Muscarinic receptor-mediated calcium signaling in spiral ganglion neurons of the mammalian cochlea

Using indo-1 microspectrofluorometry, we examined the effects of cholinergic agonists on the concentration of intracellular Ca²⁺ ions ([Ca²⁺]_i) in spiral ganglion neurons, isolated from rat cochleae at different stages of post-natal development (from P3 to P30). Extracellular application of acetylcholine (ACh) or carbamylcholine generated a rapid and transient increase in [Ca²⁺]_i. The ACh concentration-response curve indicated an apparent dissociation constant (K_d) of 8 μM and a Hill coefficient of 1.0. Removing extracellular free Ca²⁺ did not suppress the ACh-induced Ca²⁺ responses suggesting an intracellular Ca²⁺-release mechanism. When we compared the cholinergic response at different stages of postnatal development, there were no significant differences on the aspect of the Ca²⁺ response and the percentage of responsive neurons, which ranged between 50 and 65% per cochlear preparation. The application of muscarine triggered reversible Ca²⁺ responses similar to those observed with ACh, with an apparent K_d of 10 μM and a Hill coefficient of 1.0. The cholinergic-induced Ca²⁺ response was reversibly blocked by muscarinic antagonists with the following order of potency, atropine>4-DAMP>methoctramine>pirenzepine. Nicotine (10 to 100 μM) did not evoke Ca²⁺ responses and the nicotinic antagonist curare (10 μM) did not block the ACh-evoked responses. The present study is the first direct demonstration of functional muscarinic receptors (mAChRs) in spiral ganglion neurons. These mAChRs activated by the cholinergic lateral efferent system may participate in the regulation of the electrical activity of the afferent auditory fibers contacting the inner hair cells.     

Schwann cells revert to non-myelinating phenotypes in the deafened rat cochlea

Loss of sensory hair cells within the cochlea results in a permanent sensorineural hearing loss and initiates the gradual degeneration of spiral ganglion neurons (SGNs) - the primary afferent neurons of the cochlea. While these neurons are normally myelinated via Schwann cells, loss of myelin occurs as a precursor to neural degeneration. However, the relationship between demyelination and the status of Schwann cells in deafness is not well understood. We used a marker of peripheral myelin (myelin protein zero; P0) and a marker of Schwann cells (S100) to determine the temporal sequence of myelin and Schwann cell loss as a function of duration of deafness. Rat pups were systemically deafened for periods ranging from 2 weeks to greater than 6 months by co-administration of frusemide and gentamicin. Cochleae were cryosectioned and quantitative immunohistochemistry used to determine the extent of P0 and S100 labelling within the peripheral processes, SGN soma and their central processes within the modiolus. SGN density was also determined for each cochlear turn. P0 labelling decreased throughout the cochlea with increasing duration of deafness. The reduction in P0 labelling occurred at a faster rate than the SGN loss. In contrast, S100 labelling was not significantly reduced compared with age-matched controls in any cochlear region until 6 months post-deafening. These results suggest that Schwann cells may revert to non-myelinating phenotypes in response to deafness and exhibit greater survival traits than SGNs. The potential clinical significance of these findings for cochlear implants is discussed.     

What's to lose and what's to learn: development under auditory deprivation, cochlear implants and limits of cortical plasticity

Sensory and environmental manipulations affect the development of sensory systems. Higher-order auditory representations (auditory categories or “objects”) evolve with experience and via top–down influences modify representations in early auditory areas. During development of a functional auditory system, the capacity for bottom–up reorganizations is successively less well expressed due to a molecular change in synaptic properties. It is, however, complemented by top–down influences that direct and modulate the residual (adult) capacity for circuit reorganization. In a deprived condition, this developmental step is substantially affected. As higher-order representations cannot be established in absence of auditory experience, the developmental decrease in capacity for “bottom–up regulated” reorganizations (as repeatedly demonstrated in also in deprived sensory systems) cannot be complemented by an increasing influence of top–down modulations. In consequence, the ability to learn is compromised in sensory deprivation, resulting in a sensitive period for recovery.     

