Comparing the effects of transcranial alternating current and temporal interference (tTIS) electric stimulation through whole-brain mapping of c-Fos immunoreactivity.

The analysis of the topography of brain neuromodulation following transcranial alternating current (AC) stimulation is relevant for defining strategies directed to specific nuclei stimulation in patients. Among the different procedures of AC stimulation, temporal interference (tTIS) is a novel method for non-invasive neuromodulation of specific deep brain targets. However, little information is currently available about its tissue effects and its activation topography in in vivo animal models. After a single session (30 min, 0.12 mA) of transcranial alternate current (2,000 Hz; ES/AC group) or tTIS (2,000/2,010 Hz; Es/tTIS group) stimulation, rat brains were explored by whole-brain mapping analysis of c-Fos immunostained serial sections. For this analysis, we used two mapping methods, namely density-to-color processed channels (independent component analysis (ICA) and graphical representation (MATLAB) of morphometrical and densitometrical values obtained by density threshold segmentation. In addition, to assess tissue effects, alternate serial sections were stained for glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), and Nissl. AC stimulation induced a mild superficial increase in c-Fos immunoreactivity. However, tTIS stimulation globally decreased the number of c-Fos-positive neurons and increased blood brain barrier cell immunoreactivity. tTIS also had a stronger effect around the electrode placement area and preserved neuronal activation better in restricted areas of the deep brain (directional stimulation). The enhanced activation of intramural blood vessels' cells and perivascular astrocytes suggests that low-frequency interference (10 Hz) may also have a trophic effect.

A new and very spiny lizard (Gymnophthalmidae: Echinosaura) from the Andes in northwestern Ecuador.

We describe a new species of Neotropical spiny-lizard of the genus Echinosaura from the Imbabura and Carchi Provinces on the western slopes of the Andes in northwestern Ecuador. The new species mostly resembles E. horrida. However, it can be distinguished from all congeners by having keeled enlarged dorsal scales forming a paired vertebral row, two paravertebral series of short oblique rows of projecting scales, and a pair of spine-like scales on temporal and nuchal regions. We also provide a detailed description of the osteology of the skull and pectoral girdle of the new species and present a phylogenetic hypothesis for Echinosaura based on three mitochondrial genes (12S, 16S, ND4) and one nuclear gene (c-mos).

Effects of Multisession Anodal Electrical Stimulation of the Auditory Cortex on Temporary Noise-Induced Hearing Loss in the Rat.

The protective effect of the efferent system against acoustic trauma (AT) has been shown by several experimental approaches, including damage to one ear, sectioning of the olivocochlear bundle (OCB) in the floor of the IV ventricle, and knock-in mice overexpressing outer hair cell (OHC) cholinergic receptors, among others. Such effects have been related to changes in the regulation of the cholinergic efferent system and in cochlear amplification, which ultimately reverse upon protective hearing suppression. In addition to well-known circuits of the brainstem, the descending corticofugal pathway also regulates efferent neurons of the olivary complex. In this study, we applied our recently developed experimental paradigm of multiple sessions of electrical stimulation (ES) to activate the efferent system in combination with noise overstimulation. ABR thresholds increased 1 and 2 days after AT (8-16 kHz bandpass noise at 107 dB for 90 min) recovering at AT + 14 days. However, after multiple sessions of epidural anodal stimulation, no changes in thresholds were observed following AT. Although an inflammatory response was also observed 1 day after AT in both groups, the counts of reactive macrophages in both experimental conditions suggest decreased inflammation in the epidural stimulation group. Quantitative immunocytochemistry for choline acetyltransferase (ChAT) showed a significant decrease in the size and optical density of the efferent terminals 1 day after AT and a rebound at 14 days, suggesting depletion of the terminals followed by a long-term compensatory response. Such a synthesis recovery was significantly higher upon cortical stimulation. No significant correlation was found between ChAT optical density and size of the buttons in sham controls (SC) and ES/AT + 1day animals; however, significant negative correlations were shown in all other experimental conditions. Therefore, our comparative analysis suggests that cochleotopic cholinergic neurotransmission is also better preserved after multisession epidural stimulation.

