RESUMO
Disturbance of sensory input during development can have disastrous effects on the development of sensory cortical areas. To examine how moderate perturbations of hearing can impact the development of primary auditory cortex, we examined markers of excitatory synapses in mice who lacked prestin, a protein responsible for somatic electromotility of cochlear outer hair cells. While auditory brain stem responses of these mice show an approximately 40 dB increase in threshold, we found that loss of prestin produced no changes in spine density or morphological characteristics on apical dendrites of cortical layer 5 pyramidal neurons. PSD-95 immunostaining also showed no changes in overall excitatory synapse density. Surprisingly, behavioral assessments of auditory function using the acoustic startle response showed only modest changes in prestin KO animals. These results suggest that moderate developmental hearing deficits produce minor changes in the excitatory connectivity of layer 5 neurons of primary auditory cortex and surprisingly mild auditory behavioral deficits in the startle response.
Assuntos
Córtex Auditivo/metabolismo , Período Crítico Psicológico , Espinhas Dendríticas/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Proteínas Motores Moleculares/genética , Células Piramidais/metabolismo , Animais , Camundongos , Camundongos Knockout , Proteínas Motores Moleculares/metabolismo , Reflexo de Sobressalto/fisiologia , Sinapses/metabolismoRESUMO
Development of a viral vector that can infect hair cells of the cochlea without producing viral-associated ototoxic effects is crucial for utilizing gene replacement therapy as a treatment for certain forms of hereditary deafness. In the present study, cochlear function was monitored using distortion-product otoacoustic emissions (DPOAEs) in guinea pigs that received infusions of either (E1(-), E3(-)) adenovirus, or adeno-associated virus (AAV), directly into the scala tympani. Replication-deficient (E1(-), E3(-)) adenovirus-directed gene transfer, using the cytomegalovirus (CMV) promoter, drove transgene expression to inner hair cells and pillar cells of the cochlea. AAV transduction was tested with several promoters, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha) promoters; which drove transgene expression to cochlear blood vessels, nerve fibers, and certain spiral limbus cells, respectively. AAV transgene expression was visualized by green fluorescent protein immunostaining. Immunocytochemistry to heparan sulfate confirmed the absence of proteoglycans in guinea pig hair cells, indicating that the receptor for AAV was not present on these cells. However, the heparan sulfate proteoglycan expression pattern mimicked the AAV transduction pattern. An overall finding was that cochlear function was not altered throughout the infection period using AAV titers as high as 5 x 10(8) IP/infused cochlea. In contrast, cochlear function was severely compromised by 8 days postinfection with adenoviral titers of 5 x 10(8) PFU/infused cochlea, and outer hair cells were eliminated. Thus, cochlear hair cells are amenable to in vivo gene transfer using a replication-deficient (E1(-), E3(-)) adenovirus. However, replication-defective or gutted adenovirus vectors must be employed to overcome the ototoxic effects of (E1(-), E3(-)) adenovirus vectors.
Assuntos
Adenoviridae/genética , Cóclea/metabolismo , Dependovirus/genética , Vetores Genéticos/genética , Transgenes/genética , Adenoviridae/fisiologia , Animais , Cóclea/irrigação sanguínea , Cóclea/inervação , Cóclea/virologia , Dependovirus/fisiologia , Expressão Gênica , Técnicas de Transferência de Genes , Genes Reporter/genética , Terapia Genética/métodos , Cobaias , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Células Ciliadas Auditivas/virologia , Heparina/análogos & derivados , Heparina/análise , Imuno-Histoquímica , Microscopia Eletrônica de Varredura , Especificidade de Órgãos , Fator 1 de Elongação de Peptídeos/genética , Fosfopiruvato Hidratase/genética , Fator de Crescimento Derivado de Plaquetas/genética , Regiões Promotoras Genéticas/genética , Proteoglicanas/análise , Receptores Virais/química , Receptores Virais/metabolismo , Replicação ViralRESUMO
The authors studied phoria adaptation to horizontal base-out prism in 17 patients with well-documented cerebellar lesions. There was no significant difference between mean adaptation measured in the patients and ten normal controls. Individually, normal adaptation was found in 12 patients. Abnormal adaptation was found in five patients, all but one of which had other neurologic lesions. These results suggest that phoria adaptation to base-out prism is not diminished by a cerebellar lesion unless it is accompanied by another nervous system lesion(s).