Mechanism of electrical stimulation-induced neuroprotection: effects of verapamil on protection of primary auditory afferents

In order to assess the role of L-type voltage-gated calcium channels in electrical stimulation-mediated neuroprotection in vivo, we assessed survival of primary auditory afferents (spiral ganglion cells) in systemically deafened guinea pigs following chronic electrical stimulation with or without intracochlear infusion of verapamil, an L-type voltage-gated calcium channel antagonist. Continuous intracochlear drug delivery (0.5 microl/h) was provided using a delivery system developed previously in our laboratory using Alzet mini-osmotic pumps. In the absence of chronic stimulation, spiral ganglion cell survival was relatively symmetric in animals treated unilaterally with either artificial perilymph or verapamil (50 microg/ml). In the presence of unilateral chronic electrical stimulation, spiral ganglion cell survival was significantly greater in stimulated, perilymph-infused ears, relative to the contralateral ear. In contrast, spiral ganglion cell survival was bilaterally symmetric in chronically stimulated, verapamil-infused animals. The difference in symmetry of spiral ganglion cell survival between the two groups was statistically significant. In vitro, passive depolarization has been demonstrated to enhance survival of cultured neurons via activation of L-type calcium channels. The results of this study indicate that, as suggested by in vitro depolarization models, in vivo electrical stimulation-mediated neuroprotection requires the activation of L-type voltage-gated calcium channels. Chronic electrical stimulation of the deaf ear is an ideal preparation for further studies in which to extrapolate findings from in vitro depolarization models.     

Intracellular responses of the rat anteroventral cochlear nucleus to intracochlear electrical stimulation

The anteroventral cochlear nucleus (AVCN) is the first central processing site for acoustic information. The influence and extent of convergent auditory nerve input to AVCN neurons was investigated using brief (<0.2 ms) intracochlear electrical activation of spiral ganglion cells. In 40 neurons recorded in vivo, the major intracellular response to stimulation was an excitatory postsynaptic potential (EPSP) with short latency (∼1 ms) and fast rise time (<1 ms). Graduated EPSP amplitude increases were also seen with increasing stimulation strength resulting in spike generation. Hyperpolarization followed excitation in most neurons, its extent distinguished three response types: Type I showed no hyperpolarization; Type II and Type III displayed short (<10 ms) and long (>19 ms) duration hyperpolarization, respectively. Hyperpolarization was attributed to an inhibitory postsynaptic potential (IPSP) in addition to spike after hyperpolarization. Neurobiotin filling identified Type I and II neurons as stellate and Type III as bushy cells. These results suggests that AVCN neurons receive direct, possibly convergent, excitatory input from auditory nerves emanating from spiral ganglion cells with hyperpolarization resulting from polysynaptic inhibitory input.     

The cellular origin of corticofugal projections to the superior olivary complex in the rat

Corticofugal pathways originating in auditory cortex innervate most subcortical auditory nuclei in the ascending pathway [Auditory Neurosci. 1 (1995) 287-308; J. Comp. Neurol. 371 (1996) 15-40]. Our goal is to determine if these projections arise from the same neurons or if different neurons project to each of the separate structures. We also seek to identify the layers and fields of auditory cortex from which these neurons originate. In the present study, we answer these questions with respect to the projections to the superior olivary complex (SOC). Fluorescent retrograde tracers, Fast Blue (FB) or Diamidino Yellow (DiY), were injected into the SOC and the pattern of labeled cells was determined in temporal neocortex. We also injected FB into the granule cell domain (GCD) of the cochlear nucleus. Cortical projections to the GCD derive exclusively from layer V pyramidal cells in primary auditory cortex [Brain Res. 706 (1996) 97-102]. Thus the pattern of labeling produced by injections in the GCD provided a reference for interpreting the labeling after SOC injections. Layer V pyramidal cells project to the SOC, and these neurons were distributed bilaterally in primary and secondary areas of auditory cortex. The projections to the SOC from primary auditory cortex are predominantly uncrossed, whereas those from secondary auditory cortex are nearly equal for the two hemispheres. In animals that received injections of FB in the GCD and DiY in the SOC, cells labeled by each injection had a different laminar distribution and very few cells were double labeled. These data suggest that the cortical pathways ending in the cochlear nucleus and SOC are largely independent. We discuss the implications of these findings with respect to the multifunctional nature of the SOC in brainstem auditory processing.     

