Molecular dissection of I(A) in cortical pyramidal neurons reveals three distinct components encoded by Kv4.2, Kv4.3, and Kv1.4 alpha-subunits

The rapidly activating and inactivating voltage-gated K(+) (Kv) current, I(A), is broadly expressed in neurons and is a key regulator of action potential repolarization, repetitive firing, backpropagation (into dendrites) of action potentials, and responses to synaptic inputs. Interestingly, results from previous studies on a number of neuronal cell types, including hippocampal, cortical, and spinal neurons, suggest that macroscopic I(A) is composed of multiple components and that each component is likely encoded by distinct Kv channel alpha-subunits. The goals of the experiments presented here were to test this hypothesis and to determine the molecular identities of the Kv channel alpha-subunits that generate I(A) in cortical pyramidal neurons. Combining genetic disruption of individual Kv alpha-subunit genes with pharmacological approaches to block Kv currents selectively, the experiments here revealed that Kv1.4, Kv4.2, and Kv4.3 alpha-subunits encode distinct components of I(A) that together underlie the macroscopic I(A) in mouse (male and female) cortical pyramidal neurons. Recordings from neurons lacking both Kv4.2 and Kv4.3 (Kv4.2(-/-)/Kv4.3(-/-)) revealed that, although Kv1.4 encodes a minor component of I(A), the Kv1.4-encoded current was found in all the Kv4.2(-/-)/Kv4.3(-/-) cortical pyramidal neurons examined. Of the cortical pyramidal neurons lacking both Kv4.2 and Kv1.4, 90% expressed a Kv4.3-encoded I(A) larger in amplitude than the Kv1.4-encoded component. The experimental findings also demonstrate that the targeted deletion of the individual Kv alpha-subunits encoding components of I(A) results in electrical remodeling that is Kv alpha-subunit specific

Holistic corpus-based dialectology

This paper is concerned with sketching future directions for corpus-based dialectology. We advocate a holistic approach to the study of geographically conditioned linguistic variability, and we present a suitable methodology, 'corpusbased dialectometry', in exactly this spirit. Specifically, we argue that in order to live up to the potential of the corpus-based method, practitioners need to (i) abandon their exclusive focus on individual linguistic features in favor of the study of feature aggregates, (ii) draw on computationally advanced multivariate analysis techniques (such as multidimensional scaling, cluster analysis, and principal component analysis), and (iii) aid interpretation of empirical results by marshalling state-of-the-art data visualization techniques. To exemplify this line of analysis, we present a case study which explores joint frequency variability of 57 morphosyntax features in 34 dialects all over Great Britain

Determinants of English accents

In this study we investigate which factors affect the degree of non-native accent of L2 speakers of English who learned English in school and mostly lived for some time in an anglophone setting. We use data from the Speech Accent Archive containing over 700 speakers speaking almost 160 different native languages. We show that besides several important predictors, including the age of English onset and length of anglophone residence, the linguistic distance between the speaker’s native language and English is a significant predictor of the degree of non-native accent in pronunciation. This study extends an earlier study which only focused on Indo-European L2 learners of Dutch and used a general speaking proficiency measure

Interdependent roles for accessory KChIP2, KChIP3, and KChIP4 subunits in the generation of Kv4-encoded IA channels in cortical pyramidal neurons

The rapidly activating and inactivating voltage-dependent outward K(+) (Kv) current, I(A), is widely expressed in central and peripheral neurons. I(A) has long been recognized to play important roles in determining neuronal firing properties and regulating neuronal excitability. Previous work demonstrated that Kv4.2 and Kv4.3 α-subunits are the primary determinants of I(A) in mouse cortical pyramidal neurons. Accumulating evidence indicates that native neuronal Kv4 channels function in macromolecular protein complexes that contain accessory subunits and other regulatory molecules. The K(+) Channel Interacting Proteins (KChIPs) are among the identified Kv4 channel accessory subunits and are thought to be important for the formation and functioning of neuronal Kv4 channel complexes. Molecular genetic, biochemical and electrophysiological approaches were exploited in the experiments described here to examine directly the roles of KChIPs in the generation of functional Kv4-encoded I(A) channels. These combined experiments revealed that KChIP2, KChIP3 and KChIP4 are robustly expressed in adult mouse posterior (visual) cortex and that all three proteins co-immunoprecipitate with Kv4.2. In addition, in cortical pyramidal neurons from mice lacking KChIP3 (KChIP3(−/−)), mean I(A) densities were reduced modestly, whereas in mean I(A) densities in KChIP2(−/−) and WT neurons were not significantly different. Interestingly, in both KChIP3(−/−) and KChIP2(−/−) cortices the expression levels of the other KChIPs (KChIP2 and 4 or KChIP3 and 4, respectively) were increased. In neurons expressing constructs to mediate simultaneous RNA interference-induced reductions in the expression of KChIP2, 3 and 4, I(A) densities were markedly reduced and Kv current remodeling was evident

