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1.
J Assoc Res Otolaryngol ; 20(6): 565-577, 2019 12.
Article in English | MEDLINE | ID: mdl-31410614

ABSTRACT

The submillisecond acuity for detecting rapid spatial and temporal fluctuations in acoustic stimuli observed in humans and laboratory animals depends in part on select groups of auditory neurons that preserve synchrony from the ears to the binaural nuclei in the brainstem. These fibers have specialized synapses and axons that use a low-threshold voltage-activated outward current, IKL, conducted through Kv1 potassium ion channels. These are in turn coupled with HCN channels that express a mixed cation inward mixed current, IH, to support precise synchronized firing. The behavioral evidence is that their respective Kcna1 or HCN1 genes are absent in adult mice; the results are weak startle reflexes, slow responding to noise offsets, and poor sound localization. The present behavioral experiments were motivated by an in vitro study reporting increased IKL in an auditory nucleus in Kcna2-/- mice lacking the Kv1.2 subunit, suggesting that Kcna2-/- mice might perform better than Kcna2+/+ mice. Because Kcna2-/- mice have only a 17-18-day lifespan, we compared both preweanling Kcna2-/- vs. Kcna2+/+ mice and Kcna1-/- vs. Kcna1+/+ mice at P12-P17/18; then, the remaining mice were tested at P23/P25. Both null mutant strains had a stunted physique, but the Kcna1-/- mice had severe behavioral deficits while those in Kcna2-/- mice were relatively few and minor. The in vitro increase of IKL could have resulted from Kv1.1 subunits substituting for Kv1.2 units and the loss of the inhibitory "managerial" effect of Kv1.2 on Kv1.1. However, any increased neuronal synchronicity that accompanies increased IKL may not have been enough to affect behavior. All mice performed unusually well on the early spatial tests, but then, they fell towards adult levels. This unexpected effect may reflect a shift from summated independent monaural pathways to integrated binaural processing, as has been suggested for similar observations for human infants.


Subject(s)
Kv1.1 Potassium Channel/physiology , Kv1.2 Potassium Channel/physiology , Sound Localization , Acoustic Stimulation , Animals , Female , Kv1.1 Potassium Channel/genetics , Kv1.2 Potassium Channel/genetics , Male , Mice , Mice, Inbred C3H , Motor Activity , Noise , Reflex, Startle , Weaning
2.
BMC Mol Biol ; 18(1): 14, 2017 05 22.
Article in English | MEDLINE | ID: mdl-28532435

ABSTRACT

BACKGROUND: Along with sodium/calcium (Ca2+) exchangers, plasma membrane Ca2+ ATPases (ATP2Bs) are main regulators of intracellular Ca2+ levels. There are four ATP2B paralogs encoded by four different genes. Atp2b2 encodes the protein pump with the fastest activation, ATP2B2. In mice, the Atp2b2 transcript has several alternate transcriptional start site variants: α, ß, µ and δ. These variants are expressed in developmental and tissue specific manners. The α and ß Atp2b2 transcripts are equally expressed in the brain. αAtp2b2 is the only transcript found in the outer hair cells of young mice (Silverstein RS, Tempel BL. in Neuroscience 141:245-257, 2006). Mutations in the coding region of the mouse Atp2b2 gene indicate a narrow window for tolerated dysfunction of the ATP2B2 protein, specifically in the auditory system. This highlights the necessity of tight regulation of this gene for normal cell physiology. RESULTS: Although ATP2Bs are important regulators of Ca2+ in many cell types, little is known about their transcriptional regulation. This study identifies the proximal promoter of the αAtp2b2 transcript. Further investigations indicate that ATOH1 and EGR1 modulate promoter activity. Additionally, we report that EGR1 increases endogenous expression of Atp2b2 transcript in two cell lines. Electrophoretic mobility shift assays (EMSA) indicate that EGR1 binds to a specific site in the CpG island of the αAtp2b2 promoter. CONCLUSION: This study furthers our understanding of Atp2b2 regulation by: (I) elucidating transcriptional regulatory mechanisms for Atp2b2, and (II) identifying transcription factors that modulate expression of Atp2b2 in the brain and peripheral auditory system and (III) allows for future studies modulating gene expression of Atp2b2.


Subject(s)
Auditory Cortex/metabolism , Brain/metabolism , Early Growth Response Protein 1/metabolism , Gene Expression Regulation , Plasma Membrane Calcium-Transporting ATPases/genetics , Promoter Regions, Genetic , Animals , Calcium , Cell Line , Cerebellum/metabolism , CpG Islands , Haploinsufficiency , Mice , Plasma Membrane Calcium-Transporting ATPases/metabolism , Protein Binding , Transcription Factors/metabolism , Transcription, Genetic
3.
J Neurophysiol ; 117(2): 756-766, 2017 02 01.
Article in English | MEDLINE | ID: mdl-27881722