Expression and Localization of Kv1.1 and Kv3.1b Potassium Channels in the Cochlear Nucleus and Inferior Colliculus after Long-Term Auditory Deafferentation.

Deafness affects the expression and distribution of voltage-dependent potassium channels (Kvs) of central auditory neurons in the short-term, i.e., hours to days, but the consequences in the expression of Kvs after long-term deafness remain unknown. We tested expression and distribution of Kv1.1 and Kv3.1b, key for auditory processing, in the rat cochlear nucleus (CN), and in the inferior colliculus (IC), at 1, 15 and 90 days after mechanical lesion of the cochlea, using a combination of qRT-PCR and Western blot in the whole CN, along with semi-quantitative immunocytochemistry in the AVCN, where the role of both Kvs in excitability control for accurate auditory timing signal processing is well established. Neither Kv1.1/Kv3.1b mRNA or protein expression changed significantly in the CN between 1 and 15 days after deafness. At 90 days post-lesion, however, mRNA and protein expression for both Kvs increased, suggesting that expression regulation of Kv1.1 and Kv3.1b is part of cellular mechanisms for long-term adaptation to auditory input deprivation in the CN. Consistent with these findings, immunocytochemical localization showed increased labeling intensity for both Kvs in the AVCN at day 90 after cochlear lesion, further supporting that up-regulation of Kv1.1 and Kv3.1b in neurons of this CN division, over a long term after auditory deprivation, may be required to adapt intrinsic excitability to altered input. Contrary to findings in the CN, in the IC, expression levels of Kv1.1 and Kv3.1b did not undergo major changes after cochlear lesion. In particular, there was no evidence of long-term up-regulation of neither Kv1.1 or Kv3.1b, supporting that such post-lesion adaptive mechanism may not be needed in the IC. This suggests that post-lesion plastic adaptations to auditory input deprivation are not stereotypical along the auditory pathway.

Reversible Functional Changes Evoked by Anodal Epidural Direct Current Electrical Stimulation of the Rat Auditory Cortex.

Rat auditory cortex was subjected to 0.1 mA anodal direct current in seven 10-min sessions on alternate days. Based on the well-known auditory cortex control of olivocochlear regulation through corticofugal projections, auditory brainstem responses (ABRs) were recorded as an indirect test of the effectiveness and reversibility of the multisession protocol of epidural stimulation. Increases of 20-30 dB ABR auditory thresholds shown after epidural stimulation reverted back to control levels 10 min after a single session. However, increases in thresholds revert 4 days after multisession stimulation. Less changes in wave amplitudes and threshold shifts were shown in ABR recorded contralaterally to the electrically stimulated side of the brain. To assess tissue effects of epidural electric stimulation on the brain cortex, well characterized functional anatomical markers of glial cells (GFAP/astrocytes and Iba1/microglial cells) and neurons (c-Fos) were analyzed in alternate serial sections by quantitative immunocytochemistry. Restricted astroglial and microglial reactivity was observed within the cytoarchitectural limits of the auditory cortex. However, interstitial GFAP overstaining was also observed in the ventricular surface and around blood vessels, thus supporting a potential global electrolytic stimulation of the brain. These results correlate with extensive changes in the distribution of c-Fos immunoreactive neurons among layers along sensory cortices after multisession stimulation. Quantitative immunocytochemical analysis supported this idea by showing a significant increase in the number of positive neurons in supragranular layers and a decrease in layer 6 with no quantitative changes detected in layer 5. Our data indicate that epidural stimulation of the auditory cortex induces a reversible decrease in hearing sensitivity due to local, restricted epidural stimulation. A global plastic response of the sensory cortices, also reported here, may be related to electrolytic effects of electric currents.

Stereotactically-guided Ablation of the Rat Auditory Cortex, and Localization of the Lesion in the Brain.