Onnentupa Oy:n jälkihuoltosuunnitelma

Laurea-ammattikorkeakoulu Tiivistelmä Hyvinkää Sosiaalialan ko Seija Mässeli Onnentupa Oy:n jälkihuoltosuunnitelma Vuosi 2013 Sivumäärä 58 Tämän opinnäytetyön toimeksiantona oli tuottaa yksityiselle lastensuojelun yrittäjälle, Onnentupa Oy:lle jälkihuollon ohjeistus. Onnentupa Oy koostuu neljästä Tuusulan alueella sijaitsevasta perhekodista, jotka kuuluvat Ammatillisten perhekotien liittoon. Jälkihuolto on noussut koko ajan ajankohtaisemmaksi aiheeksi, ja yksityisiltä perhekodeilta kysytään koko ajan enemmän jälkihuollon mahdollisuuksista. Tämän kehityksen tueksi tarvittiin jälkihuollon ohjeistus, josta muodostuisi toimiva arjen työväline. Työ on toteutettu kvalitatiivisena aineistotutkimuksena, jossa on käytetty kirjallisten lähteiden lisäksi Onnentupa Oy:n työntekijöiden haastatteluja. Näin on pyritty varmistamaan, että lopputulos on yksityisyrittäjän toiveiden mukainen ja tietyille yksiköille sopiva. Opinnäytetyö sisältää raportin, jossa käsitellään aiheeseen liittyvä teoriaosuus, jossa kuvataan lastensuojelu kokonaisena prosessina ja tarkastellaan etenkin perhekotien tarjoamaa sijaishuoltoa. Nuoruus herkkänä ikävaiheena on myös otettu tässä raportissa erilliseksi tarkastelun kohteeksi. Varsinaisen raportin jälkeen esittelen opinnäytetyössäni konkreettisen jälkihuoltosuunnitelman Onnentupa Oy:lle. Jälkihuolto on osa lastensuojelun kenttää, mutta sitä ei varsinaisesti pidetä kokonaan omana alueenaan vaan enemmänkin sijaishuollon jatkeena. Jälkihuollon perusasioista, kuten sen kestosta ja järjestämisvelvollisuudesta määrää lastensuojelulaki, mutta laki ei määrittele jälkihuollolle tarkkaa sisältöä. Näin ollen tutkimuskysymyksekseni muodostui pohdinta siitä, millainen on hyvä jälkihuoltosuunnitelma. Lopputuloksena syntyi konkreettinen ohjeistus, joka käsittelee jälkihuollon prosessin seikkaperäisesti aina jälkihuollon suunnittelusta sen arviointiin ja päättymiseen. Ohjeistus on pidetty yleisluontoisena, jotta sen voi yksilöidä jokaiselle itsenäistyvälle nuorelle sopivaksi. Avainsanat: Lastensuojelu, nuoruus, sijaishuolto, perhekoti, jälkihuolto.Laurea University of Applied Sciences Abstract Hyvinkää Bachelor of social services Seija Mässeli The after-care plan for Onnentupa Oy Year 2013 Pages 58 The assignment for this thesis was to produce an after-care plan for a private entrepreneur in child-protective services; Onnentupa Oy. Onnentupa Oy consists of four family care units located in the area of Tuusula. All these family care units belong to the Ammatillisten perhekotien liitto (Union of professional family care units). After-care has consistently risen to be more and more topical issue, and private family care units get constantly asked more about the chances of after-care. In support of this development an after-care plan was needed that could provide a working tool in the business’s weekday. The work has been executed as a qualitative material research, in which literary sources have been used in addition of the interviews of Onnentupa Oy’s employees. This has been done to ensure that the product fits the wishes of the private entrepreneur and is fitting for specific care units. This thesis includes a report, which goes through the important theories related to the subject, like child-protective services as a whole process. Special attention is brought to the foster care that the family care units provide. A separate analysis of youth as a delicate age is also included in this report. After the actual report I go on to model the concrete after-care plan for Onnentupa Oy. After-care is part of the field of child-protective services, but it’s not really thought of as a whole separate entirety. Instead it is viewed as a continuation for foster care. Finland’s laws of child-protective services determine the basics of after-care, like how long it lasts and who is obliges to offer it as a service, but the law does not define the precise content of what should be included in an after-care plan. This leads to my research problem: what does a good after-care plan look like? As a final result of this thesis a concrete instruction was produced. It goes through the process of after-care in detail from the planning of after-care to its evaluation and ending the after-care. This instruction has been held general in nature, so that it can be individualized to every young man and woman who are in the process of gaining independence. Keywords child-protective services, youth, foster care, family care unit, after care