Absence of hearing loss in a mouse model for DFNA17 and MYH9-related disease: the use of public gene-targeted ES cell resources

Multiple mouse embryonic stem (ES) cell banks expand the capability to characterize functions of genes implicated in human disease and to develop mouse models for the further understanding of disease pathology. Genetic diseases that result in hearing loss can provide insight into causative molecular mechanisms for deafness. We utilized BayGenomics, the public mouse ES cell bank, to identify gene-trapped ES cell lines associated with hearing loss. We identified two gene-trapped ES cell lines specific for the non-muscle myosin heavy chain class IIA or myosin heavy chain IX (Myh9). Inherited mutations in the Myh9 gene have been linked to non-syndromic hereditary hearing impairment DFNA17 as well as 'MYH9-related disease' characterized by macrothrombocytopenia, leukocyte inclusions, and in some patients deafness. Mutant Myh9 mice were derived from one of these ES cell lines that underwent germline transmission for in-depth otological examination. No homozygous mice however were identified at birth, consistent with recently published data describing the embryonic lethality of homozygous mutations in Myh9. We provide evidence that adult heterozygous Myh9 mouse inner ears contain half wild-type levels of Myh9 mRNA. Hearing loss however was not observed in heterozygous Myh9 mice in contrast to human Myh9-related diseases. Aged heterozygous Myh9 mice also did not show signs of cochleosaccular degeneration common in DFNA17. Although inheritance of Myh9 mutations in humans is dominant, we conclude that heterozygous loss of Myh9 is not critical to hearing function in mice by itself.     

Cell transplantation to the auditory nerve and cochlear duct

We have developed a technique to deliver cells to the inner ear without injuring the membranes that seal the endolymphatic and perilymphatic chambers. The integrity of these membranes is essential for normal hearing, and the technique should significantly reduce surgical trauma during cell transplantation. Embryonic stem cells transplanted at the internal auditory meatal portion of an atrophic auditory nerve migrated extensively along it. Four-five weeks after transplantation, the cells were found not only throughout the auditory nerve, but also in Rosenthal's canal and the scala media, the most distal portion of the auditory nervous system where the hair cells reside. Migration of the transplanted cells was more extensive following damage to the auditory nerve. In the undamaged nerve, migration was more limited, but the cells showed more signs of neuronal differentiation. This highlights an important balance between tissue damage and the potential for repair.     

Morphological evidence for supporting cell to hair cell conversion in the mammalian utricular macula

The possible origin of the immature hair cells that appear in the utricular maculae of guinea pigs following gentamicin-induced hair cell death was investigated. Guinea pigs were continuously infused with bromodeoxyuridine, to label proliferating cells and their progeny, for 2 weeks after inducing damage to the inner ear on one side with gentamicin. The opposite ear in each animal served as control. Serial sections were cut through the entire utricular maculae of both ears of each animal and the number of labelled cells in the epithelium and underlying connective tissue was counted. Label was present in cells in the sensory epithelium in the utricles from the drug exposed ears but not in the controls. The nuclei of cells in the underlying connective tissue were also labelled in both ears. Some of the labelled nuclei in the epithelium were at the level normally occupied by hair cells, but most were at the level of supporting cell nuclei. However, the total number of labelled nuclei in the sensory epithelium was small; the maximum was 12 in one animal. The number of labelled nuclei in the connective tissue of the treated ears was significantly greater than the number in the untreated ear. This confirms that cell proliferation is stimulated in the mature mammalian utricular macula after hair cell loss, but the extent to which it occurs appears to be insufficient to explain the recovery in hair cell numbers which is observed. Detailed thin section studies of the utricular maculae of gentamicin-treated animals over a prolonged post-treatment period were also performed. In utricles which had suffered damage, there were cells which, like supporting cells but unlike hair cells, were resting on basement membrane, but which possessed at their apical ends organized bundles of microvilli similar to immature hair cell stereocilia. Other cells with more obvious stereocilia remained in contact with the basement membrane via and a small foot process. In still other cells, where a stereociliary bundle was obvious and almost mature in appearance, there was a foot process extending towards the basement membrane but not quite in contact, suggesting it had just detached. All these cells were contacted by nerve endings and specialization of the membranes were apparent at the site of cell-neurone contact. The morphological characteristics of these cells are consistent with phenotypic conversion of supporting cells into hair cells and this may account for some of the hair cell production in the mature mammalian vestibular sensory epithelia after hair cell death.     