ABSTRACT

The medial nucleus of the trapezoid body (MNTB) is an important source of inhibition during the computation of sound location. It transmits fast and precisely timed action potentials at high frequencies; this requires an efficient calcium clearance mechanism, in which plasma membrane calcium ATPase 2 (PMCA2) is a key component. Deafwaddler (dfw2J ) mutant mice have a null mutation in PMCA2 causing deafness in homozygotes (dfw2J /dfw2J ) and high-frequency hearing loss in heterozygotes (+/dfw2J ). Despite the deafness phenotype, no significant differences in MNTB volume or cell number were observed in dfw2J homozygous mutants, suggesting that PMCA2 is not required for MNTB neuron survival. The MNTB tonotopic axis encodes high to low sound frequencies across the medial to lateral dimension. We discovered a cell size gradient along this axis: lateral neuronal somata are significantly larger than medially located somata. This size gradient is decreased in +/dfw2J and absent in dfw2J /dfw2J The lack of acoustically driven input suggests that sound-evoked activity is required for maintenance of the cell size gradient. This hypothesis was corroborated by selective elimination of auditory hair cell activity with either hair cell elimination in Pou4f3 DTR mice or inner ear tetrodotoxin (TTX) treatment. The change in soma size was reversible and recovered within 7 days of TTX treatment, suggesting that regulation of the gradient is dependent on synaptic activity and that these changes are plastic rather than permanent.NEW & NOTEWORTHY Neurons of the medial nucleus of the trapezoid body (MNTB) act as fast-spiking inhibitory interneurons within the auditory brain stem. The MNTB is topographically organized, with low sound frequencies encoded laterally and high frequencies medially. We discovered a cell size gradient along this axis: lateral neurons are larger than medial neurons. The absence of this gradient in deaf mice lacking plasma membrane calcium ATPase 2 suggests an activity-dependent, calcium-mediated mechanism that controls neuronal soma size.


Subject(s)
Cochlear Nucleus/pathology , Deafness/pathology , Deafness/physiopathology , Evoked Potentials, Auditory/physiology , Neurons/pathology , Sound , 2-Amino-5-phosphonovalerate/pharmacology , Animals , Deafness/genetics , Diphtheria Toxin/pharmacology , Evoked Potentials, Auditory/genetics , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Mice, Inbred CBA , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Mutation/genetics , Neurons/physiology , Plasma Membrane Calcium-Transporting ATPases/genetics , Plasma Membrane Calcium-Transporting ATPases/metabolism , Presynaptic Terminals/physiology , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Transcription Factor Brn-3C/genetics , Transcription Factor Brn-3C/metabolism
4.
Hear Res ; 330(Pt B): 213-20, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26119177

ABSTRACT

The sense of hearing is the fastest of our senses and provides the first all-or-none action potential in the auditory nerve in less than four milliseconds. Short stimulus evoked latencies and their minimal variability are hallmarks of auditory processing from spiral ganglia to cortex. Here, we review how even small changes in first spike latencies (FSL) and their variability (jitter) impact auditory temporal processing. We discuss a number of mouse models with degraded FSL/jitter whose mutations occur exclusively in the central auditory system and therefore might serve as candidates to investigate the cellular mechanisms underlying auditory processing disorders (APD).


Subject(s)
Auditory Pathways/physiopathology , Auditory Perception , Auditory Perceptual Disorders/physiopathology , Hearing , Synaptic Transmission , Acoustic Stimulation , Animals , Auditory Perceptual Disorders/genetics , Auditory Perceptual Disorders/psychology , Disease Models, Animal , Evoked Potentials, Auditory, Brain Stem , Genetic Predisposition to Disease , Humans , Mice , Mutation , Nerve Tissue Proteins/genetics , Phenotype , Reaction Time , Speech Perception , Time Factors
5.
J Assoc Res Otolaryngol ; 16(4): 459-71, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25940139

ABSTRACT

The 129S6/SvEvTac (129S6) inbred mouse is known for its resistance to noise-induced hearing loss (NIHL). However, less is understood of its unique age-related hearing loss (AHL) phenotype and its potential relationship with the resistance to NIHL. Here, we studied the physiological characteristics of hearing loss in 129S6 and asked if noise resistance (NR) and AHL are genetically linked to the same chromosomal region. We used auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) to examine hearing sensitivity between 1 and 13 months of age of recombinant-inbred (congenic) mice with an NR phenotype. We identified a region of proximal chromosome (Chr) 17 (D17Mit143-D17Mit100) that contributes to a sensory, non-progressive hearing loss (NPHL) affecting exclusively the high-frequencies (>24 kHz) and maps to the nr1 locus on Chr 17. ABR experiments showed that 129S6 and CBA/CaJ F1 (CBACa) hybrid mice exhibit normal hearing, indicating that the hearing loss in 129S6 mice is inherited recessively. An allelic complementation test between the 129S6 and 101/H (101H) strains did not rescue hearing loss, suggesting genetic allelism between the nphl and phl1 loci of these strains, respectively. The hybrids had a milder hearing loss than either parental strain, which indicate a possible interaction with other genes in the mouse background or a digenic interaction between different genes that reside in the same genomic region. Our study defines a locus for nphl on Chr 17 affecting frequencies greater than 24 kHz.