I(A) channels encoded by Kv1.4 and Kv4.2 regulate neuronal firing in the suprachiasmatic nucleus and circadian rhythms in locomotor activity

Neurons in the suprachiasmatic nucleus (SCN) display coordinated circadian changes in electrical activity that are critical for daily rhythms in physiology, metabolism, and behavior. SCN neurons depolarize spontaneously and fire repetitively during the day and hyperpolarize, drastically reducing firing rates, at night. To explore the hypothesis that rapidly activating and inactivating A-type (I(A)) voltage-gated K(+) (Kv) channels, which are also active at subthreshold membrane potentials, are critical regulators of the excitability of SCN neurons, we examined locomotor activity and SCN firing in mice lacking Kv1.4 (Kv1.4(-/-)), Kv4.2 (Kv4.2(-/-)), or Kv4.3 (Kv4.3(-/-)), the pore-forming (α) subunits of I(A) channels. Mice lacking either Kv1.4 or Kv4.2 α subunits have markedly shorter (0.5 h) periods of locomotor activity than wild-type (WT) mice. In vitro extracellular multi-electrode recordings revealed that Kv1.4(-/-) and Kv4.2(-/-) SCN neurons display circadian rhythms in repetitive firing, but with shorter periods (0.5 h) than WT cells. In contrast, the periods of wheel-running activity in Kv4.3(-/-) mice and firing in Kv4.3(-/-) SCN neurons were indistinguishable from WT animals and neurons. Quantitative real-time PCR revealed that the transcripts encoding all three Kv channel α subunits, Kv1.4, Kv4.2, and Kv4.3, are expressed constitutively throughout the day and night in the SCN. Together, these results demonstrate that Kv1.4- and Kv4.2-encoded I(A) channels regulate the intrinsic excitability of SCN neurons during the day and night and determine the period and amplitude of circadian rhythms in SCN neuron firing and locomotor behavior

The sodium channel accessory subunit Navβ1 regulates neuronal excitability through modulation of repolarizing voltage-gated K+ channels

The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (I(A)) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting and cell surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na(+) channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases I(A) densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of heterologously expressed Kv4.2 protein, resulting in greater total and cell surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Taken together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded I(A) channel complexes and a novel regulator of I(A) channel densities and neuronal excitability

Proteomic analyses of native brain KV4.2 channel complexes

Somatodendritic A-type (I(A)) voltage-gated K(+) (K(V)) channels are key regulators of neuronal excitability, functioning to control action potential waveforms, repetitive firing and the responses to synaptic inputs. Rapidly activating and inactivating somatodendritic I(A) channels are encoded by K(V)4 α subunits and accumulating evidence suggests that these channels function as components of macromolecular protein complexes. Mass spectrometry (MS)-based proteomic approaches were developed and exploited here to identify potential components and regulators of native brain K(V)4.2-encoded I(A) channel complexes. Using anti-K(V)4.2 specific antibodies, K(V)4.2 channel complexes were immunoprecipitated from adult wild type mouse brain. Parallel control experiments were performed on brain samples isolated from (K(V)4.2(−/−)) mice harboring a targeted disruption of the KCND2 (K(V)4.2) locus. Three proteomic strategies were employed: an in-gel approach, coupled to one-dimensional liquid chromatography-tandem MS (1D-LC-MS/MS), and two in-solution approaches, followed by 1D-or 2D-LC-MS/MS. The targeted in-gel 1D-LC-MS/MS analyses demonstrated the presence of the K(V)4 α subunits (K(V)4.2, K(V)4.3 and K(V)4.1) and the K(V)4 accessory, KChIP (KChIPI-4) and DPP (DPP6 and 10), proteins in native brain K(V)4.2 channel complexes. The more comprehensive, in-solution approach, coupled to 2D-LC-MS/MS, also called Multidimensional Protein Identification Technology (MudPIT), revealed that additional regulatory proteins, including the K(V) channel accessory subunit K(V)β1, are also components of native brain K(V)4.2 channel complexes. Additional biochemical and functional approaches will be required to elucidate the physiological roles of these newly identified K(V)4 interacting proteins