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1.  Functional effects of spinocerebellar ataxia type 13 mutations are conserved in zebrafish Kv3.3 channels 
BMC Neuroscience  2010;11:99.
Background
The zebrafish has been suggested as a model system for studying human diseases that affect nervous system function and motor output. However, few of the ion channels that control neuronal activity in zebrafish have been characterized. Here, we have identified zebrafish orthologs of voltage-dependent Kv3 (KCNC) K+ channels. Kv3 channels have specialized gating properties that facilitate high-frequency, repetitive firing in fast-spiking neurons. Mutations in human Kv3.3 cause spinocerebellar ataxia type 13 (SCA13), an autosomal dominant genetic disease that exists in distinct neurodevelopmental and neurodegenerative forms. To assess the potential usefulness of the zebrafish as a model system for SCA13, we have characterized the functional properties of zebrafish Kv3.3 channels with and without mutations analogous to those that cause SCA13.
Results
The zebrafish genome (release Zv8) contains six Kv3 family members including two Kv3.1 genes (kcnc1a and kcnc1b), one Kv3.2 gene (kcnc2), two Kv3.3 genes (kcnc3a and kcnc3b), and one Kv3.4 gene (kcnc4). Both Kv3.3 genes are expressed during early development. Zebrafish Kv3.3 channels exhibit strong functional and structural homology with mammalian Kv3.3 channels. Zebrafish Kv3.3 activates over a depolarized voltage range and deactivates rapidly. An amino-terminal extension mediates fast, N-type inactivation. The kcnc3a gene is alternatively spliced, generating variant carboxyl-terminal sequences. The R335H mutation in the S4 transmembrane segment, analogous to the SCA13 mutation R420H, eliminates functional expression. When co-expressed with wild type, R335H subunits suppress Kv3.3 activity by a dominant negative mechanism. The F363L mutation in the S5 transmembrane segment, analogous to the SCA13 mutation F448L, alters channel gating. F363L shifts the voltage range for activation in the hyperpolarized direction and dramatically slows deactivation.
Conclusions
The functional properties of zebrafish Kv3.3 channels are consistent with a role in facilitating fast, repetitive firing of action potentials in neurons. The functional effects of SCA13 mutations are well conserved between human and zebrafish Kv3.3 channels. The high degree of homology between human and zebrafish Kv3.3 channels suggests that the zebrafish will be a useful model system for studying pathogenic mechanisms in SCA13.
doi:10.1186/1471-2202-11-99
PMCID: PMC2933717  PMID: 20712895
2.  Frequency of KCNC3 DNA Variants as Causes of Spinocerebellar Ataxia 13 (SCA13) 
PLoS ONE  2011;6(3):e17811.
Background
Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers.
Methodology
DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes.
Principal Findings
Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel.
Conclusions
Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. The p.Arg423His mutation is recurrent in different populations and associated with early onset. In contrast to previous p.Arg423His mutation carriers, we now observed seizures and mild mental retardation in one individual. This study confirms the wide phenotypic spectrum in SCA13.
doi:10.1371/journal.pone.0017811
PMCID: PMC3066194  PMID: 21479265
3.  Rescue of Motor Coordination by Purkinje Cell-Targeted Restoration of Kv3.3 Channels in Kcnc3-Null Mice Requires Kcnc1 
The Journal of Neuroscience  2009;29(50):15735-15744.
The role of cerebellar Kv3.1 and Kv3.3 channels in motor coordination was examined with an emphasis on the deep cerebellar nuclei (DCN). Kv3 channel subunits encoded by Kcnc genes are distinguished by rapid activation and deactivation kinetics that support high-frequency, narrow action potential firing. Previously we reported that increased lateral deviation while ambulating and slips while traversing a narrow beam of ataxic Kcnc3-null mice were corrected by restoration of Kv3.3 channels specifically to Purkinje cells, whereas Kcnc3-mutant mice additionally lacking one Kcnc1 allele were partially rescued. Here, we report mice lacking all Kcnc1 and Kcnc3 alleles exhibit no such rescue. For Purkinje cell output to reach the rest of the brain it must be conveyed by neurons of the DCN or vestibular nuclei. As Kcnc1, but not Kcnc3, alleles are lost, mutant mice exhibit increasing gait ataxia accompanied by spike broadening and deceleration in DCN neurons, suggesting the facet of coordination rescued by Purkinje-cell-restricted Kv3.3 restoration in mice lacking just Kcnc3 is hypermetria, while gait ataxia emerges when additionally Kcnc1 alleles are lost. Thus, fast repolarization in Purkinje cells appears important for normal movement velocity, whereas DCN neurons are a prime candidate locus where fast repolarization is necessary for normal gait patterning.
doi:10.1523/JNEUROSCI.4048-09.2009
PMCID: PMC3849660  PMID: 20016089
4.  Ablation of Kv3.1 and Kv3.3 Potassium Channels Disrupts Thalamocortical Oscillations In Vitro and In Vivo 
The Journal of Neuroscience  2008;28(21):5570-5581.