Psychological therapies for bipolar disorder: the role of model-driven approaches to therapy integration

OBJECTIVES: The psychological and social aspects of bipolar disorder are receiving increasing recognition. Recently, there have been promising developments in psychological interventions, but there is scope for further improvement of therapeutic outcomes. This paper argues for the use of more detailed psychological models of bipolar disorder to inform the further development of therapeutic approaches. METHOD: Evidence for psychological, family and social factors in bipolar disorder is reviewed. The efficacy of current individual and family interventions are discussed. A model, which has potential to synthesize group and individual approaches, is outlined. RESULTS: Psychological, social and family factors have important influences on the onset, course and outcome of bipolar disorder. Interventions based on vulnerability stress models have proved effective. However, to enhance efficacy future developments need to be based on models that integrate current understandings of the multiple levels at which mood fluctuations occur. A particular recent model is discussed which leads to specific proposals for future intervention research. CONCLUSIONS: Psychological and family approaches to BD have much potential. They clearly have a role in conjunction with appropriate pharmacological treatment. If this potential is to be fully realized future developments need to be based on psychological models that can accommodate the complexity of this illness.     

Evidence for NMDA receptor in the afferent synaptic transmission of the vestibular system

This study aimed to define the pharmacology and physiological role of the N-methyl-d-aspartate (NMDA) receptor in the synapse between the hair cells and primary afferent neurons in the vestibular system. The spontaneous and mechanically evoked spike discharges of vestibular nerve fibers were extracellularly recorded in isolated inner ear from the axolotl (Ambystoma tigrinum). Pressure ejection of NMDA (10⁻⁶ to 10⁻³ M) elicited a dose-dependent increase of the basal spike discharge from the vestibular nerve fibers. Extracellular magnesium antagonized the NMDA effect in a dose-dependent manner.d(-)-2-amino-5-phosphonovaleric acid (AP5, 10⁻⁵ to 10⁻³ M) and 7-chloro-kynurenic acid (7ClKyn, 10⁻⁶ to 10⁻³ M) inhibited the basal activity of the vestibular nerve fibers. 7ClKyn also diminished the responses elicited by the mechanical stimulation of the preparation. Glycine (10⁻⁹ to 10⁻⁶ M) applied by bath substitution enhanced the NMDA responses, and the glycine agonistd-serine partially reversed the 7ClKyn inhibitory action. These results suggest that NMDA receptors participate in the generation of the basal spike discharge of vestibular system primary afferent neurons, but its activation is not critical for the response to brief mechanical stimuli.     

Modification of the brain-derived neurotrophic factor gene: a portal to transform mesenchymal stem cells into advantageous engineering cells for neuroregeneration and neuroprotection