Subject(s)
Evoked Potentials, Auditory, Brain Stem , Presbycusis/genetics , Animals , Chromosomes, Mammalian , Female , Genes, Recessive , Hair Cells, Auditory, Outer/physiology , Male , Mice, 129 Strain , Mice, Inbred CBA , Presbycusis/physiopathology
6.
J Assoc Res Otolaryngol ; 15(5): 721-38, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24952082

ABSTRACT

Noise-induced hearing loss (NIHL) is a prevalent health risk. Inbred mouse strains 129S6/SvEvTac (129S6) and MOLF/EiJ (MOLF) show strong NIHL resistance (NR) relative to CBA/CaJ (CBACa). In this study, we developed quantitative trait locus (QTL) maps for NR. We generated F1 animals by intercrossing (129S6 × CBACa) and (MOLF × CBACa). In each intercross, NR was recessive. N2 animals were produced by backcrossing F1s to their respective parental strain. The 232 N2-129S6 and 225 N2-MOLF progenies were evaluated for NR using auditory brainstem response. In 129S6, five QTL were identified on chromosomes (Chr) 17, 18, 14, 11, and 4, referred to as loci nr1, nr2, nr3, nr4, and nr5, respectively. In MOLF, four QTL were found on Chr 4, 17, 6, and 12, referred to as nr7, nr8, nr9, and nr10, respectively. Given that NR QTL were discovered on Chr 4 and 17 in both the N2-129S6 and N2-MOLF cross, we generated two consomic strains by separately transferring 129S6-derived Chr 4 and 17 into an otherwise CBACa background and a double-consomic strain by crossing the two strains. Phenotypic analysis of the consomic strains indicated that whole 129S6 Chr 4 contributes strongly to mid-frequency NR, while whole 129S6 Chr 17 contributes markedly to high-frequency NR. Therefore, we anticipated that the double-consomic strain containing Chr 4 and 17 would demonstrate NR across the mid- and high-frequency range. However, whole 129S6 Chr 17 masks the expression of mid-frequency NR from whole 129S6 Chr 4. To further dissect NR on 129S6 Chr 4 and 17, CBACa.129S6 congenic strains were generated for each chromosome. Phenotypic analysis of the Chr 17 CBACa.129S6 congenic strains further defined the NR region on proximal Chr 17, uncovered another NR locus (nr6) on distal Chr 17, and revealed an epistatic interaction between proximal and distal 129S6 Chr 17.


Subject(s)
Hearing Loss, Noise-Induced/genetics , Quantitative Trait Loci , Animals , Chromosome Mapping , Mice , Mice, Inbred CBA
7.
J Assoc Res Otolaryngol ; 15(4): 543-54, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24799196

ABSTRACT

The plasma membrane Ca(2+) ATPase 2 (PMCA2) is necessary for auditory transduction and serves as the primary Ca(2+) extrusion mechanism in auditory stereocilia bundles. To date, studies examining PMCA2 in auditory function using mutant mice have focused on the phenotype of late adolescent and adult mice. Here, we focus on the changes of PMCA2 in the maturation of auditory sensitivity by comparing auditory responses to RNA and protein expression levels in haploinsufficient PMCA2 and wild-type mice from P16 into adulthood. Auditory sensitivity in wild-type mice improves between P16 and 3 weeks of age, when it becomes stable through adolescence. In haploinsufficient mice, there are frequency-dependent loss of sensitivity and subsequent recovery of thresholds between P16 and adulthood. RNA analysis demonstrates that α-Atp2b2 transcript levels increase in both wild-type and heterozygous cochleae between P16 and 5 weeks. The increases reported for the α-Atp2b2 transcript type during this stage in development support the requisite usage of this transcript for mature auditory transduction. PMCA2 expression also increases in wild-type cochleae between P16 and 5 weeks suggesting that this critical auditory protein may be involved in normal maturation of auditory sensitivity after the onset of hearing. We also characterize expression levels of two long noncoding RNA genes, Gm15082 (lnc82) and Gm15083 (lnc83), which are transcribed on the opposite strand in the 5' region of Atp2b2 and propose that the lnc83 transcript may be involved in regulating α-Atp2b2 expression.


Subject(s)
Aging/metabolism , Auditory Pathways/growth & development , Auditory Pathways/metabolism , Cochlea/metabolism , Plasma Membrane Calcium-Transporting ATPases/metabolism , Animals , Calcium/metabolism , Gene Expression Regulation, Developmental , Hearing/physiology , Hearing Tests , Mice , Mice, Inbred CBA , Mice, Mutant Strains , Models, Animal , Plasma Membrane Calcium-Transporting ATPases/genetics
8.
Hear Res ; 304: 41-8, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23792079

ABSTRACT

Tight regulation of calcium (Ca2+) concentrations in the stereocilia bundles of auditory hair cells of the inner ear is critical to normal auditory transduction. The plasma membrane Ca2+ ATPase 2 (PMCA2), encoded by the Atp2b2 gene, is the primary mechanism for clearance of Ca2+ from auditory stereocilia, keeping intracellular levels low, and also contributes to maintaining adequate levels of extracellular Ca2+ in the endolymph. This study characterizes a novel null Atp2b2 allele, dfw(i5), by examining cochlear anatomy, vestibular function and auditory physiology in mutant mice. Loss of auditory function in PMCA2 mutants can be attributed to dysregulation of intracellular Ca2+ inside the stereocilia bundles. However, extracellular Ca2+ ions surrounding the stereocilia are also required for rigidity of cadherin 23, a component of the stereocilia tip-link encoded by the Cdh23 gene. This study further resolves the interaction between Atp2b2 and Cdh23 in a gene dosage and frequency-dependent manner, and finds that low frequencies are significantly affected by the interaction. In +/dfw(i5) mice, one mutant copy of Cdh23 is sufficient to cause broad frequency hearing impairment. Additionally, we report another modifying interaction with Atp2b2 on auditory sensitivity, possibly caused by an unidentified hearing loss gene in mice.