The genes Kcnc1 and Kcnc3 encode the subunits for the fast-activating/fast-deactivating, voltage-gated potassium channels Kv3.1 and Kv3.3, which are expressed in several brain regions known to be involved in the regulation of the sleep–wake cycle. When these genes are genetically eliminated, Kv3.1/Kv3.3-deficient mice display severe sleep loss as a result of unstable slow-wave sleep. Within the thalamocortical circuitry, Kv3.1 and Kv3.3 subunits are highly expressed in the thalamic reticular nucleus (TRN), which is thought to act as a pacemaker at sleep onset and to be involved in slow oscillatory activity (spindle waves) during slow-wave sleep. We showed that in cortical electroencephalographic recordings of freely moving Kv3.1/Kv3.3-deficient mice, spectral power is reduced up to 70% at frequencies <15 Hz. In addition, the number of sleep spindles in vivo as well as rhythmic rebound firing of TRN neurons in vitro is diminished in mutant mice. Kv3.1/Kv3.3-deficient TRN neurons studied in vitro show ∼60% increase in action potential duration and a reduction in high-frequency firing after depolarizing current injections and during rebound burst firing. The results support the hypothesis that altered electrophysiological properties of TRN neurons contribute to the reduced EEG power at slow frequencies in the thalamocortical network of Kv3-deficient mice.
doi:10.1523/JNEUROSCI.0747-08.2008
PMCID: PMC3844809  PMID: 18495891
parvalbumin; fast-spiking interneurons; thalamic reticular nucleus; spindle waves; rhythmic rebound bursts; cortical activation
5.  Potassium Channel Gene Expression in the Rat Cochlear Nucleus 
Hearing research  2007;228(1-2):31-43.
Potassium channels play a critical role in defining the electrophysiological properties accounting for the unique response patterns of auditory neurons. Serial analysis of gene expression (SAGE), microarrays, RT-PCR, and real-time RT-PCR were used to generate a broad profile of potassium channel expression in the rat cochlear nucleus. This study identified mRNAs for 51 different potassium channel subunits or channel interacting proteins. The relative expression levels of 27 of these transcripts among the AVCN, PVCN, and DCN were determined by real-time RT-PCR. Four potassium channel transcripts showed substantial levels of differential expression. Kcnc2 was expressed more than 15-fold higher in the DCN as compared to AVCN and PVCN. In contrast, Kcnj13 had an approximate 10-fold higher expression in AVCN and PVCN than in DCN. Two subunits that modify the activity of other channels were inversely expressed between ventral and dorsal divisions. Kcns1 was over 15-fold higher in DCN than AVCN or PVCN, while Kcns3 was about 25-fold higher in AVCN than in DCN. The expression patterns of potassium channels in the subdivisions of the cochlear nucleus provide a basis for understanding the electrophysiological mechanisms which sub-serve central auditory processing and provide targets for further investigations into neural plastic changes that occur with hearing loss.
doi:10.1016/j.heares.2007.01.024
PMCID: PMC1995076  PMID: 17346910
cochlear nucleus; gene expression; SAGE; potassium channels; Kv3.2; Kir7.1
6.  KCNC3: Phenotype, mutations, channel biophysics – a study of 260 familial ataxia patients 
Human mutation  2010;31(2):191-196.
We recently identified KCNC3, encoding the Kv3.3 voltage-gated potassium channel, as the gene mutated in SCA13. One g.10684G>A (p.Arg420His) mutation caused late-onset ataxia resulting in a non-functional channel subunit with dominant-negative properties. A French early-onset pedigree with mild mental retardation segregated a g.10767T>C (p.Phe448Leu) mutation. This mutation changed the relative stability of the channel’s open conformation. Coding exons were amplified and sequenced in 260 autosomal-dominant ataxia index cases of European descent. Functional analyses were performed using expression in Xenopus oocytes. The previously identified p.Arg420His mutation occurred in three families with late-onset ataxia. A novel mutation g.10693G>A (p.Arg423His) was identified in two families with early-onset. In one pedigree, a novel g.10522G>A (p.Arg366His) sequence variant was seen in one index case but did not segregate with affected status in the respective family. In a heterologous expression system, the p.Arg423His mutation exhibited dominant negative properties. The p.Arg420His mutation, results in a non-functional channel subunit was recurrent and associated with late-onset progressive ataxia. In two families the p.Arg423His mutation was associated with early-onset slow progressive ataxia. Despite a phenotype reminiscent of the p.Phe448Leu mutation, segregating in a large early-onset French pedigree, the p.Arg423His mutation resulted in a nonfunctional subunit with a strong dominant-negative effect.
doi:10.1002/humu.21165
PMCID: PMC2814913  PMID: 19953606
Ion Channel gene defects; Spinocerebellar Ataxia; SCA13; KCNC3
7.  Connections of the Auditory Brainstem in a Songbird, Taeniopygia guttata. I. Projections of Nucleus Angularis and Nucleus Laminaris to the Auditory Torus 
The Journal of comparative neurology  2010;518(11):10.1002/cne.22334.