The rat auditory cortex (AC) is becoming popular among auditory neuroscience investigators who are interested in experience-dependence plasticity, auditory perceptual processes, and cortical control of sound processing in the subcortical auditory nuclei. To address new challenges, a procedure to accurately locate and surgically expose the auditory cortex would expedite this research effort. Stereotactic neurosurgery is routinely used in pre-clinical research in animal models to engraft a needle or electrode at a pre-defined location within the auditory cortex. In the following protocol, we use stereotactic methods in a novel way. We identify four coordinate points over the surface of the temporal bone of the rat to define a window that, once opened, accurately exposes both the primary (A1) and secondary (Dorsal and Ventral) cortices of the AC. Using this method, we then perform a surgical ablation of the AC. After such a manipulation is performed, it is necessary to assess the localization, size, and extension of the lesions made in the cortex. Thus, we also describe a method to easily locate the AC ablation postmortem using a coordinate map constructed by transferring the cytoarchitectural limits of the AC to the surface of the brain.The combination of the stereotactically-guided location and ablation of the AC with the localization of the injured area in a coordinate map postmortem facilitates the validation of information obtained from the animal, and leads to a better analysis and comprehension of the data.

Ablation of the auditory cortex results in changes in the expression of neurotransmission-related mRNAs in the cochlea.

The auditory cortex (AC) dynamically regulates responses of the Organ of Corti to sound through descending connections to both the medial (MOC) and lateral (LOC) olivocochlear efferent systems. We have recently provided evidence that AC has a reinforcement role in the responses to sound of the auditory brainstem nuclei. In a molecular level, we have shown that descending inputs from AC are needed to regulate the expression of molecules involved in outer hair cell (OHC) electromotility control, such as prestin and the α10 nicotinic acetylcholine receptor (nAchR). In this report, we show that descending connections from AC to olivocochlear neurons are necessary to regulate the expression of molecules involved in cochlear afferent signaling. RT-qPCR was performed in rats at 1, 7 and 15 days after unilateral ablation of the AC, and analyzed the time course changes in gene transcripts involved in neurotransmission at the first auditory synapse. This included the glutamate metabolism enzyme glutamate decarboxylase 1 (glud1) and AMPA glutamate receptor subunits GluA2-4. In addition, gene transcripts involved in efferent regulation of type I spiral ganglion neuron (SGN) excitability mediated by LOC, such as the α7 nAchR, the D2 dopamine receptor, and the α1, and γ2 GABAA receptor subunits, were also investigated. Unilateral AC ablation induced up-regulation of GluA3 receptor subunit transcripts, whereas both GluA2 and GluA4 mRNA receptors were down-regulated already at 1 day after the ablation. Unilateral removal of the AC also resulted in up-regulation of the transcripts for α7 nAchR subunit, D2 dopamine receptor, and α1 GABAA receptor subunit at 1 day after the ablation. Fifteen days after the injury, AC ablations induced an up-regulation of glud1 transcripts.

Neurotología e implantación coclear / Neurotology and cochlear implants

En esta revisión se analiza la implantación coclear en relación con los fundamentos del funcionamiento del sistema auditivo. Se revisan los conceptos sobre plasticidad neuronal aplicados a la estimulación eléctrica en la hipoacusia profunda perinatal y del adulto, y se comentan las bases científicas actuales que justifican la implantación precoz tras el cribado de la sordera congénita. Finalmente, se propone esta revisión como un ejemplo de la importancia de fomentar la subespecialidad de neurotología en nuestro medio, a fin de tender puentes entre especialidades que mejoren tanto el conocimiento en el campo de la investigación de la patología auditiva como en el cuidado de los pacientes. Los objetivos de esta revisión, dirigida sobre todo a especialistas del campo de la otorrinolaringología, son los de analizar algunos fundamentos neurológicos de relevancia para comprender mejor los eventos clínicos que condicionan las indicaciones y la rehabilitación de los pacientes con implantes cocleares, así como estimular por este medio la potenciación de la subespecialidad en neurotología (AU)

In this review we analyse cochlear implantation in terms of the fundamental aspects of the functioning of the auditory system. Concepts concerning neuronal plasticity applied to electrical stimulation in perinatal and adult deep hypoacusis are reviewed, and the latest scientific bases that justify early implantation following screening for congenital deafness are discussed. Finally, this review aims to serve as an example of the importance of fostering the sub-specialty of neurotology in our milieu, with the aim of bridging some of the gaps between specialties and thus improving both the knowledge in the field of research on auditory pathologies and in the screening of patients. The objectives of this review, targeted above all towards specialists in the field of otorhinolaryngology, are to analyse some significant neurological foundations in order to reach a better understanding of the clinical events that condition the indications and the rehabilitation of patients with cochlear implants, as well as to use this means to foster the growth of the sub-specialty of neurotology (AU)

[Neurotology and cochlear implants]. / Neurotologia e implantacion coclear.