Subject(s)
Cadherins/genetics , Cadherins/physiology , Hearing/genetics , Hearing/physiology , Plasma Membrane Calcium-Transporting ATPases/genetics , Plasma Membrane Calcium-Transporting ATPases/physiology , Alleles , Amino Acid Sequence , Animals , Base Sequence , Calcium Signaling , Evoked Potentials, Auditory, Brain Stem , Female , Hair Cells, Auditory/physiology , Hearing Loss/genetics , Hearing Loss/physiopathology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Mutant Strains , Mutation , Plasma Membrane Calcium-Transporting ATPases/deficiency , Stereocilia/physiology
9.
Epilepsia ; 53 Suppl 1: 134-41, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22612818

ABSTRACT

Voltage-gated K(+) channels (Kv) represent the largest family of genes in the K(+) channel family. The Kv1 subfamily plays an essential role in the initiation and shaping of action potentials, influencing action potential firing patterns and controlling neuronal excitability. Overlapping patterns with differential expression and precise localization of Kv1.1 and Kv1.2 channels targeted to specialized subcellular compartments contribute to distinctive patterns of neuronal excitability. Dynamic regulation of the components in these subcellular domains help to finely tune the cellular and regional networks. Disruption of the expression, distribution, and density of these channels through deletion or mutation of the genes encoding these channels, Kcna1 and Kcna2, is associated with neurologic pathologies including epilepsy and ataxia in humans and in rodent models. Kv1.1 and Kv1.2 knockout mice both have seizures beginning early in development; however, each express a different seizure type (pathway), although the channels are from the same subfamily and are abundantly coexpressed. Voltage-gated ion channels clustered in specific locations may present a novel therapeutic target for influencing excitability in neurologic disorders associated with some channelopathies.


Subject(s)
Kv1.1 Potassium Channel/genetics , Kv1.2 Potassium Channel/genetics , Seizures/genetics , Animals , Axons/metabolism , Brain/growth & development , Brain Chemistry/genetics , Brain Chemistry/physiology , Epilepsy/genetics , Epilepsy/physiopathology , Humans , Mice , Mice, Knockout , Mutation/genetics , Mutation/physiology , Neuronal Plasticity/genetics , Neuronal Plasticity/physiology , Ranvier's Nodes/metabolism , Seizures/physiopathology
10.
Neuron ; 71(5): 911-25, 2011 Sep 08.
Article in English | MEDLINE | ID: mdl-21903083

ABSTRACT

Offset responses upon termination of a stimulus are crucial for perceptual grouping and gap detection. These gaps are key features of vocal communication, but an ionic mechanism capable of generating fast offsets from auditory stimuli has proven elusive. Offset firing arises in the brainstem superior paraolivary nucleus (SPN), which receives powerful inhibition during sound and converts this into precise action potential (AP) firing upon sound termination. Whole-cell patch recording in vitro showed that offset firing was triggered by IPSPs rather than EPSPs. We show that AP firing can emerge from inhibition through integration of large IPSPs, driven by an extremely negative chloride reversal potential (E(Cl)), combined with a large hyperpolarization-activated nonspecific cationic current (I(H)), with a secondary contribution from a T-type calcium conductance (I(TCa)). On activation by the IPSP, I(H) potently accelerates the membrane time constant, so when the sound ceases, a rapid repolarization triggers multiple offset APs that match onset timing accuracy.


Subject(s)
Action Potentials/physiology , Neurons/physiology , Reaction Time/physiology , Acoustic Stimulation/methods , Action Potentials/drug effects , Animals , Animals, Newborn , Auditory Pathways/physiology , Biophysics , Calcium/metabolism , Calcium Channel Blockers/pharmacology , Calcium Channels, T-Type/metabolism , Chlorides/metabolism , Computer Simulation , Cyclic Nucleotide-Gated Cation Channels/deficiency , Electric Stimulation , Functional Laterality , Furosemide/pharmacology , Gene Expression Regulation/genetics , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , In Vitro Techniques , Ion Channel Gating/genetics , Ion Channel Gating/physiology , Mibefradil/pharmacology , Mice , Mice, Inbred CBA , Mice, Knockout , Models, Neurological , Neurons/drug effects , Olivary Nucleus/cytology , Patch-Clamp Techniques/methods , Potassium Channels/deficiency , Psychoacoustics , Pyrimidines/pharmacology , Reaction Time/drug effects , Reaction Time/genetics , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Stilbamidines/metabolism , Symporters/metabolism , Synaptic Potentials/drug effects , Synaptic Potentials/physiology , K Cl- Cotransporters
11.
J Physiol ; 589(Pt 5): 1143-57, 2011 Mar 01.
Article in English | MEDLINE | ID: mdl-21224222