Auditory information is important for social and reproductive behaviors in birds generally, but is crucial for oscine species (songbirds), in particular because in these species auditory feedback ensures the learning and accurate maintenance of song. While there is considerable information on the auditory projections through the forebrain of songbirds, there is no information available for projections through the brainstem. At the latter levels the prevalent model of auditory processing in birds derives from an auditory specialist, the barn owl, which uses time and intensity parameters to compute the location of sounds in space, but whether the auditory brainstem of songbirds is similarly functionally organized is unknown. To examine the songbird auditory brainstem we charted the projections of the cochlear nuclei angularis (NA) and magnocellularis (NM) and the third-order nucleus laminaris (NL) in zebra finches using standard tract-tracing techniques. As in other avian species, the projections of NM were found to be confined to NL, and NL and NA provided the ascending projections. Here we report on differential projections of NA and NL to the torus semicircularis, known in birds as nucleus mesencephalicus lateralis, pars dorsalis (MLd), and in mammals as the central nucleus of the inferior colliculus (ICc). Unlike the case in nonsongbirds, the projections of NA and NL to MLd in the zebra finch showed substantial overlap, in agreement with the projections of the cochlear nuclei to the ICc in mammals. This organization could suggest that the “what” of auditory stimuli is as important as “where.”
doi:10.1002/cne.22334
PMCID: PMC3862038  PMID: 20394061
cochlear nuclei; central nucleus of inferior colliculus; MLd; zebra finch; avian
8.  Developmental Changes Underlying the Formation of the Specialized Time Coding Circuits in Barn Owls (Tyto alba) 
The Journal of Neuroscience  2002;22(17):7671-7679.
Barn owls are capable of great accuracy in detecting the interaural time differences (ITDs) that underlie azimuthal sound localization. They compute ITDs in a circuit in nucleus laminaris (NL) that is reorganized with respect to birds like the chicken. The events that lead to the reorganization of the barn owl NL take place during embryonic development, shortly after the cochlear and laminaris nuclei have differentiated morphologically. At first the developing owl’s auditory brainstem exhibits morphology reminiscent of that of the developing chicken. Later, the two systems diverge, and the owl’s brainstem auditory nuclei undergo a secondary morphogenetic phase during which NL dendrites retract, the laminar organization is lost, and synapses are redistributed. These events lead to the restructuring of the ITD coding circuit and the consequent reorganization of the hindbrain map of ITDs and azimuthal space.
PMCID: PMC3260528  PMID: 12196590
avian development; morphogenesis; auditory; laminaris; evolution; interaural time difference
9.  Development of N-Methyl-D-Aspartate Receptor Subunits in Avian Auditory Brainstem 
N-methyl-D-aspartate (NMDA) receptor subunit-specific probes were used to characterize developmental changes in the distribution of excitatory amino acid receptors in the chicken’s auditory brainstem nuclei. Although NR1 subunit expression does not change greatly during the development of the cochlear nuclei in the chicken (Tang and Carr [2004] Hear. Res 191:79 – 89), there are significant developmental changes in NR2 subunit expression. We used in situ hybridization against NR1, NR2A, NR2B, NR2C, and NR2D to compare NR1 and NR2 expression during development. All five NMDA subunits were expressed in the auditory brainstem before embryonic day (E) 10, when electrical activity and synaptic responses appear in the nucleus magnocellularis (NM) and the nucleus laminaris (NL). At this time, the dominant form of the receptor appeared to contain NR1 and NR2B. NR2A appeared to replace NR2B by E14, a time that coincides with synaptic refinement and evoked auditory responses. NR2C did not change greatly during auditory development, whereas NR2D increased from E10 and remained at fairly high levels into adulthood. Thus changes in NMDA NR2 receptor subunits may contribute to the development of auditory brainstem responses in the chick.
doi:10.1002/cne.21303
PMCID: PMC3268522  PMID: 17366608
cochlear nucleus; magnocellularis; laminaris; angularis; tonotopic gradient
10.  Development of N-Methyl-D-Aspartate Receptor Subunits in Avian Auditory Brainstem 
Hearing Research  2004;191(1-2):79-89.