In this review we analyse cochlear implantation in terms of the fundamental aspects of the functioning of the auditory system. Concepts concerning neuronal plasticity applied to electrical stimulation in perinatal and adult deep hypoacusis are reviewed, and the latest scientific bases that justify early implantation following screening for congenital deafness are discussed. Finally, this review aims to serve as an example of the importance of fostering the sub-specialty of neurotology in our milieu, with the aim of bridging some of the gaps between specialties and thus improving both the knowledge in the field of research on auditory pathologies and in the screening of patients. The objectives of this review, targeted above all towards specialists in the field of otorhinolaryngology, are to analyse some significant neurological foundations in order to reach a better understanding of the clinical events that condition the indications and the rehabilitation of patients with cochlear implants, as well as to use this means to foster the growth of the sub-specialty of neurotology.

TITLE: Neurotologia e implantacion coclear.En esta revision se analiza la implantacion coclear en relacion con los fundamentos del funcionamiento del sistema auditivo. Se revisan los conceptos sobre plasticidad neuronal aplicados a la estimulacion electrica en la hipoacusia profunda perinatal y del adulto, y se comentan las bases cientificas actuales que justifican la implantacion precoz tras el cribado de la sordera congenita. Finalmente, se propone esta revision como un ejemplo de la importancia de fomentar la subespecialidad de neurotologia en nuestro medio, a fin de tender puentes entre especialidades que mejoren tanto el conocimiento en el campo de la investigacion de la patologia auditiva como en el cuidado de los pacientes. Los objetivos de esta revision, dirigida sobre todo a especialistas del campo de la otorrinolaringologia, son los de analizar algunos fundamentos neurologicos de relevancia para comprender mejor los eventos clinicos que condicionan las indicaciones y la rehabilitacion de los pacientes con implantes cocleares, asi como estimular por este medio la potenciacion de la subespecialidad en neurotologia.

Acoustic input and efferent activity regulate the expression of molecules involved in cochlear micromechanics.

Electromotile activity in auditory outer hair cells (OHCs) is essential for sound amplification. It relies on the highly specialized membrane motor protein prestin, and its interactions with the cytoskeleton. It is believed that the expression of prestin and related molecules involved in OHC electromotility may be dynamically regulated by signals from the acoustic environment. However little is known about the nature of such signals and how they affect the expression of molecules involved in electromotility in OHCs. We show evidence that prestin oligomerization is regulated, both at short and relatively long term, by acoustic input and descending efferent activity originating in the cortex, likely acting in concert. Unilateral removal of the middle ear ossicular chain reduces levels of trimeric prestin, particularly in the cochlea from the side of the lesion, whereas monomeric and dimeric forms are maintained or even increased in particular in the contralateral side, as shown in Western blots. Unilateral removal of the auditory cortex (AC), which likely causes an imbalance in descending efferent activity on the cochlea, also reduces levels of trimeric and tetrameric forms of prestin in the side ipsilateral to the lesion, whereas in the contralateral side prestin remains unaffected, or even increased in the case of trimeric and tetrameric forms. As far as efferent inputs are concerned, unilateral ablation of the AC up-regulates the expression of α10 nicotinic Ach receptor (nAChR) transcripts in the cochlea, as shown by RT-Quantitative real-time PCR (qPCR). This suggests that homeostatic synaptic scaling mechanisms may be involved in dynamically regulating OHC electromotility by medial olivocochlear efferents. Limited, unbalanced efferent activity after unilateral AC removal, also affects prestin and ß-actin mRNA levels. These findings support that the concerted action of acoustic and efferent inputs to the cochlea is needed to regulate the expression of major molecules involved in OHC electromotility, both at the transcriptional and posttranscriptional levels.

