Spiral ganglion neuron degeneration in aminoglycoside-deafened rats involves innate and adaptive immune responses not requiring complement.

Spiral ganglion neurons (SGNs) transmit auditory information from cochlear hair cells to the brain. SGNs are thus not only important for normal hearing, but also for effective functioning of cochlear implants, which stimulate SGNs when hair cells are missing. SGNs slowly degenerate following aminoglycoside-induced hair cell loss, a process thought to involve an immune response.

Cyclic AMP signaling promotes regeneration of cochlear synapses after excitotoxic or noise trauma.

Introduction: Cochlear afferent synapses connecting inner hair cells to spiral ganglion neurons are susceptible to excitotoxic trauma on exposure to loud sound, resulting in a noise-induced cochlear synaptopathy (NICS). Here we assessed the ability of cyclic AMP-dependent protein kinase (PKA) signaling to promote cochlear synapse regeneration, inferred from its ability to promote axon regeneration in axotomized CNS neurons, another system refractory to regeneration.

Methods: We mimicked NICS by applying a glutamate receptor agonist, kainic acid (KA) to organotypic cochlear explant cultures and experimentally manipulated cAMP signaling to determine whether PKA could promote synapse regeneration.

Anti-inflammatory Therapy Protects Spiral Ganglion Neurons After Aminoglycoside Antibiotic-Induced Hair Cell Loss.

Destruction of cochlear hair cells by aminoglycoside antibiotics leads to gradual death of the spiral ganglion neurons (SGNs) that relay auditory information to the brain, potentially limiting the efficacy of cochlear implants. Because the reasons for this cochlear neurodegeneration are unknown, there are no neuroprotective strategies for patients. To investigate this problem, we assessed transcriptomic changes in the rat spiral ganglion following aminoglycoside antibiotic (kanamycin)-induced hair cell destruction.

Protection of cochlear synapses from noise-induced excitotoxic trauma by blockade of Ca-permeable AMPA receptors.

Exposure to loud sound damages the postsynaptic terminals of spiral ganglion neurons (SGNs) on cochlear inner hair cells (IHCs), resulting in loss of synapses, a process termed synaptopathy. Glutamatergic neurotransmission via α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type receptors is required for synaptopathy, and here we identify a possible involvement of GluA2-lacking Ca-permeable AMPA receptors (CP-AMPARs) using IEM-1460, which has been shown to block GluA2-lacking AMPARs. In CBA/CaJ mice, a 2-h exposure to 100-dB sound pressure level octave band (8 to 16 kHz) noise results in no permanent threshold shift but does cause significant synaptopathy and a reduction in auditory brainstem response (ABR) wave-I amplitude.

Postnatal expression of neurotrophic factors accessible to spiral ganglion neurons in the auditory system of adult hearing and deafened rats.

Spiral ganglion neurons (SGNs) receive input from cochlear hair cells and project from the cochlea to the cochlear nucleus. After destruction of hair cells with aminoglycoside antibiotics or noise, SGNs gradually die. It has been assumed that SGN death is attributable to loss of neurotrophic factors (NTFs) derived from hair cells or supporting cells in the organ of Corti (OC).

Intracochlear electrical stimulation suppresses apoptotic signaling in rat spiral ganglion neurons after deafening in vivo.

Objective: To establish the intracellular consequences of electrical stimulation to spiral ganglion neurons after deafferentation. Here we use a rat model to determine the effect of both low and high pulse rate acute electrical stimulation on activation of the proapoptotic transcription factor Jun in deafferented spiral ganglion neurons in vivo.

Study Design: Experimental animal study.

The Trk A, B, C's of neurotrophins in the cochlea.

The spiral ganglion neurons (SGNs) are the afferent neurons of the cochlea, connecting the auditory sensory cells-hair cells-to the brainstem cochlear nuclei. The neurotrophins neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) are expressed in the cochlea and both support SGN survival during development. These neurotrophins remain expressed in the postnatal cochlea and continue to play additional roles for SGNs, contributing to maintenance of hair cell-SGN synapses and regulating expression of ion channels, presynaptic and postsynaptic proteins, and SGN membrane electrical properties in a physiologically important spatial pattern.