ABSTRACT

Voltage-gated potassium (Kv) channels containing Kv1.1 subunits are strongly expressed in neurons that fire temporally precise action potentials (APs). In the auditory system, AP timing is used to localize sound sources by integrating interaural differences in time (ITD) and intensity (IID) using sound arriving at both cochleae. In mammals, the first nucleus to encode IIDs is the lateral superior olive (LSO), which integrates excitation from the ipsilateral ventral cochlear nucleus and contralateral inhibition mediated via the medial nucleus of the trapezoid body. Previously we reported that neurons in this pathway show reduced firing rates, longer latencies and increased jitter in Kv1.1 knockout (Kcna1−/−) mice. Here, we investigate whether these differences have direct impact on IID processing by LSO neurons. Single-unit recordings were made from LSO neurons of wild-type (Kcna1+/+) and from Kcna1−/− mice. IID functions were measured to evaluate genotype-specific changes in integrating excitatory and inhibitory inputs. In Kcna1+/+ mice, IID sensitivity ranged from +27 dB (excitatory ear more intense) to −20 dB (inhibitory ear more intense), thus covering the physiologically relevant range of IIDs. However, the distribution of IID functions in Kcna1−/− mice was skewed towards positive IIDs, favouring ipsilateral sound positions. Our computational model revealed that the reduced performance of IID encoding in the LSO of Kcna1−/− mice is mainly caused by a decrease in temporal fidelity along the inhibitory pathway. These results imply a fundamental role for Kv1.1 in temporal integration of excitation and inhibition during sound source localization.


Subject(s)
Auditory Pathways/physiology , Kv1.1 Potassium Channel/metabolism , Neurons/physiology , Olivary Nucleus/physiology , Sound Localization/physiology , Acoustic Stimulation , Action Potentials/physiology , Animals , Electrophysiology , Immunohistochemistry , Kv1.1 Potassium Channel/genetics , Mice , Mice, Knockout , Models, Neurological
12.
J Biol Chem ; 286(11): 9360-72, 2011 Mar 18.
Article in English | MEDLINE | ID: mdl-21233214

ABSTRACT

Impairments in axonal dopamine release are associated with neurological disorders such as schizophrenia and attention deficit hyperactivity disorder and pathophysiological conditions promoting drug abuse and obesity. The D2 dopamine autoreceptor (D2-AR) exerts tight regulatory control of axonal dopamine (DA) release through a mechanism suggested to involve K(+) channels. To evaluate the contribution of Kv1 voltage-gated potassium channels of the Shaker gene family to the regulation of axonal DA release by the D2-AR, the present study employed expression analyses, real time measurements of striatal DA overflow, K(+) current measurements and immunoprecipitation assays. Kv1.1, -1.2, -1.3, and -1.6 mRNA and protein were detected in midbrain DA neurons purified by fluorescence-activated cell sorting and in primary DA neuron cultures. In addition, Kv1.1, -1.2, and -1.6 were localized to DA axonal processes in the dorsal striatum. By means of fast scan cyclic voltammetry in striatal slice preparations, we found that the inhibition of stimulation-evoked DA overflow by a D2 agonist was attenuated by Kv1.1, -1.2, and -1.6 toxin blockers. A particular role for the Kv1.2 subunit in the process whereby axonal D2-AR inhibits DA overflow was established with the use of a selective Kv1.2 blocker and Kv1.2 knock-out mice. Moreover, we demonstrate the ability of D2-AR activation to increase Kv1.2 currents in co-transfected cells and its reliance on Gßγ subunit signaling along with the physical coupling of D2-AR and Kv1.2-containing channels in striatal tissue. These findings underline the contribution of Kv1.2 in the regulation of nigrostriatal DA release by the D2-AR and thereby offer a novel mechanism by which DA release is regulated.


Subject(s)
Axons/metabolism , Corpus Striatum/metabolism , Dopamine/metabolism , Kv1.2 Potassium Channel/metabolism , Receptors, Dopamine D2/metabolism , Signal Transduction/physiology , Animals , Dopamine/genetics , Dopamine Agonists/pharmacology , Kv1.2 Potassium Channel/genetics , Male , Mice , Mice, Knockout , Receptors, Dopamine D2/genetics , Signal Transduction/drug effects
13.
Dev Neurobiol ; 70(4): 253-67, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20095043

ABSTRACT

Usher syndrome is the leading cause of combined deaf-blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction, and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in Exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock-in mice. Absent auditory-evoked brainstem responses indicated that the mutant mice are deaf at 1 month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock-in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Deafness/physiopathology , Disease Models, Animal , Retinal Degeneration/physiopathology , Usher Syndromes/physiopathology , Adaptor Proteins, Signal Transducing/metabolism , Aging , Animals , Cell Cycle Proteins , Cochlea/pathology , Cochlea/physiopathology , Cochlea/ultrastructure , Cytoskeletal Proteins , Deafness/pathology , Electroretinography , Evoked Potentials, Auditory, Brain Stem , Exons , Gene Knock-In Techniques , Hair Cells, Auditory/pathology , Hair Cells, Auditory/physiology , Louisiana , Mice , Mice, Transgenic , Mutation, Missense , RNA Splice Sites , RNA, Messenger/metabolism , Retina/pathology , Retina/physiopathology , Retinal Degeneration/pathology , Retinal Rod Photoreceptor Cells/pathology , Retinal Rod Photoreceptor Cells/physiology , Usher Syndromes/pathology
14.
J Comp Neurol ; 514(6): 624-40, 2009 Jun 20.
Article in English | MEDLINE | ID: mdl-19365819