Long-term evolution of brainstem electrical evoked responses to sound after restricted ablation of the auditory cortex.

INTRODUCTION: This study aimed to assess the top-down control of sound processing in the auditory brainstem of rats. Short latency evoked responses were analyzed after unilateral or bilateral ablation of auditory cortex. This experimental paradigm was also used towards analyzing the long-term evolution of post-lesion plasticity in the auditory system and its ability to self-repair. METHOD: Auditory cortex lesions were performed in rats by stereotactically guided fine-needle aspiration of the cerebrocortical surface. Auditory Brainstem Responses (ABR) were recorded at post-surgery day (PSD) 1, 7, 15 and 30. Recordings were performed under closed-field conditions, using click trains at different sound intensity levels, followed by statistical analysis of threshold values and ABR amplitude and latency variables. Subsequently, brains were sectioned and immunostained for GAD and parvalbumin to assess the location and extent of lesions accurately. RESULTS: Alterations in ABR variables depended on the type of lesion and post-surgery time of ABR recordings. Accordingly, bilateral ablations caused a statistically significant increase in thresholds at PSD1 and 7 and a decrease in waves amplitudes at PSD1 that recover at PSD7. No effects on latency were noted at PSD1 and 7, whilst recordings at PSD15 and 30 showed statistically significant decreases in latency. Conversely, unilateral ablations had no effect on auditory thresholds or latencies, while wave amplitudes only decreased at PSD1 strictly in the ipsilateral ear. CONCLUSION: Post-lesion plasticity in the auditory system acts in two time periods: short-term period of decreased sound sensitivity (until PSD7), most likely resulting from axonal degeneration; and a long-term period (up to PSD7), with changes in latency responses and recovery of thresholds and amplitudes values. The cerebral cortex may have a net positive gain on the auditory pathway response to sound.

Cortical Auditory Deafferentation Induces Long-Term Plasticity in the Inferior Colliculus of Adult Rats: Microarray and qPCR Analysis.

The cortico-collicular pathway is a bilateral excitatory projection from the cortex to the inferior colliculus (IC). It is asymmetric and predominantly ipsilateral. Using microarrays and RT-qPCR we analyzed changes in gene expression in the IC after unilateral lesions of the auditory cortex, comparing the ICs ipsi- and contralateral to the lesioned side. At 15 days after surgery there were mainly changes in gene expression in the IC ipsilateral to the lesion. Regulation primarily involved inflammatory cascade genes, suggesting a direct effect of degeneration rather than a neuronal plastic reorganization. Ninety days after the cortical lesion the ipsilateral IC showed a significant up-regulation of genes involved in apoptosis and axonal regeneration combined with a down-regulation of genes involved in neurotransmission, synaptic growth, and gap junction assembly. In contrast, the contralateral IC at 90 days post-lesion showed an up-regulation in genes primarily related to neurotransmission, cell proliferation, and synaptic growth. There was also a down-regulation in autophagy and neuroprotection genes. These findings suggest that the reorganization in the IC after descending pathway deafferentation is a long-term process involving extensive changes in gene expression regulation. Regulated genes are involved in many different neuronal functions, and the number and gene rearrangement profile seems to depend on the density of loss of the auditory cortical inputs.

Transient Down-Regulation of Sound-Induced c-Fos Protein Expression in the Inferior Colliculus after Ablation of the Auditory Cortex.

