Prenatal cocaine exposure accelerates morphological changes and transient expression of tyrosine hydroxylase in the cochlea of developing rats.

Prenatal cocaine exposure causes alterations in auditory brainstem response in children and experimental animals and has adverse effects on auditory information processing and language skills in children. These effects may result from lesions in the cochlea since this organ is particularly sensitive to chemical insults during the development. We have thus studied here the effect of prenatal cocaine exposure on the maturation of the rat cochlea using the transient non-catecholaminergic expression of tyrosine hydroxylase in spiral ganglion neurons as an index of cochlear maturation and morphometry to evaluate the maturation of primary auditory neurons and the organ of Corti. We showed that prenatal cocaine exposure accelerated the cochlear maturation. In the basal coil of cochleas from PND8 cocaine-treated pups, the Kölliker's organ had disappeared, the tunnel of Corti was opened, and the stria vascularis no longer contained undifferentiated marginal cells. The maximum expression of tyrosine hydroxylase in type I primary auditory neurons occurred at PND8 instead of PND12 in pair-fed controls. On the other hand, the prenatal cocaine exposure had no effect on the width and height of the organ of Corti, spiral ganglion volume and number and size of primary auditory neurons. In conclusion, our data suggest that prenatal cocaine exposure, though not lethal to primary auditory neurons, accelerates aspects of the cochlear sensorineural maturation. This accelerated cochlear maturation in cocaine-treated rat pups could cause auditory dysfunctions by desynchronizing the development of the whole auditory pathway.

Expression of the Opa1 mitochondrial protein in retinal ganglion cells: its downregulation causes aggregation of the mitochondrial network.

PURPOSE: Mutations in the mitochondrial dynamin-related GTPase OPA1 cause autosomal dominant optic atrophy (ADOA), but the pathophysiology of this disease is unknown. As a first step in functional studies, this study was conducted to evaluate the expression of Opa1 in whole retina and in isolated retinal ganglion cells (RGCs) and to test the effects of Opa1 downregulation in cultured RGCs. METHODS: Opa1 mRNA isoforms from total retina and from RGCs freshly isolated by immunopanning were determined by RT-PCR. Protein expression was examined by immunohistochemistry and Western blot with antibodies against Opa1 and cytochrome c, and the mitochondrial network was visualized with a mitochondrial marker. Short interfering (si)RNA targeting OPA1 mRNAs were transfected to cultured RGCs and mitochondrial network phenotypes were followed for 15 days, in comparison with those of cerebellar granule cells (CGCs). RESULTS: Opa1 expression did not predominate in rat postnatal RGCs as found by immunohistochemistry and Western blot analysis. The pattern of mRNA isoforms was similar in whole retina and RGCs. After a few days in culture, isolated RGCs showed fine mitochondrial punctiform structures in the soma and neurites that colocalized with cytochrome c and Opa1. Opa1 knockdown in RGCs induced mitochondrial network aggregation at a higher rate than in CGCs. CONCLUSIONS: Results suggest that the level of expression and the mRNA isoforms do not underlie the vulnerability of RGCs to OPA1 mutations. However, aggregation of the mitochondrial network induced by the downregulation of Opa1 appears more frequent in RGCs than in control CGCs.

Cysteine-string protein in inner hair cells of the organ of Corti: synaptic expression and upregulation at the onset of hearing.

Cysteine-string protein is a vesicle-associated protein that plays a vital function in neurotransmitter release. We have studied its expression and regulation during cochlear maturation. Both the mRNA and the protein were found in primary auditory neurons and the sensory inner hair cells. More importantly, cysteine-string protein was localized on synaptic vesicles associated with the synaptic ribbon in inner hair cells and with presynaptic differentiations in lateral and medial olivocochlear terminals -- the cell bodies of which lie in the auditory brainstem. No cysteine-string protein was expressed by the sensory outer hair cells suggesting that the distinct functions of the two cochlear hair cell types imply different mechanisms of neurotransmitter release. In developmental studies in the rat, we observed that cysteine-string protein was present beneath the inner hair cells at birth and beneath outer hair cells by postnatal day 2 only. We found no expression in the inner hair cells before about postnatal day 12, which corresponds to the period during which the first cochlear action potentials could be recorded. In conclusion, the close association of cysteine-string protein with synaptic vesicles tethered to synaptic ribbons in inner hair cells and its synchronized expression with the appearance and maturation of the cochlear potentials strongly suggest that this protein plays a fundamental role in sound-evoked glutamate release by inner hair cells. This also suggests that this role may be common to ribbon synapses and conventional central nervous system synapses.