N-methyl-D-aspartate (NMDA) receptor subunit-specific probes were used to characterize developmental changes in the distribution of excitatory amino acid receptors in the chicken’s auditory brainstem nuclei. Although NR1 subunit expression does not change greatly during the development of the cochlear nuclei in the chicken (Tang and Carr [2004] Hear. Res 191:79–89), there are significant developmental changes in NR2 subunit expression. We used in situ hybridization against NR1, NR2A, NR2B, NR2C, and NR2D to compare NR1 and NR2 expression during development. All five NMDA subunits were expressed in the auditory brainstem before embryonic day (E) 10, when electrical activity and synaptic responses appear in the nucleus magnocellularis (NM) and the nucleus laminaris (NL). At this time, the dominant form of the receptor appeared to contain NR1 and NR2B. NR2A appeared to replace NR2B by E14, a time that coincides with synaptic refinement and evoked auditory responses. NR2C did not change greatly during auditory development, whereas NR2D increased from E10 and remained at fairly high levels into adulthood. Thus changes in NMDA NR2 receptor subunits may contribute to the development of auditory brainstem responses in the chick.
doi:10.1016/j.heares.2004.01.007
PMCID: PMC3269632  PMID: 15109707
cochlear nucleus; magnocellularis; laminaris; angularis; tonotopic gradient
11.  Chronic deficit in the expression of voltage-gated potassium channel Kv3.4 subunit in the hippocampus of pilocarpine-treated epileptic rats 
Brain research  2010;1368:308-316.
Voltage gated K+ channels (Kv) are a highly diverse group of channels critical in determining neuronal excitability. Deficits of Kv channel subunit expression and function have been implicated in the pathogenesis of epilepsy. In this study, we investigate whether the expression of the specific subunit Kv3.4 is affected during epileptogenesis following pilocarpine-induced status epilepticus. For this purpose, we used immunohistochemistry, Western blotting assays and comparative analysis of gene expression using TaqMan-based probes and delta-delta cycle threshold (Δ ΔCT) method of quantitative real-time polymerase chain reaction (qPCR) technique in samples obtained from age-matched control and epileptic rats. A marked down-regulation of Kv3.4 immunoreactivity was detected in the stratum lucidum and hilus of dentate gyrus in areas corresponding to the mossy fiber system of chronically epileptic rats. Correspondingly, a 20% reduction of Kv3.4 protein levels was detected in the hippocampus of chronic epileptic rats. Real-time quantitative PCR analysis of gene expression revealed that a significant 33% reduction of transcripts for Kv3.4 (gene Kcnc4) occurred after 1 month of pilocarpine-induced status epilepticus and persisted during the chronic phase of the model. These data indicate a reduced expression of Kv3.4 channels at protein and transcript levels in the epileptic hippocampus. Down-regulation of Kv3.4 in mossy fibers may contribute to enhanced presynaptic excitability leading to recurrent seizures in the pilocarpine model of temporal lobe epilepsy.
doi:10.1016/j.brainres.2010.10.047
PMCID: PMC3014430  PMID: 20971086
Pilocarpine; status epilepticus; mossy fibers; TaqMan; potassium channels; temporal lobe epilepsy
12.  Novel phenotype associated with a mutation in the KCNA1(Kv1.1) gene 
Episodic ataxia type 1 (EA1) is an autosomal dominant K+ channelopathy which manifests with short attacks of cerebellar ataxia and dysarthria, and may also show interictal myokymia. Episodes can be triggered by emotional or physical stress, startle response, sudden postural change or fever. Here we describe a 31-year-old man displaying markedly atypical symptoms, including long-lasting attacks of jerking muscle contractions associated with hyperthermia, severe migraine, and a relatively short-sleep phenotype. A single nucleotide change in KCNA1 (c.555C>G) was identified that changes a highly conserved residue (p.C185W) in the first transmembrane segment of the voltage-gated K+ channel Kv1.1. The patient is heterozygous and the mutation was inherited from his asymptomatic mother. Next generation sequencing revealed no variations in the CACNA1A, CACNB4, KCNC3, KCNJ10, PRRT2 or SCN8A genes of either the patient or mother, except for a benign variant in SLC1A3. Functional analysis of the p.C185W mutation in KCNA1 demonstrated a deleterious dominant-negative phenotype where the remaining current displayed slower activation kinetics, subtle changes in voltage-dependence and faster recovery from slow inactivation. Structural modeling also predicts the C185W mutation to be functionally deleterious. This description of novel clinical features, associated with a Kv1.1 mutation highlights a possibly unrecognized relationship between K+ channel dysfunction, hyperthermia and migraine in EA1, and suggests that thorough assessments for these symptoms should be carefully considered for all patients affected by EA1.