Assuntos
Adaptação Ocular , Doenças Cerebelares/fisiopatologia , Estrabismo/fisiopatologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Diplopia/fisiopatologia , Humanos , Pessoa de Meia-Idade , Testes Visuais , Visão Binocular , Percepção VisualRESUMO
A cDNA was isolated from the nematode Caenorhabditis elegans that encodes an endoprotease which is a member of the Kex2 family of serine endoproteases. Degenerate oligonucleotide primers were designed based on conserved regions within the active sites of known Kex2-like endoproteases, and were used for reverse transcription-polymerase chain reaction (RT-PCR) of poly(A)+RNA isolated from C. elegans. A PCR product was isolated that had homology to the active sites of known furin endoproteases, and was used as a probe to screen a C. elegans cDNA library. A Kex2-like endoprotease (CelfurPC) which encoded a 692-amino-acid pre-proendoprotease, was identified. The deduced amino acid sequence for the catalytic domain of CelfurPC is homologous to the known Kex2-like endoproteases, with strongest structural homology to the furin/PACE4 family. However, all furins and PACE4 proteins contain a characteristic cysteine-rich domain, and all furins contain a transmembrane domain, neither of which is present in the CelfurPC protein. CelfurPC may thus represent a new class of Kex2-like endoprotease.
Assuntos
Caenorhabditis elegans/genética , DNA Complementar/genética , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Subtilisinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/enzimologia , DNA Complementar/isolamento & purificação , Furina , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Hair cells of the vertebrate inner ear are subject to efferent control by the release of acetylcholine (ACh) from brainstem neurons. While ACh ultimately causes the hair cell to hyperpolarize through the activation of small conductance Ca(2+)-activated K(+) channels, the initial effect is to open a ligand-gated cation channel that briefly depolarizes the hair cell. The hair cell's ligand-gated cation channel has unusual pharmacology that is well matched to that of the nicotinic subunit alpha9 expressed in Xenopus oocytes. We used sequence-specific amplification to identify the ortholog of alpha9 in the chick's cochlea (basilar papilla). Chick alpha9 is 73% identical to rat alpha9 at the amino acid level. A second transcript was identified that differed by the loss of 132 base pairs coding for 44 amino acids near the putative ligand-binding site. RT-PCR on whole cochlear ducts suggested that this short variant is less abundant than the full length alpha9 mRNA. In situ hybridization revealed alpha9 mRNA in sensory hair cells of the chick cochlea. The pattern of expression was consistent with the efferent innervation pattern. The alpha9 label was strongest in short (outer) hair cells on which large calyciform efferent endings are found. Tall (inner) hair cells receiving little or no efferent innervation had substantially less label. The cochlear ganglion neurons were not labeled, consistent with the absence of axo-dendritic efferent innervation in birds. These findings suggest that alpha9 contributes to the ACh receptor of avian hair cells and supports the generality of this hypothesis among all vertebrates.
Assuntos
Galinhas , Cóclea/metabolismo , Células Ciliadas Auditivas/metabolismo , Processamento Alternativo , Animais , Sequência de Bases , Clonagem Molecular , Cóclea/citologia , Cobaias , Células Ciliadas Auditivas/citologia , Hibridização In Situ , Dados de Sequência Molecular , Neurônios Eferentes/metabolismo , Especificidade de Órgãos/genética , RNA Mensageiro/biossíntese , Ratos , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The offset of smooth pursuit eye movements is very different from the onset. The onset eye velocity is characterized by overshoot and ringing before settling to steady-state velocity. Yet, at pursuit offset, the eye velocity returns smoothly to zero. One reason for this difference may be that the pursuit system is nonlinear and behaves differently during acceleration and deceleration. After testing four subjects, we found no difference between acceleration and deceleration ringing dynamics, except for decelerations to 0 +/- 2 deg/sec, suggesting that around zero velocity, the central nervous system switches off pursuit and adopts fixation. This supports the idea that fixation and pursuit represent different neurological systems.