We tested whether lesions of the excitatory glutamatergic projection from the auditory cortex (AC) to the inferior colliculus (IC) induce plastic changes in neurons of this nucleus. Changes in neuronal activation in the IC deprived unilaterally of the cortico-collicular projection were assessed by quantitative c-Fos immunocytochemistry. Densitometry and stereology measures of sound-induced c-Fos immunoreactivity in the IC showed diminished labeling at 1, 15, 90, and 180 days after lesions to the AC suggesting protein down-regulation, at least up to 15 days post-lesion. Between 15 and 90 days after the lesion, c-Fos labeling recovers, approaching control values at 180 days. Thus, glutamatergic excitation from the cortex maintains sound-induced activity in neurons of the IC. Subdivisions of this nucleus receiving a higher density of cortical innervation such as the dorsal cortex showed greater changes in c-Fos immunoreactivity, suggesting that the anatomical strength of the projection correlates with effect strength. Therefore, after damage of the corticofugal projection, neurons of the IC down-regulate and further recover sound-induced c-Fos protein expression. This may be part of cellular mechanisms aimed at balancing or adapting neuronal responses to altered synaptic inputs.

Long-term regulation in calretinin staining in the rat inferior colliculus after unilateral auditory cortical ablation.

In this study we analyzed the effects in the inferior colliculus of a unilateral ablation of the auditory cortex in rats. Variations in both calretinin immunoreactivity and protein levels determined by Western blot suggest that such lesions induce changes in the regulation of this calcium-binding protein. Stereological counts of calretinin-immunoreactive neurons in the inferior colliculus 15, 90, and 180 days after the lesion showed a progressive increase in the number of immunoreactive neurons, with a parallel increase in the intensity of staining. Two hundred forty days after the cortical lesion, both the number of immunoreactive neurons and the staining intensity had returned to control values. The effects of the cortical lesion on calretinin regulation are more intense in those inferior colliculus subdivisions more densely innervated by the corticocollicular projection. This finding, along with the time course of calretinin regulation suggests that degeneration of the descending projection is linked to calretinin regulation in the inferior colliculus. We hypothesize, based on the role of calretinin, that the observed increase in immunoreactivity levels seen in the inferior colliculus after lesioning of the auditory cortex may be related to altered excitability in deafferented neurons. Our finding, may reflect adaptive mechanisms to changes in calcium influx and excitability in inferior colliculus neurons induced by lesions of the descending projection from the cortex to the inferior colliculus.

Long-term functional recovery in the rat auditory system after unilateral auditory cortex ablation.

CONCLUSIONS: An intact bilateral auditory corticofugal projection is necessary for the auditory system, and above all its main targets, to start working correctly after an acoustic stimulation. After restricted unilateral cortical lesions, the auditory system is able to recover its function in adult animals at 90 days after surgery. (Post-lesion plasticity in adults.) OBJECTIVES: To study the influence of the cortex on the auditory system functionality and to asses its ability for post-lesion recovery. METHODS: Restricted unilateral lesions were made in the auditory cortex of adult rats. To evaluate the functionality of the auditory pathway after corticofugal deafferentation, the acoustic startle reflex and prepulse inhibition, together with the auditory brainstem response (ABR), were tested along the survival time. RESULTS: All the three tests showed a decrease in their responses at 15 days post lesion, and a full recovery at 90 days post lesion for the ABR and at 180 days post lesion for the acoustic startle reflex and prepulse inhibition.

Direct projections from cochlear nuclear complex to auditory thalamus in the rat.

It is known that the dorsal cochlear nucleus and medial geniculate body in the auditory system receive significant inputs from somatosensory and visual-motor sources, but the purpose of such inputs is not totally understood. Moreover, a direct connection of these structures has not been demonstrated, because it is generally accepted that the inferior colliculus is an obligatory relay for all ascending input. In the present study, we have used auditory neurophysiology, double labeling with anterograde tracers, and retrograde tracers to investigate the ascending projections of the cochlear nuclear complex. We demonstrate that the dorsal cochlear nucleus and the small cell cap of the ventral cochlear nucleus have a direct projection to the medial division of the medial geniculate body. These direct projections from the cochlear nucleus complex bypass the inferior colliculus and are widely distributed within the medial division of the medial geniculate, suggesting that the projection is not topographic. As a nonlemniscal auditory pathway that parallels the conventional auditory lemniscal pathway, its functions may be distinct from the perception of sound. Because this pathway links the parts of the auditory system with prominent nonauditory, multimodal inputs, it may form a neural network through which nonauditory sensory and visual-motor systems may modulate auditory information processing.