doi:10.3389/fphys.2014.00525
PMCID: PMC4295438  PMID: 25642194
episodic ataxia type 1; hyperthermia; sleep; migraine; Shaker potassium channels; C185W
13.  Interaural timing difference circuits in the auditory brainstem of the emu (Dromaius novaehollandiae) 
In the auditory system, precise encoding of temporal information is critical for sound localization, a task with direct behavioral relevance. Interaural timing differences are computed using axonal delay lines and cellular coincidence detectors in nucleus laminaris (NL). We present morphological and physiological data on the timing circuits in the emu, Dromaius novaehollandiae, and compare these results with those from the barn owl (Tyto alba) and the domestic chick (Gallus gallus). Emu NL was composed of a compact monolayer of bitufted neurons whose two thick primary dendrites were oriented dorsoventrally. They showed a gradient in dendritic length along the presumed tonotopic axis. The NL and nucleus magnocellularis (NM) neurons were strongly immunoreactive for parvalbumin, a calcium-binding protein. Antibodies against synaptic vesicle protein 2 and glutamic acid decarboxlyase revealed that excitatory synapses terminated heavily on the dendritic tufts, while inhibitory terminals were distributed more uniformly. Physiological recordings from brainstem slices demonstrated contralateral delay lines from NM to NL. During whole-cell patch-clamp recordings, NM and NL neurons fired single spikes and were doubly-rectifying. NL and NM neurons had input resistances of 30.0 ± 19.9 MΩ and 49.0 ± 25.6 MΩ, respectively, and membrane time constants of 12.8 ± 3.8 ms and 3.9 ± 0.2 ms. These results provide further support for the Jeffress model for sound localization in birds. The emu timing circuits showed the ancestral (plesiomorphic) pattern in their anatomy and physiology, while differences in dendritic structure compared to chick and owl may indicate specialization for encoding ITDs at low best frequencies.
doi:10.1002/cne.20862
PMCID: PMC2948976  PMID: 16435285
avian; nucleus laminaris; nucleus magnocellularis; dendrite; coincidence detection; sound localization
14.  Biophysical basis of the sound analog membrane potential that underlies coincidence detection in the barn owl 
Interaural time difference (ITD), or the difference in timing of a sound wave arriving at the two ears, is a fundamental cue for sound localization. A wide variety of animals have specialized neural circuits dedicated to the computation of ITDs. In the avian auditory brainstem, ITDs are encoded as the spike rates in the coincidence detector neurons of the nucleus laminaris (NL). NL neurons compare the binaural phase-locked inputs from the axons of ipsi- and contralateral nucleus magnocellularis (NM) neurons. Intracellular recordings from the barn owl's NL in vivo showed that tonal stimuli induce oscillations in the membrane potential. Since this oscillatory potential resembled the stimulus sound waveform, it was named the sound analog potential (Funabiki et al., 2011). Previous modeling studies suggested that a convergence of phase-locked spikes from NM leads to an oscillatory membrane potential in NL, but how presynaptic, synaptic, and postsynaptic factors affect the formation of the sound analog potential remains to be investigated. In the accompanying paper, we derive analytical relations between these parameters and the signal and noise components of the oscillation. In this paper, we focus on the effects of the number of presynaptic NM fibers, the mean firing rate of these fibers, their average degree of phase-locking, and the synaptic time scale. Theoretical analyses and numerical simulations show that, provided the total synaptic input is kept constant, changes in the number and spike rate of NM fibers alter the ITD-independent noise whereas the degree of phase-locking is linearly converted to the ITD-dependent signal component of the sound analog potential. The synaptic time constant affects the signal more prominently than the noise, making faster synaptic input more suitable for effective ITD computation.
doi:10.3389/fncom.2013.00102
PMCID: PMC3821004  PMID: 24265615
phase-locking; sound localization; auditory brainstem; periodic signals; oscillation; owl
15.  Astrocyte-Secreted Factors Modulate the Developmental Distribution of Inhibitory Synapses in Nucleus Laminaris of the Avian Auditory Brainstem 
The Journal of comparative neurology  2012;520(6):1262-1277.
Nucleus laminaris (NL) neurons in the avian auditory brainstem are coincidence detectors necessary for the computation of interaural time differences used in sound localization. In addition to their excitatory inputs from nucleus magnocellularis, NL neurons receive inhibitory inputs from the superior olivary nucleus (SON) that greatly improve coincidence detection in mature animals. The mechanisms that establish mature distributions of inhibitory inputs to NL are not known. We used the vesicular GABA transporter (VGAT) as a marker for inhibitory presynaptic terminals to study the development of inhibitory inputs to NL between embryonic day 9 (E9) and E17. VGAT immunofluorescent puncta were first seen sparsely in NL at E9. The density of VGAT puncta increased with development, first within the ventral NL neuropil region and subsequently throughout both the ventral and dorsal dendritic neuropil, with significantly fewer terminals in the cell body region. A large increase in density occurred between E13–15 and E16–17, at a developmental stage when astrocytes that express glial fibrillary acidic protein (GFAP) become mature. We cultured E13 brainstem slices together with astrocyte-conditioned medium (ACM) obtained from E16 brainstems and found that ACM, but not control medium, increased the density of VGAT puncta. This increase was similar to that observed during normal development. Astrocyte-secreted factors interact with the terminal ends of SON axons to increase the number of GABAergic terminals. These data suggest that factors secreted from GFAP-positive astrocytes promote maturation of inhibitory pathways in the auditory brainstem.
doi:10.1002/cne.22786
PMCID: PMC3926803  PMID: 22020566
astrocytes; inhibitory synapses; auditory brainstem; nucleus laminaris; synaptogenesis; superior olivary nucleus
16.  Maps of interaural time difference in the chicken’s brainstem nucleus laminaris 
Biological cybernetics  2008;98(6):541-559.