Spine formation and maturation in the developing rat auditory cortex.

The rat auditory cortex is organized as a tonotopic map of sound frequency. This map is broadly tuned at birth and is refined during the first 3 weeks postnatal. The structural correlates underlying tonotopic map maturation and reorganization during development are poorly understood.

p75(NTR) expression and nuclear localization of p75(NTR) intracellular domain in spiral ganglion Schwann cells following deafness correlate with cell proliferation.

Spiral ganglion Schwann cells (SGSCs) myelinate spiral ganglion neurons (SGNs) and represent a potential source of neurotrophic support for SGNs. Deafening due to loss of hair cells results in gradual degeneration and death of SGNs. Successful efforts to maintain or regenerate a functional auditory nerve may depend on a healthy population of SGSCs, yet the responses of SGSCs to neural injury remain largely unknown.

A kinase anchor protein 150 (AKAP150)-associated protein kinase A limits dendritic spine density.

The A kinase anchor protein AKAP150 recruits the cAMP-dependent protein kinase (PKA) to dendritic spines. Here we show that in AKAP150 (AKAP5) knock-out (KO) mice frequency of miniature excitatory post-synaptic currents (mEPSC) and inhibitory post-synaptic currents (mIPSC) are elevated at 2 weeks and, more modestly, 4 weeks of age in the hippocampal CA1 area versus litter mate WT mice. Linear spine density and ratio of AMPAR to NMDAR EPSC amplitudes were also increased.

Functional role of neurotrophin-3 in synapse regeneration by spiral ganglion neurons on inner hair cells after excitotoxic trauma in vitro.

Spiral ganglion neurons (SGNs) are postsynaptic to hair cells and project to the brainstem. The inner hair cell (IHC) to SGN synapse is susceptible to glutamate excitotoxicity and to acoustic trauma, with potentially adverse consequences to long-term SGN survival. We used a cochlear explant culture from P6 rat pups consisting of a portion of organ of Corti maintained intact with the corresponding portion of spiral ganglion to investigate excitotoxic damage to IHC-SGN synapses in vitro.

Activity of all JNK isoforms contributes to neurite growth in spiral ganglion neurons.

Jun N-terminal kinase (JNK) is a multifunctional protein kinase crucial for neuronal apoptosis as well as neurite growth. We have previously shown that JNK activity is correlated with spiral ganglion neuron (SGN) apoptosis following hair cell loss in rats (Alam et al., 2007) implying that JNK inhibition may have therapeutic potential to protect SGNs in deaf individuals.

Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1.

Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network.

Role of Ca2+/calmodulin-dependent protein kinase II in dendritic spine remodeling during epileptiform activity in vitro.

Epileptiform activity (EA) in vivo and in vitro induces a loss of dendritic spines and synapses. Because CaMKII has been implicated in synaptogenesis and synaptic plasticity, we investigated the role of CaMKII in the effects of EA on spines, using rat hippocampal slice cultures. To visualize dendrites and postsynaptic densities (PSDs) in pyramidal neurons in the slices, we used biolistic transfection to express either free GFP or a PSD95-YFP construct that specifically labels PSDs.

Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain.

The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites.

CaMKII and CaMKIV mediate distinct prosurvival signaling pathways in response to depolarization in neurons.

By fusing the CaMKII-inhibitory peptide AIP to GFP, we constructed a specific and effective CaMKII inhibitor, GFP-AIP. Expression of GFP-AIP and/or dominant-inhibitory CaMKIV in cultured neonatal rat spiral ganglion neurons (SGNs) shows that CaMKII and CaMKIV act additively and in parallel to mediate the prosurvival effect of depolarization. Depolarization or expression of constitutively active CaMKII functionally inactivates Bad, indicating that this is one means by which CaMKII promotes neuronal survival.

Prosurvival and proapoptotic intracellular signaling in rat spiral ganglion neurons in vivo after the loss of hair cells.

Neurons depend on afferent input for survival. Rats were given daily kanamycin injections from P8 to P16 to destroy hair cells, the sole afferent input to spiral ganglion neurons (SGNs). Most SGNs die over an approximately 14-week period after deafferentation.

Overexpression of Bcl-2 or Bcl-xL prevents spiral ganglion neuron death and inhibits neurite growth.