ABSTRACT

Calcium signaling plays a role in synaptic regulation of dendritic structure, usually on the time scale of hours or days. Here we use immunocytochemistry to examine changes in expression of plasma membrane calcium ATPase type 2 (PMCA2), a high-affinity calcium efflux protein, in the chick nucleus laminaris (NL) following manipulations of synaptic inputs. Dendrites of NL neurons segregate into dorsal and ventral domains, receiving excitatory input from the ipsilateral and contralateral ears, respectively, via nucleus magnocellularis (NM). Deprivation of the contralateral projection from NM to NL leads to rapid retraction of ventral, but not the dorsal, dendrites of NL neurons. Immunocytochemistry revealed symmetric distribution of PMCA2 in two neuropil regions of normally innervated NL. Electron microscopy confirmed that PMCA2 localizes in both NM terminals and NL dendrites. As early as 30 minutes after transection of the contralateral projection from NM to NL or unilateral cochlea removal, significant decreases in PMCA2 immunoreactivity were seen in the deprived neuropil of NL compared with the other neuropil that continued to receive normal input. The rapid decrease correlated with reductions in the immunoreactivity for microtubule-associated protein 2, which affects cytoskeleton stabilization. These results suggest that PMCA2 is regulated independently in ventral and dorsal NL dendrites and/or their inputs from NM in a way that is correlated with presynaptic activity. This provides a potential mechanism by which deprivation can change calcium transport that, in turn, may be important for rapid, compartment-specific dendritic remodeling.


Subject(s)
Auditory Pathways/enzymology , Brain Stem/enzymology , Plasma Membrane Calcium-Transporting ATPases/metabolism , Animals , Auditory Pathways/ultrastructure , Blotting, Western , Brain Stem/ultrastructure , Chickens , Cochlea/physiology , Immunohistochemistry , Mice , Mice, Inbred BALB C , Mice, Mutant Strains , Microscopy, Electron, Transmission , Microtubule-Associated Proteins/metabolism , Neurons/enzymology , Neurons/physiology , Neurons/ultrastructure , Neuropil/enzymology , Photomicrography , Synaptosomal-Associated Protein 25/metabolism
15.
J Neurophysiol ; 98(3): 1501-25, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17634333

ABSTRACT

Genes Kcna1 and Kcna2 code for the voltage-dependent potassium channel subunits Kv1.1 and Kv1.2, which are coexpressed in large axons and commonly present within the same tetramers. Both contribute to the low-voltage-activated potassium current I Kv1, which powerfully limits excitability and facilitates temporally precise transmission of information, e.g., in auditory neurons of the medial nucleus of the trapezoid body (MNTB). Kcna1-null mice lacking Kv1.1 exhibited seizure susceptibility and hyperexcitability in axons and MNTB neurons, which also had reduced I Kv1. To explore whether a lack of Kv1.2 would cause a similar phenotype, we created and characterized Kcna2-null mice (-/-). The -/- mice exhibited increased seizure susceptibility compared with their +/+ and +/- littermates, as early as P14. The mRNA for Kv1.1 and Kv1.2 increased strongly in +/+ brain stems between P7 and P14, suggesting the increasing importance of these subunits for limiting excitability. Surprisingly, MNTB neurons in brain stem slices from -/- and +/- mice were hypoexcitable despite their Kcna2 deficit, and voltage-clamped -/- MNTB neurons had enlarged I Kv1. This contrasts strikingly with the Kcna1-null MNTB phenotype. Toxin block experiments on MNTB neurons suggested Kv1.2 was present in every +/+ Kv1 channel, about 60% of +/- Kv1 channels, and no -/- Kv1 channels. Kv1 channels lacking Kv1.2 activated at abnormally negative potentials, which may explain why MNTB neurons with larger proportions of such channels had larger I Kv1. If channel voltage dependence is determined by how many Kv1.2 subunits each contains, neurons might be able to fine-tune their excitability by adjusting the Kv1.1:Kv1.2 balance rather than altering Kv1 channel density.