Animals, including humans, use interaural time differences (ITDs) that arise from different sound path lengths to the two ears as a cue of horizontal sound source location. The nature of the neural code for ITD is still controversial. Current models differentiate between two population codes: either a map-like rate-place code of ITD along an array of neurons, consistent with a large body of data in the barn owl, or a population rate code, consistent with data from small mammals. Recently, it was proposed that these different codes reflect optimal coding strategies that depend on head size and sound frequency. The chicken makes an excellent test case of this proposal because its physical pre-requisites are similar to small mammals, yet it shares a more recent common ancestry with the owl. We show here that, like in the barn owl, the brainstem nucleus laminaris in mature chickens displayed the major features of a place code of ITD. ITD was topographically represented in the maximal responses of neurons along each isofrequency band, covering approximately the contralateral acoustic hemisphere. Furthermore, the represented ITD range appeared to change with frequency, consistent with a pressure gradient receiver mechanism in the avian middle ear. At very low frequencies, below400 Hz, maximal neural responses were symmetrically distributed around zero ITD and it remained unclear whether there was a topographic representation. These findings do not agree with the above predictions for optimal coding and thus revive the discussion as to what determines the neural coding strategies for ITDs.
doi:10.1007/s00422-008-0220-6
PMCID: PMC3170859  PMID: 18491165
Auditory; Hearing; Sound localization; Sensory
17.  Avian Adeno-Associated Virus Vector Efficiently Transduces Neurons in the Embryonic and Post-Embryonic Chicken Brain 
PLoS ONE  2012;7(11):e48730.
The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits.
doi:10.1371/journal.pone.0048730
PMCID: PMC3492410  PMID: 23144948
18.  Transgenic Quail as a Model for Research in the Avian Nervous System – A Comparative Study of the Auditory Brainstem 
Research performed on transgenic animals has led to numerous advances in biological research. However, using traditional retroviral methods to generate transgenic avian research models has proven problematic. As a result, experiments aimed at genetic manipulations on birds remained difficult for this popular research tool. Recently, lentiviral methods have enabled production of transgenic birds, including a transgenic Japanese quail (Coturnix coturnix japonica) line showing neuronal-specificity and stable expression of eGFP across generations (termed here as GFP quail). To test whether the GFP quail may serve as a viable alternative to the popular chicken model system, with the additional benefit of gene manipulation, we compared the development, organization, structure and function of a specific neuronal circuit in chicken (Gallus gallus domesticus) to that of the GFP quail. This study focuses on a well-defined avian brain region, the principal nuclei of the sound localization circuit in the auditory brainstem, nucleus magnocellularis (NM) and nucleus laminaris (NL). Our results demonstrate that structural and functional properties of NM and NL neurons in the GFP quail, as well as their dynamic properties in response to changes in the environment, are nearly identical to those in chickens. These similarities demonstrate that the GFP quail, as well as other transgenic quail lines, can serve as an attractive avian model system, with the advantage of being able to build on the wealth of information already available from the chicken.
doi:10.1002/cne.23187
PMCID: PMC3488602  PMID: 22806400
transgenic quail; auditory brainstem
19.  Theoretical foundations of the sound analog membrane potential that underlies coincidence detection in the barn owl 
A wide variety of neurons encode temporal information via phase-locked spikes. In the avian auditory brainstem, neurons in the cochlear nucleus magnocellularis (NM) send phase-locked synaptic inputs to coincidence detector neurons in the nucleus laminaris (NL) that mediate sound localization. Previous modeling studies suggested that converging phase-locked synaptic inputs may give rise to a periodic oscillation in the membrane potential of their target neuron. Recent physiological recordings in vivo revealed that owl NL neurons changed their spike rates almost linearly with the amplitude of this oscillatory potential. The oscillatory potential was termed the sound analog potential, because of its resemblance to the waveform of the stimulus tone. The amplitude of the sound analog potential recorded in NL varied systematically with the interaural time difference (ITD), which is one of the most important cues for sound localization. In order to investigate the mechanisms underlying ITD computation in the NM-NL circuit, we provide detailed theoretical descriptions of how phase-locked inputs form oscillating membrane potentials. We derive analytical expressions that relate presynaptic, synaptic, and postsynaptic factors to the signal and noise components of the oscillation in both the synaptic conductance and the membrane potential. Numerical simulations demonstrate the validity of the theoretical formulations for the entire frequency ranges tested (1–8 kHz) and potential effects of higher harmonics on NL neurons with low best frequencies (<2 kHz).
