate that such convergence, like that in the corticothalamic system, is mediated by 2 distinct morphological types of axon terminals. Nevertheless, and despite the conservation of axonal morphologies across different subcortical systems, it is not yet clear if the concomitant physiological attributes described in the thalamus are directly applicable to multisensory integration.

Unilateral cochlear ablation in adult ferrets results in upregulation in calretinin immunostaining in the central nucleus of the inferior colliculus.

In the present study, unilateral cochlear ablations were performed in adult ferrets in order to determine whether an upregulation of the calretinin immunostained plexus in the central nucleus of the inferior colliculus occurs and if so, what the time course of this upregulation is. Accordingly, the mean gray level and the calretinin-immunostained area of the axonal plexus in the central nucleus of the inferior colliculus were evaluated at 1, 20 and 90 days after cochlear ablation. In unoperated animals, the calretinin-immunostained plexus was bilaterally symmetric. In ablated animals, both the mean gray level and the immunostained area of the plexus increased in the central nucleus of the inferior colliculus contralateral to the lesion compared with both the ipsilateral side and unoperated animals. This upregulation was present 24 h after the ablation and did not change at the two subsequent time points. In a previous study in young ferrets, the immunostained area of the plexus in the central nucleus of the inferior colliculus contralateral to the lesion increased 200% compared with control ferrets [J Comp Neurol 460 (2003) 585], whereas it increased only 33% in adult ferrets. These findings suggest that 1) calretinin upregulation in the contralateral central nucleus of the inferior colliculus following cochlear ablation occurs by 24 h after cochlear ablation and 2) there is an age-related decline in the magnitude of this upregulation after cochlear ablation.

Morphologic and Neurochemical Abnormalities in the Auditory Brainstem of the Genetically Epilepsy‐prone Hamster (GPG/Vall)

Summary:  Purpose: This study was performed to evaluate whether audiogenic seizures, in a strain of genetically epilepsy‐prone hamsters (GPG/Vall), might be associated with morphologic alterations in the cochlea and auditory brainstem. In addition, we used parvalbumin as a marker of neurons with high levels of activity to examine changes within neurons.

Quantitative measurement of afferent layers in the ferret inferior colliculus: DNLL projections to sublayers.

In the central nucleus of the inferior colliculus (IC), afferent projections are aligned with dendritic arbors of disk-shaped cells, forming fibrodendritic layers. One feature that may serve as a guide for study of the intrinsic organization of the IC layers is the segregation of certain inputs to bands and patches within the layers of the central nucleus. In this study, we used Phaseolus leucoagglutinin as an anterograde tracer to examine the projections from the dorsal nucleus of the lateral lemniscus to the contralateral IC in adult ferrets. The labeled afferent projections distributed along the IC layers in a series of bands where there were dense endings and interband spaces where there were few if any endings. Branches of individual labeled axons that were reconstructed distributed within a single afferent band. Measurements of both the terminal density distribution and the optical density across the band were similar indicating that afferent bands were approximately 85 microm thick. Quantitative measurements of the labeled afferent bands will enhance comparison with other afferent projections and analysis of afferent development and plasticity.

Morphologic and neurochemical alterations in the superior colliculus of the genetically epilepsy-prone hamster (GPG/Vall).

The GPG/Vall hamster is an animal model that exhibits seizures in response to sound stimulation. Since the superior colliculus (SC) is implicated in the neuronal network of audiogenic seizures (AGS) in other forms of AGS, this study evaluated seizure-related anatomical or neurochemical abnormalities in the SC of the GPG/Vall hamster. This involved calbindin (CB) and parvalbumin (PV) immunohistochemistry, densitometric analysis and high performance liquid chromatography in the superficial and deep layers of the SC in control and epileptic animals. Compared to control animals, a reduction in SC volume and a hypertrophy of neurons located in the deep layers of the SC were observed in the epileptic hamster. Although, analysis of CB-immunohistochemistry in the superficial layers did not show differences between groups, analysis of PV-immunostaining in the deep SC revealed an increase in the mean gray level within immunostained neurons as well as a decreased immunostained neuropil in the GPG/Vall hamster as compared to control animals. These alterations were accompanied by a decrease in the levels of GABA and increased levels of taurine in the epileptic animal. These data indicate that the deep SC of the GPG/Vall hamster is structurally abnormal; suggesting its involvement in the neuronal network for AGS.

Neurofilament proteins are preferentially expressed in descending output neurons of the cat the superior colliculus: a study using SMI-32.

Physiological studies indicate that the output neurons in the multisensory (i.e. intermediate and deep) laminae of the cat superior colliculus receive converging information from widespread regions of the neuraxis, integrate this information, and then relay the product to regions of the brainstem involved in the control of head and eye movements. Yet, an understanding of the neuroanatomy of these converging afferents has been hampered because many terminals contact distal dendrites that are difficult to label with the neurochemical markers generally used to visualize superior colliculus output neurons. Here we show that the SMI-32 antibody, directed at the non-phosphorylated epitopes of high molecular weight neurofilament proteins, is an effective marker for these superior colliculus output neurons. It is also one that can label their distal dendrites. Superior colliculus sections processed for SMI-32 revealed numerous labeled neurons with varying morphologies within the deep laminae. In contrast, few labeled neurons were observed in the superficial laminae. Neurons with large somata in the lateral aspects of the deep superior colliculus were particularly well labeled, and many of their secondary and tertiary dendrites were clearly visible. Injections of the fluorescent biotinylated dextran amine into the pontine reticular formation revealed that approximately 80% of the SMI-32 immunostained neurons also contained retrogradely transported biotinylated dextran amine, indicating that SMI-32 is a common cytoskeletal component expressed in descending output neurons. Superior colliculus output neurons also are known to express the calcium-binding protein parvalbumin, and many SMI-32 immunostained neurons also proved to be parvalbumin immunostained. These studies suggest that SMI-32 can serve as a useful immunohistochemical marker for detailing the somatic and dendritic morphology of superior colliculus output neurons and for facilitating evaluations of their input/output relationships.