Subject(s)
Kv1.2 Potassium Channel/deficiency , Kv1.2 Potassium Channel/physiology , Seizures/genetics , Shaker Superfamily of Potassium Channels/physiology , Aging , Animals , Brain Stem/physiology , Brain Stem/physiopathology , Genetic Vectors , Genome , Genotype , Life Expectancy , Mice , Mice, Knockout , Neurons/physiology , Open Reading Frames , Restriction Mapping
16.
Epilepsia ; 48(11): 2023-46, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17651419

ABSTRACT

PURPOSE: Mice lacking the Kv1.1 potassium channel alpha subunit encoded by the Kcna1 gene develop recurrent behavioral seizures early in life. We examined the neuropathological consequences of seizure activity in the Kv1.1(-/-) (knock-out) mouse, and explored the effects of injecting a viral vector carrying the deleted Kcna1 gene into hippocampal neurons. METHODS: Morphological techniques were used to assess neuropathological patterns in hippocampus of Kv1.1(-/-) animals. Immunohistochemical and biochemical techniques were used to monitor ion channel expression in Kv1.1(-/-) brain. Both wild-type and knockout mice were injected (bilaterally into hippocampus) with an HSV1 amplicon vector that contained the rat Kcna1 subunit gene and/or the E. coli lacZ reporter gene. Vector-injected mice were examined to determine the extent of neuronal infection. RESULTS: Video/EEG monitoring confirmed interictal abnormalities and seizure occurrence in Kv1.1(-/-) mice. Neuropathological assessment suggested that hippocampal damage (silver stain) and reorganization (Timm stain) occurred only after animals had exhibited severe prolonged seizures (status epilepticus). Ablation of Kcna1 did not result in compensatory changes in expression levels of other related ion channel subunits. Vector injection resulted in infection primarily of granule cells in hippocampus, but the number of infected neurons was quite variable across subjects. Kcna1 immunocytochemistry showed "ectopic" Kv1.1 alpha channel subunit expression. CONCLUSIONS: Kcna1 deletion in mice results in a seizure disorder that resembles--electrographically and neuropathologically--the patterns seen in rodent models of temporal lobe epilepsy. HSV1 vector-mediated gene transfer into hippocampus yielded variable neuronal infection.


Subject(s)
Gene Deletion , Gene Transfer Techniques , Hippocampus/pathology , Kv1.1 Potassium Channel/genetics , Seizures/genetics , Seizures/pathology , Animals , Coloring Agents , Electroencephalography/statistics & numerical data , Gene Expression , Genetic Vectors/genetics , Herpesvirus 1, Human/genetics , Hippocampus/chemistry , Hippocampus/metabolism , Immunohistochemistry , Kv1.1 Potassium Channel/deficiency , Kv1.1 Potassium Channel/metabolism , Mice , Mice, Knockout , Monitoring, Physiologic , Seizures/diagnosis , Severity of Illness Index
17.
Hear Res ; 224(1-2): 51-60, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17208398

ABSTRACT

Deletions affecting the terminal end of chromosome 3p result in a characteristic set of clinical features termed 3p-- syndrome. Bilateral, sensorineural hearing loss (SNHL) has been found in some but not all cases, suggesting the possibility that it is due to loss of a critical gene in band 3p25. To date, no genetic locus in this region has been shown to cause human hearing loss. However, the ATP2B2 gene is located in 3p25.3, and haploinsufficiency of the mouse homolog results in SNHL with similar severity. We compared auditory test results with fine deletion mapping in seven previously unreported 3p-- syndrome patients and identified a 1.38Mb region in 3p25.3 in which deletions were associated with moderate to severe, bilateral SNHL. This novel hearing loss locus contains 18 genes, including ATP2B2. ATP2B2 encodes the plasma membrane calcium pump PMCA2. We used immunohistochemistry in human cochlear sections to show that PMCA2 is located in the stereocilia of hair cells, suggesting its function in the auditory system is conserved between humans and mice. Although other genes in this region remain candidates, we conclude that haploinsufficiency of ATP2B2 is the most likely cause of SNHL in 3p-- syndrome.


Subject(s)
Chromosome Deletion , Chromosomes, Human, Pair 3/genetics , Hearing Loss, Bilateral/genetics , Hearing Loss, Sensorineural/genetics , Animals , Base Sequence , Child , Child, Preschool , Chromosome Mapping , Cochlea/metabolism , DNA Primers/genetics , Disease Models, Animal , Female , Hearing Loss, Bilateral/metabolism , Hearing Loss, Bilateral/physiopathology , Hearing Loss, Sensorineural/metabolism , Hearing Loss, Sensorineural/physiopathology , Humans , Immunohistochemistry , Male , Mice , Mutation , Plasma Membrane Calcium-Transporting ATPases/deficiency , Plasma Membrane Calcium-Transporting ATPases/genetics , Plasma Membrane Calcium-Transporting ATPases/metabolism , Species Specificity , Syndrome
18.
J Neurosci ; 26(27): 7201-11, 2006 Jul 05.
Article in English | MEDLINE | ID: mdl-16822977

ABSTRACT

Transmission of visual signals at the first retinal synapse is associated with changes in calcium concentration in photoreceptors and bipolar cells. We investigated how loss of plasma membrane Ca2+ ATPase isoform 2 (PMCA2), the calcium transporter isoform with the highest affinity for Ca2+/calmodulin, affects transmission of rod- and cone-mediated responses. PMCA2 expression in the neuroblast layer was observed soon after birth; in the adult, PMCA2 was expressed in inner segments and synaptic terminals of rod photoreceptors, in rod bipolar cells, and in most inner retinal neurons but was absent from cones. To determine the role of PMCA2 in retinal signaling, we compared morphology and light responses of retinas from control mice and deafwaddler dfw2J mice, which lack functional PMCA2 protein. The cytoarchitecture of retinas from control and dfw2J mice was indistinguishable at the light microscope level. Suction electrode recordings revealed no difference in the sensitivity or amplitude of outer segment light responses of control and dfw2J rods. However, rod-mediated ERG b-wave responses in dfw2J mice were approximately 45% smaller and significantly slower than those of control mice. Furthermore, recordings from individual rod bipolar cells showed that the sensitivity of transmission at the rod output synapse was reduced by approximately 50%. No changes in the amplitude or timing of cone-mediated ERG responses were observed. These results suggest that PMCA2-mediated Ca2+ extrusion modulates the amplitude and timing of the high-sensitivity rod pathway to a much greater extent than that of the cone pathway.