Assuntos
Movimentos Oculares , Fixação Ocular , Acompanhamento Ocular Uniforme , Humanos , Fatores de TempoRESUMO
A number of studies have shown that the ear can be protected from sound over-exposure, either by activating the cochlear efferent system, or by sound 'conditioning' in which the role of the efferent system is less certain. To study more definitively the molecular basis of deliberately induced cochlear protection from excessive sounds, it is advantageous to determine, for an inbred mouse strain, a range of noise exposure parameters that effectively alter cochlear function. As an initial step towards this goal, young CBA/CaJ mice were exposed to a 105-dB SPL octave-band noise (OBN), centered at 10 kHz, for various lengths of time consisting of 10 min, or 0.5, 1, 3, or 6 h. Distortion product otoacoustic emissions (DPOAEs) at the 2f1-f2 frequency, in response to equilevel primary tones of low to moderate levels, were used to quantify the damaging effects of these sound over-exposures on cochlear function. In addition, staining for acetylcholinesterase (AChE) activity to assess for noise-induced changes in the pattern of efferent-nerve innervation to the cochlea was also performed in a subset of mice that were exposed to the longest-lasting 6-h OBN. The 10-min OBN resulted in only temporary reductions in DPOAE levels, which recovered to pre-exposure values within 5 days. Increasing the exposure to 0.5 h resulted in permanent DPOAE losses that, for low primary-tone levels, were still present at 31 days post-exposure. Additionally, the 1-h and longer exposures caused permanent reductions in DPOAEs for all test levels, which were measurable at 31 days following exposure. Light-microscopic observations restricted to the 11-18-kHz frequency region of the organ of Corti, for a subset of mice exposed to the 6-h OBN, uncovered a significant loss of outer hair cells (OHCs). However, despite the OHC loss in this region, the AChE activity associated with the related pattern of efferent innervation remained largely intact.
Assuntos
Ruído , Emissões Otoacústicas Espontâneas/fisiologia , Distorção da Percepção/fisiologia , Acetilcolinesterase/metabolismo , Animais , Morte Celular , Cóclea/inervação , Vias Eferentes/fisiologia , Células Ciliadas Auditivas Externas/patologia , Células Ciliadas Auditivas Externas/fisiologia , Camundongos , Camundongos Endogâmicos CBA , Fatores de TempoRESUMO
Motor learning can be demonstrated in the vestibulo-ocular reflex (VOR) by changing its gain (eye velocity/head velocity) with goggles and optokinetic (OK) drums. It is known that the flocculus is essential for this plasticity but there is controversy about whether the modifiable synapses mainly responsible are in the flocculus. To investigate this further we utilized the known reciprocal relationship between complex spikes and simple spikes in Purkinje cell discharges. By stimulating climbing fibers from the olive to the flocculus at 7 Hz, the simple spike rate of almost all recorded floccular cells could be driven to zero. This was termed floccular shutdown and it felt to effect a functional, reversible flocculectomy. Sixty single units in the flocculi of four cats were recorded. Stimulation of the climbing fibers at 7 Hz caused the discharge rate to decrease to zero in 95% of these cells. The gain of the horizontal VOR in three cats was driven repeatedly to twice or half its normal value by rotation within a moving OK drum and also by wearing magnifying or fixed-field goggles; this process required 3 days. If, on the 4th day, the cat was exposed to an OK drum rotating in the opposite direction, the gain was driven back to normal in 30 min. If, however, the climbing fibers were stimulated at 7 Hz during these 30 min, the gain did not return--learning was blocked. This verified that loss of floccular activity by this method abolishes VOR gain plasticity. Moreover, when 7 Hz stimulation first began, after 3 days of adaptation, the adapted gain remained at its adapted value, either half or twice normal, even in the face of floccular shutdown. This result appears incompatible with the hypothesis that the modifiable synapses are in the flocculus.