Transient Ca2+-permeable AMPA receptors in postnatal rat primary auditory neurons.

Fast excitatory transmission in the nervous system is mostly mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors whose subunit composition governs physiological characteristics such as ligand affinity and ion conductance properties. Here, we report that AMPA receptors at inner hair cell (IHC) synapses lack the GluR2 subunit and are transiently Ca2+-permeable before hearing onset as evidenced using agonist-induced Co2+ accumulation, Western blots and GluR2 confocal microscopy in the rat cochlea. AMPA (100 microM) induced Co2+ accumulation in primary auditory neurons until postnatal day (PND) 10. This accumulation was concentration-dependent, strengthened by cyclothiazide (50 microM) and blocked by GYKI 52466 (80 microM) and Joro spider toxin (1 microM). It was unaffected by D-AP5 (50 microM), and it could not be elicited by 56 mM K+ or 1 mM NMDA + 10 microM glycine. Western blots showed that GluR1 immunoreactivity, present in homogenates of immature cochleas, had disappeared by PND12. GluR2 immunoreactivity was not detected until PND10 and GluR3 and GluR4 immunoreactivities were detected at all the ages examined. Confocal microscopy confirmed that the GluR2 immunofluorescence was not located postsynaptically to IHCs before PND10. In conclusion, AMPA receptors on maturing primary auditory neurons differ from those on adult neurons. They are probably composed of GluR1, GluR3 and GluR4 subunits and have a high Ca2+ permeability. The postsynaptic expression of GluR2 subunits may be continuously regulated by the presynaptic activity allowing for variations in the Ca2+ permeability and physiological properties of the receptor.

Catecholamine-independent transient expression of tyrosine hydroxylase in primary auditory neurons is coincident with the onset of hearing in the rat cochlea.

During the last stages of neuronal maturation, tyrosine hydroxylase is transiently expressed in the absence of the other catecholamine-synthesizing enzymes. We show here that it is expressed in rat spiral ganglion neurons between postnatal days 8 and 20, with a peak of expression at postnatal day 12. These tyrosine hydroxylase-immunoreactive neurons did not display aromatic amino acid decarboxylase- or dopamine-beta-hydroxylase-immunoreactivities, ruling out the possibilities of dopamine or noradrenaline synthesis. They also did not display peripherin- or intense neurofilament 200-kDa-immunoreactivities, two indicators of type II primary auditory neurons. Tyrosine hydroxylase-immunoreactive dendrites were seen in synaptic contact with the inner hair cells and expressed the GluR2 subunit of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, further confirming the type I nature of the neurons transiently expressing the enzyme. The end of the tyrosine hydroxylase expression was not due to cell death because the immunoreactive neurons did not show TUNEL-labelled nuclei. Finally, all the type I neurons expressed the tyrosine hydroxylase mRNA at postnatal day 12, suggesting that the expression of the enzyme is a maturational step common to all these neurons and that the expression of the protein is not synchronized. Because the period of transient expression of tyrosine hydroxylase in type I neurons parallels the periods of maturation of evoked exocytosis in inner hair cells and of appearance and maturation of the cochlear potentials, we propose that the expression of the enzyme indicates the onset of hearing in individual type I primary auditory neurons. This enzyme expression could rely on a Ca2+ activation of its encoding gene subsequent to a sudden and massive Ca2+ entry through voltage-activated Ca2+ channels.