Multipotential mesenchymal stem cells (MSCs) are ideal seed cells for recruiting the loss of neural cells due to their strong proliferative capacity, easy acquisition, and considerable tolerance of genetic modifications. After transduction of brain-derived neurotrophic factor (BDNF) gene via recombinant retroviral vectors into the human MSCs, nearly 100% of cells expressed BDNF (which were therefore transformed into BNDF-MSCs) as detected by immunocytochemistry, and the quantity of BDNF in the culture medium was increased by approximately 20,000-fold. In spite of the genomic integration of an exogenous gene, BDNF-MSCs did not present any structural aberration in the chromosomes. All-trans-retinoic acid (RA) induction caused the BDNF-MSCs to differentiate into neural cells with significantly increased expressions of such neural-specific proteins as nestin, NeuN, O4, and glial fibrillary acidic protein (GFAP). The voltage-dependent K+/Ca2+ currents were recorded from the induced BDNF-MSCs using patch-clamp technique. Compared with the MSCs induced by both RA and BDNF, BDNF-MSCs survived in significantly greater number in the induction medium, and also more cells were induced into neuron-like cells (NeuN, P < 0.01) and oligodendrocyte-like cells (O4, P < 0.05). We suppose that, once engrafted into human central nervous system, the BDNF-MSCs would not only recruit the neuronal losses, but also provide, by way of paracrine, large quantities of BDNF that effectively perform the functions of neuroprotection and neuroregeneration, promoting the activation of endogenous neural stem/progenitor cells and their chemotactic migration. On the other hand, the BDNF-MSCs that can survive in the host environment and differentiate subsequently into functional mature cells may also serve as specifically targeting vectors for ex vivo gene therapy.     

Targeted Intraparenchymal Delivery of Human NGF by Gene Transfer to the Primate Basal Forebrain for 3 Months Does Not Accelerate β-Amyloid Plaque Deposition

Nerve growth factor therapy has been proposed as a potential means of preventing degeneration of basal forebrain cholinergic neurons in Alzheimer's disease and thereby improving cognition. However, NGF has been reported to upregulate expression of the β-amyloid precursor protein, which in turn could accelerate deposition of “mature” β-amyloid in the brain. To address this possibility, the brains of 16 adult and aged rhesus monkeys were examined for β-amyloid plaque deposition in the presence or absence of NGF treatment. Six aged monkeys received intraparenchymal grafts into the cholinergic basal forebrain of autologous cells genetically modified to secrete NGF, six aged monkeys received intraparenchymal grafts of autologous control cells expressing the reporter gene β-galactosidase, and four adult nonoperated monkeys served as additional controls. All brains were examined for expression of mature β-amyloid using an antibody recognizing amino acids 1–40 of the β-amyloid peptide. Amyloid plaques were systematically quantified in representative sections of the temporal, frontal, cingulate, insular, and parietal cortices and in the amygdala and hippocampus. Results disclosed that aging resulted in an increase in amyloid plaque formation: no plaques at all were detected in nonaged monkeys, whereas a mean of 20 ± 13 plaques per section were present in control-aged monkeys. Aged subjects with intraparenchymal NGF-secreting grafts for 3 months contained a mean of 29 ± 14 plaques per section, an amount that did not differ significantly from control-aged monkeys (P = 0.66). Thus, 3 months of intraparenchymal NGF delivery did not significantly increase β-amyloid deposition.     

Analysis of connexin expression during mouse Schwann cell development identifies connexin29 as a novel marker for the transition of neural crest to precursor cells

Connexins are transmembrane proteins forming gap junction channels for direct intercellular and, for example in myelinating glia cells, intracellular communication. In mature myelin-forming Schwann cells, expression of multiple connexins, i.e. connexin (Cx) 43, Cx29, Cx32, and Cx46 (after nerve injury) has been detected. However, little is known about connexin protein expression during Schwann cell development. Here we use histochemical methods on wildtype and Cx29lacZ transgenic mice to investigate the developmental expression of connexins in the Schwann cell lineage. Our data demonstrate that in the mouse Cx43, Cx29, and Cx32 protein expression is activated in a developmental sequence that is clearly correlated with major developmental steps in the lineage. Only Cx43 was expressed from neural crest cells onwards. Cx29 protein expression was absent from neural crest cells but appeared as neural crest cells generated precursors (embryonic day 12) both in vivo and in vitro. This identifies Cx29 as a novel marker for cells of the defined Schwann cell lineage. The only exception to this were dorsal roots, where the expression of Cx29 was delayed four days relative to ventral roots and spinal nerves. Expression of Cx32 commenced postnatally, coinciding with the onset of myelination. Thus, the coordinated expression of connexin proteins in cells of the embryonic and postnatal Schwann cell lineage might point to a potential role in peripheral nerve development and maturation.