doi:10.3389/fncom.2013.00151
PMCID: PMC3821005  PMID: 24265616
phase-locking; sound localization; auditory brainstem; periodic signals; oscillation; owl
20.  Heterogeneous kinetics and pharmacology of synaptic inhibition in the chick auditory brainstem 
Identification of shared features between avian and mammalian auditory brainstem circuits has provided much insight into the mechanisms underlying early auditory processing. However, previous studies have highlighted an apparent difference in inhibitory systems; synaptic inhibition is thought to be slow and GABAergic in birds, but to have fast kinetics and be predominantly glycinergic in mammals. Using patch-clamp recordings in chick brainstem slices, we found this distinction is not exclusively true. Consistent with previous work, inhibitory postsynaptic currents (IPSCs) in nucleus magnocellularis (NM) were slow and mediated by GABAA receptors. However, IPSCs in nucleus laminaris (NL) and a subset of neurons in nucleus angularis (NA) had rapid time courses two to three-fold faster than those in NM. Further, we found IPSCs in NA were mediated by both glycine and GABAA receptors, demonstrating for the first time a role for fast glycinergic transmission in the avian auditory brainstem. Although NM, NL and NA have unique roles in auditory processing, the majority of inhibitory input to each nucleus arises from the same source, ipsilateral superior olivary nucleus (SON). Our results demonstrate remarkable diversity of inhibitory transmission among the avian brainstem nuclei and suggest differential glycine and GABAA receptor activity tailors inhibition to the specific functional roles of NM, NL, and NA despite common SON input. We additionally observed that glycinergic/GABAergic activity in NA was usually depolarizing and could elicit spiking activity in NA neurons. Because NA projects to SON, these excitatory effects may influence the recruitment of inhibitory activity in the brainstem nuclei.
doi:10.1523/JNEUROSCI.0103-09.2009
PMCID: PMC2894706  PMID: 19641125
Auditory; GABA; Glycine; Patch Clamp; Inhibition; Synapse
21.  Regulation of glutamatergic and GABAergic neurotransmission in the chick nucleus laminaris: role of N-type calcium channels 
Neuroscience  2009;164(3):1009-1019.
Neurons in the chicken nucleus laminaris (NL), the third-order auditory nucleus involved in azimuth sound localization, receive bilaterally segregated (ipsilateral vs. contralateral) glutamatergic excitation from the cochlear nucleus magnocellularis and GABAergic inhibition from the ipsilateral superior olivary nucleus. Here, I investigate the voltage-gated calcium channels (VGCCs) that trigger the excitatory and the inhibitory transmission in the NL. Whole-cell recordings were performed in acute brainstem slices. The excitatory transmission was predominantly mediated by N-type VGCCs, as the specific N-type blocker ω-Conotoxin-GVIA (1-2.5 μM) inhibited excitatory postsynaptic currents (EPSCs) by ∼90%. Blockers for P/Q- and L-type VGCCs produced no inhibition, and blockade of R-type VGCCs produced a small inhibition. In individual cells, the effect of each VGCC blocker on the EPSC elicited by activation of the ipsilateral input was the same as that on the EPSC elicited by activation of the contralateral input, and the two EPSCs had similar kinetics, suggesting physiological symmetry between the two glutamatergic inputs to single NL neurons. The inhibitory transmission in NL neurons was almost exclusively mediated by N-type VGCCs, as ω-Conotoxin-GVIA (1 μM) produced a ∼90% reduction of inhibitory postsynaptic currents, whereas blockers for other VGCCs produced no inhibition. In conclusion, N-type VGCCs play a dominant role in triggering both the excitatory and the inhibitory transmission in the NL, and the presynaptic VGCCs that mediate the two bilaterally segregated glutamatergic inputs to individual NL neurons are identical. These features may play a role in optimizing coincidence detection in NL neurons.
doi:10.1016/j.neuroscience.2009.09.013
PMCID: PMC2784256  PMID: 19751802
voltage-gated calcium channel; excitatory postsynaptic current; inhibitory postsynaptic current; coincidence detection; sound localization
22.  Alteration of CaBP Expression Pattern in the Nucleus Magnocellularis following Unilateral Cochlear Ablation in Adult Zebra Finches 
PLoS ONE  2013;8(11):e79297.
Songbirds have the rare ability of auditory-vocal learning and maintenance. Up to now, the organization and function of the nucleus magnocellularis (NM), the first relay of the avian ascending auditory pathway is largely based on studies in non-vocal learning species, such as chickens and owls. To investigate whether NM exhibits different histochemical properties associated with auditory processing in songbirds, we examined the expression patterns of three calcium-binding proteins (CaBPs), including calretinin (CR), parvalbumin (PV) and calbindin-D28k (CB), and their relations to auditory inputs in NM in adult zebra finches. We found enriched and co-localized immunostaining of CR, PV and CB in the majority of NM neurons, without neuronal population preference. Furthermore, they were sensitive to adult deafferentation with differential plasticity patterns. After unilateral cochlear removal, CR staining in the ipsilateral NM decreased appreciably at 3 days after surgery, and continued to decline thereafter. PV staining showed down-regulation first at 3 days, but subsequently recovered slightly. CB staining did not significantly decrease until 7 days after surgery. Our findings suggest that the three CaBPs might play distinct roles in association with auditory processing in zebra finches. These results are in contrast to the findings in the NM of chickens where CR is the predominant CaBP and deafferentation had no apparent effect on its expression. Further extended studies in other avian species are required to establish whether the difference in CaBP patterns in NM is functionally related to the different auditory-vocal behaviors.
doi:10.1371/journal.pone.0079297
PMCID: PMC3828381  PMID: 24244471
23.  Modeling coincidence detection in nucleus laminaris 
Biological Cybernetics  2003;89(5):388-396.