Spiral ganglion neurons (SGNs) provide afferent innervation to the cochlea and rely on contact with hair cells (HCs) for their survival. Following deafferentation due to hair cell loss, SGNs gradually die. In a rat culture model, we explored the ability of prosurvival members of the Bcl-2 family of proteins to support the survival and neurite outgrowth of SGNs.

Acid-sensing ion channel 1a is a postsynaptic proton receptor that affects the density of dendritic spines.

Extracellular proton concentrations in the brain may be an important signal for neuron function. Proton concentrations change both acutely when synaptic vesicles release their acidic contents into the synaptic cleft and chronically during ischemia and seizures. However, the brain receptors that detect protons and their physiologic importance remain uncertain.

Constitutive neuregulin-1/ErbB signaling contributes to human vestibular schwannoma proliferation.

Vestibular schwannomas (VSs) are benign tumors that arise from the Schwann cells (SCs) lining the vestibular nerve. VS cells survive and proliferate far from neurons and axonally derived growth factors. We have previously shown that VSs produce the glial growth factor, neuregulin-1 (NRG1), and its receptors, ErbB2 and ErbB3.

Synaptic activity and F-actin coordinately regulate CaMKIIalpha localization to dendritic postsynaptic sites in developing hippocampal slices.

We examined the timing and mechanisms of CaMKIIalpha recruitment to nascent synapses of developing rat hippocampal pyramidal neurons in slice culture. Time-lapse confocal imaging shows that GFP-CaMKIIalpha in transfected neurons accumulates in spines as they are forming, and loss of CaMKIIalpha coincides with spine destabilization. Immunolabeling shows that endogenous CaMKIIalpha is concentrated at postsynaptic sites in spines under ambient slice culture conditions, and this is not disrupted by short-term (3 h) synaptic activity blockade or Latrunculin-induced F-actin depolymerization.

Lentiviral transduction of murine oligodendrocytes in vivo.

Lentiviral vectors are used widely to direct efficient gene transfer in vivo. We examined cell-specific expression in adult murine white matter after stereotaxic microinjection of four lentiviral constructs. We synthesized vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviruses with combinations of two promoters, cytomegalovirus (CMV) or myelin basic protein (MBP), and two reporter sequences, cytosolic enhanced green fluorescent protein (eGFP) or a plasma membrane-targeted eGFP (human lymphocyte-specific protein tyrosine kinase [Lck]-eGFP).

Regulation of hippocampal synapse remodeling by epileptiform activity.

We examined the regulation of dendritic spines and synapses by epileptiform activity (EA) in rat hippocampal slice cultures. EA, which was induced by a GABA(A) receptor inhibitor, gabazine, reduced pyramidal neuron spine density by approximately 50% after 48 h and also caused an increase in the average length of remaining spines. To directly determine the effects of EA on synapses, we used fluorescent protein-tagged PSD95, which marks postsynaptic densities.

Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures.

Protoplasmic astrocytes in mammalian CNS tissues in vivo have a highly complex 3D morphology, but in dissociated cell cultures they often assume a flattened, fibroblast-like morphology bearing only a few, simple processes. By fluorescent labeling and confocal reconstruction we show that many astrocytes in organotypic hippocampal slice cultures exhibit a more native complex cytoarchitecture. Although astrocytes at the surface of slice cultures show a reactive form with several thick glial fibrillary acidic protein (GFAP)-positive processes, astrocytes situated in deeper portions of tissue slices retain a highly complex 3D morphology with many fine spine- or veil-like protrusions.

Forskolin, 8-Br-3',5'-cyclic adenosine 5'-monophosphate, and catalytic protein kinase A expression in the nucleus increase radioiodide uptake and sodium/iodide symporter protein levels in RET/PTC1-expressing cells.

RET/PTC1, a thyroid-specific oncogene, has been reported to down-regulate sodium/iodide symporter (NIS) expression and function in vitro and in vivo. Recently, RET/PTC1 has been shown to interfere with TSH signaling at multiple levels in thyroid cells. The objective of this study was to investigate whether RET/PTC1-mediated NIS reduction can be rescued by activating cAMP-protein kinase A (PKA) pathways.