Subject(s)
Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Cation Transport Proteins/metabolism , Dark Adaptation/physiology , Retina/physiology , Vision, Ocular/physiology , Animals , Calcium-Transporting ATPases/genetics , Cation Transport Proteins/genetics , Cell Membrane/metabolism , Evoked Potentials, Visual/physiology , Female , Gene Expression Regulation, Developmental/physiology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Neurologic Mutants , Photic Stimulation , Plasma Membrane Calcium-Transporting ATPases , Retina/cytology , Retina/growth & development , Retinal Bipolar Cells/physiology , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/physiology , Synapses/physiology
19.
J Neurophysiol ; 96(3): 1203-14, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16672305

ABSTRACT

Low threshold, voltage-gated potassium currents (Ikl) are widely expressed in auditory neurons that can fire temporally precise action potentials (APs). In the medial nucleus of the trapezoid body (MNTB), channels containing the Kv1.1 subunit (encoded by the Kcna1 gene) underlie Ikl. Using pharmacology, genetics and whole cell patch-clamp recordings in mouse brain slices, we tested the role of Ikl in limiting AP latency-variability (jitter) in response to trains of single inputs at moderate to high stimulation rates. With dendrotoxin-K (DTX-K, a selective blocker of Kv1.1-containing channels), we blocked Ikl maximally (approximately 80% with 100 nM DTX-K) or partially (approximately 50% with 1-h incubation in 3 nM DTX-K). Ikl was similar in 3 nM DTX-K-treated cells and cells from Kcna1(-/-) mice, allowing a comparison of these two different methods of Ikl reduction. In response to current injection, Ikl reduction increased the temporal window for AP initiation and increased jitter in response to the smallest currents that were able to drive APs. While 100 nM DTX-K caused the largest increases, latency and jitter in Kcna1(-/-) cells and in 3 nM DTX-K-treated cells were similar to each other but increased compared with +/+. The near-phenocopy of the Kcna1(-/-) cells with 3 nM DTX-K shows that acute blockade of a subset of the Kv1.1-containing channels is functionally similar to the chronic elimination of all Kv1.1 subunits. During rapid stimulation (100-500 Hz), Ikl reduction increased jitter in response to both large and small inputs. These data show that Ikl is critical for maintaining AP temporal precision at physiologically relevant firing rates.


Subject(s)
Action Potentials/physiology , Brain/physiology , Kv1.1 Potassium Channel/physiology , Neurons/physiology , Action Potentials/drug effects , Animals , In Vitro Techniques , Kv1.1 Potassium Channel/drug effects , Mice , Patch-Clamp Techniques , Peptides/pharmacology , Potassium Channels/physiology
20.
J Assoc Res Otolaryngol ; 5(2): 99-110, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15357414

ABSTRACT

In vertebrates, transduction of sound into an electrochemical signal is carried out by hair cells that rely on calcium to perform specialized functions. The apical surfaces of hair cells are surrounded by endolymphatic fluid containing calcium at concentrations that must be maintained by active transport. The mechanism of this transport is unknown, but an ATP-dependent pump is believed to participate. Mutation of the Atp2b2 gene that encodes plasma membrane calcium ATPase type 2 (PMCA2) produces the deaf, ataxic mouse: deafwaddler2J (dfw2J). We hypothesized that PMCA2 might transport calcium into the endolymph and that dfw2J mice would have low endolymph calcium concentrations, possibly contributing to their deafness and ataxia. First, using immunocytochemistry, we demonstrated that PMCA2 is present in control mice inner and outer hair cell stereocilia where it could pump calcium into the endolymph and that PMCA2 is absent in dfw2J stereocilia. Second, using an aspirating microelectrode and calcium-sensitive fluorescent dye, we found that dfw2J mice endolymph calcium concentrations are significantly lower than those of control mice. These findings suggest that PMCA2, located in hair cell stereocilia, contributes significantly to endolymph calcium maintenance.


Subject(s)
Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/metabolism , Calcium/metabolism , Deafness/metabolism , Endolymph/metabolism , Animals , Cation Transport Proteins , Cochlea/physiology , Deafness/genetics , Deafness/physiopathology , Evoked Potentials, Auditory , Female , Hair Cells, Auditory, Inner/metabolism , Hair Cells, Auditory, Inner/pathology , Hair Cells, Auditory, Outer/metabolism , Hair Cells, Auditory, Outer/pathology , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Neurologic Mutants , Plasma Membrane Calcium-Transporting ATPases
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