A biologically detailed model of the binaural avian nucleus laminaris is constructed, as a two-dimensional array of multicompartment, conductance-based neurons, along tonotopic and interaural time delay (ITD) axes. The model is based primarily on data from chick nucleus laminaris. Typical chick-like parameters perform ITD discrimination up to 2 kHz, and enhancements for barn owl perform ITD discrimination up to 6 kHz. The dendritic length gradient of NL is explained concisely. The response to binaural out-of-phase input is suppressed well below the response to monaural input (without any spontaneous activity on the opposite side), implicating active potassium channels as crucial to good ITD discrimination.
doi:10.1007/s00422-003-0444-4
PMCID: PMC3269635  PMID: 14669019
24.  Global analysis of aberrant pre-mRNA splicing in glioblastoma using exon expression arrays 
BMC Genomics  2008;9:216.
Background
Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples.
Results
In total, we confirmed 14 genes with glioma-specific splicing; seven were novel events identified by the exon expression array (A2BP1, BCAS1, CACNA1G, CLTA, KCNC2, SNCB, and TPD52L2). Our data indicate that large changes (> 5-fold) in alternative splicing are infrequent in gliomagenesis (< 3% of interrogated RefSeq entries). The lack of splicing changes may derive from the small number of splicing factors observed to be aberrantly expressed.
Conclusion
While we observed some tumor-specific alternative splicing, the number of genes showing exclusive tumor-specific isoforms was on the order of tens, rather than the hundreds suggested previously by in silico mining. Given the important role of alternative splicing in neural differentiation, there may be selective pressure to maintain a majority of splicing events in order to retain glial-like characteristics of the tumor cells.
doi:10.1186/1471-2164-9-216
PMCID: PMC2410136  PMID: 18474104
25.  Next generation sequencing for molecular diagnosis of neurological disorders using ataxias as a model 
Brain  2013;136(10):3106-3118.
Many neurological conditions are caused by immensely heterogeneous gene mutations. The diagnostic process is often long and complex with most patients undergoing multiple invasive and costly investigations without ever reaching a conclusive molecular diagnosis. The advent of massively parallel, next-generation sequencing promises to revolutionize genetic testing and shorten the ‘diagnostic odyssey’ for many of these patients. We performed a pilot study using heterogeneous ataxias as a model neurogenetic disorder to assess the introduction of next-generation sequencing into clinical practice. We captured 58 known human ataxia genes followed by Illumina Next-Generation Sequencing in 50 highly heterogeneous patients with ataxia who had been extensively investigated and were refractory to diagnosis. All cases had been tested for spinocerebellar ataxia 1–3, 6, 7 and Friedrich’s ataxia and had multiple other biochemical, genetic and invasive tests. In those cases where we identified the genetic mutation, we determined the time to diagnosis. Pathogenicity was assessed using a bioinformatics pipeline and novel variants were validated using functional experiments. The overall detection rate in our heterogeneous cohort was 18% and varied from 8.3% in those with an adult onset progressive disorder to 40% in those with a childhood or adolescent onset progressive disorder. The highest detection rate was in those with an adolescent onset and a family history (75%). The majority of cases with detectable mutations had a childhood onset but most are now adults, reflecting the long delay in diagnosis. The delays were primarily related to lack of easily available clinical testing, but other factors included the presence of atypical phenotypes and the use of indirect testing. In the cases where we made an eventual diagnosis, the delay was 3–35 years (mean 18.1 years). Alignment and coverage metrics indicated that the capture and sequencing was highly efficient and the consumable cost was ∼£400 (€460 or US$620). Our pathogenicity interpretation pathway predicted 13 different mutations in eight different genes: PRKCG, TTBK2, SETX, SPTBN2, SACS, MRE11, KCNC3 and DARS2 of which nine were novel including one causing a newly described recessive ataxia syndrome. Genetic testing using targeted capture followed by next-generation sequencing was efficient, cost-effective, and enabled a molecular diagnosis in many refractory cases. A specific challenge of next-generation sequencing data is pathogenicity interpretation, but functional analysis confirmed the pathogenicity of novel variants showing that the pipeline was robust. Our results have broad implications for clinical neurology practice and the approach to diagnostic testing.
doi:10.1093/brain/awt236
PMCID: PMC3784284  PMID: 24030952
ataxia; genetics; autosomal dominant cerebellar ataxia; autosomal recessive cerebellar ataxia; diagnosis

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