Assuntos
Cerebelo/fisiologia , Reflexo Vestíbulo-Ocular/fisiologia , Adaptação Fisiológica , Animais , Gatos , Denervação , Estimulação Elétrica , Feminino , Aprendizagem/fisiologia , Atividade Motora , Plasticidade Neuronal , Núcleo Olivar/fisiologia , Células de Purkinje/fisiologia , Fatores de TempoRESUMO
Laminin, which strongly stimulates axon outgrowth in vitro, appears transiently within the central nervous system (CNS) in embryos. After CNS injury, laminin reportedly reappears along axonal pathways only in animal species in which central axon regeneration is successful, including the leech Hirudo medicinalis. Although glia have been suspected of making CNS laminin, in adult leeches glia are not required for laminin synthesis and evidently microglia, not present in the early embryo, produce laminin. To determine which embryonic cells make laminin, a 1.2 kb DNA fragment of leech laminin B1 chain, with homology to Drosophila, human, and mouse B1 laminins and rat S laminin, was isolated using reverse-transcription and degenerate polymerase chain reaction (PCR) cloning. In situ hybridization revealed that laminin expression began before embryonic day 8, and by days 8 and 9 it was seen in paired CNS muscle cells. By late day 9, the two neuropil glial cells began to express laminin. Lucifer Yellow dye was injected intracellularly and muscle cells stimulated to contract, confirming the identities of muscle and glial cells. Packet glial cells began to express B1 laminin by embryonic day 12. By day 15, the cells of the perineurial sheath expressed B1 laminin, whereas it was no longer detectable in CNS muscle and glia. The results agree with published immunohistochemistry showing laminin within the CNS among growing axons by day 8, and only later in the perineurial sheath, by which time laminin disappears from within the CNS. Therefore, different cells synthesize laminin in the embryo and during repair in adults.
Assuntos
Sistema Nervoso Central/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Laminina/química , Proteínas do Tecido Nervoso/química , Fragmentos de Peptídeos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Nervoso Central/citologia , Embrião não Mamífero/metabolismo , Hibridização In Situ , Sanguessugas/embriologia , Dados de Sequência Molecular , Neuroglia/metabolismo , Junção Neuromuscular/fisiologia , Fragmentos de Peptídeos/isolamento & purificação , Especificidade da Espécie , Fatores de TempoRESUMO
The loss of cochlear hair cells, or the loss of their capacity to transduce acoustic signals, is believed to be the underlying mechanism in many forms of hearing loss. To develop viral vectors that allow for the introduction of genes directly into the cochleae of adult animals, replication-deficient (E1(-), E3(-)) and replication-defective (E1(-), E3(-), pol(-)) adenovirus vectors were used to transduce the bacterial beta-galactosidase gene into the hair cells of the guinea pig cochlea in vivo. Distortion product otoacoustic emissions, which monitor the functional status of outer hair cells, were measured throughout the viral infection periods to identify hair cell ototoxicity. The results demonstrated that the use of the (E1(-), E3(-)) adenovirus vectors containing CMV-driven LacZ, compromised cochlear function when gradually introduced into scala tympani via an osmotic pump. However, when (E1(-), E3(-), pol(-)) adenoviral vectors containing CMV-driven LacZ were used to transduce cochlear hair cells, there was no loss of cochlear function over the frequency regions tested, and beta-galactosidase (beta-gal) was detected in over 80% of all hair cells. Development of a viral vector that infects cochlear hair cells without virus-induced ototoxic effects is crucial for gene replacement strategies to treat certain forms of inherited deafness and for otoprotective strategies to prevent hair cell losses to treat progressive hearing disorders. Moreover, in vivo (E1(-), E3(-), pol(-)) adenovirus mediated gene-transfer techniques applied to adult guinea pig cochleae may be useful in testing several hypotheses concerning what roles specific genes play in normal cochlear function.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Células Ciliadas Auditivas/enzimologia , Transtornos da Audição/terapia , Transfecção/métodos , Animais , Cóclea/fisiologia , Expressão Gênica , Cobaias , Bombas de Infusão Implantáveis , Emissões Otoacústicas Espontâneas , beta-Galactosidase/genéticaRESUMO
An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes. A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases. A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay. A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin. In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals.