T-type calcium channel:from physiological function to therapeutical targeting – Exploring neuronal development

Tämän tutkimuksen tarkoitus oli tutkia T-tyypin kalsiumkanavan toimintaa ja sen mahdollista roolia neuronaalisten kantasolujen migraatiossa. T-tyypin kalsiumkanavan tehtävän kehittyneissä aivoissa tiedetään olevan elektroenkefalografisten oskillaatioiden tuottaminen. Nämä taas ovat eräiden fysiologisten ja patofysiologisten tapahtumien säätelyssä avainasemassa. Tällaisia tapahtumia ovat uni, muisti, oppiminen ja epileptiset poissaolokohtaukset. Näiden lisäksi T-tyypin kalsiumkanavalla on myös periferaalisia vaikutuksia, mutta tämä tutkielma keskittyy sen neuronaalisiin toimintoihin. Tämän matalan jännitteen säätelemän kanavan toiminta neurogeneesin aikana on vähemmän tutkittua ja tunnettua kuin sen vaikutukset kehittyneissä aivoissa. T-tyypin kalsiumkanavan tiedetään edistävän kantasolujen proliferaatiota ja erilaistumista neurogeneesiksen aikana, mutta vaikutukset niiden migraatioon ovat vähemmän tunnetut. Tämä tutkimus näyttää T-tyypin kalsiumkanavan todennäköisesti osallistuvan neuronaaliseen migraatioon hiiren alkion subventrikkeli alueelta eristetyillä kanta- tai progeniittorisoluilla tehdyissä kokeissa. Selektiiviset T-tyypin kalsiumkanavan antagonistit, etosuksimidi, nikkeli ja skorpionitoksiini, kurtoxin hidastivat migraatiota erilaistuvissa progeniittorisoluissa. Tämä tutkimus koostuu kirjallisuuskatsauksesta ja kokeellisesta osasta. Tämän tutkimuksen toinen tarkoitus oli esitellä vaihtoehtoinen lähestymistapa invasiiviselle kantasoluterapialle, joka vaatii kantasolujen viljelyä ja siirtämistä ihmiseen. Tämä toinen tapa on endogeenisten kantasolujen eiinvasiivinen stimulointi, jolla ne saadaan migratoitumaan kohdekudokseen, erilaistumaan siellä ja tehtävänsä suoritettuaan lopettamaan jakaantumisen. Non-invasiivinen kantasoluterapia on vasta tiensä alussa, ja tarvitsee farmakologista osaamista kehittyäkseen. Joitain onnistuneita ei-invasiivisia hoitoja on jo tehty selkärangan vaurioiden korjaamisessa. Vastaavanlaisia menetelmiä voitaisiin käyttää myös keskushermoston vaurioiden ja neurodegeneratiivisten sairauksien hoidossa. Näiden menetelmien kehittäminen vaatii endogeenisten kantasoluja inhiboivien ja indusoivien mekanismien tuntemista. Yksi tärkeä kantasolujen erilaistumista stimuloiva tekijä on kalsiumioni. Jänniteherkät kalsiumkanavat osallistuvat kaikkiin neurogeneesiksen eri vaiheisiin. T-tyypin kalsiumkanava, joka ekspressoituu suuressa määrin keskushermoston kehityksen alkuvaiheessa ja vähenee neuronaalisen kehityksen edetessä, saattaa olla oleellisessa asemassa progeniittorisolujen ohjaamisessa.

Loud calls of male Nilgiri langurs (Presbytis johnii): Age-, individual-, and population-specific differences

Adult male Nilgiri langurs (Presbytis johnii) utter loud call bouts consisting of one or more phrases. Phrases are made up of several units showing similar or different structural features. The units involved differ with respect to not only their physical structure but also their overall utilization: three vocal patterns are uttered exclusively by mature males living in bisexual groups or all-male bands and, in addition to being part of loud call bouts, are given during encounters with terrestrial predators; two vocal patterns are uttered by males and females, again not just as constituents of loud calls; and one vocal pattern is given exclusively by mature males living in bisexual groups. Within a given bout, phrases differ not only with respect to their composition but also in their temporal organization. In addition to the acoustic components, loud calls are regularly accompanied by stereotyped motoric displays. The motoric and acoustic components of loud call displays appear independently of each other and at different times during ontogeny. The development of the display is characterized by combination of units with different structural features and synchronization of vocal and motoric components. Although more evidence is needed, our observations suggest that the development of loud call displays coincides with the aquisitation of social maturation and competence and requires not only social experience but also a certain amount of motoric training. In spite of the high degree of ritualization, loud call displays are not completely fixed in form, but instead are open to individual- and population-specific variation.

Substance misuse problems during pregnancy with special emphasis on buprenorphine

Maternal drug abuse during pregnancy endangers the future health and wellbeing of the infant and growing child. On the other hand, via maternal abstinence, these problems would never occur; so the problems would be totally preventable. Buprenorphine is widely used in opioid maintenance treatment as a substitute medication. In Finland, during 2000 s buprenorphine misuse has steadily increased. In 2009 almost one third of clientele of substance treatment units were in treatment because of buprenorphine dependence. At Helsinki Women s Clinic the first child with prenatal buprenorphine exposure was born in 2001. During 1992-2001 in the three capital area maternity hospitals (Women s clinic, Maternity hospital, Jorvi hospital) 524 women were followed at special antenatal clinics due to substance abuse problems. Three control women were drawn from birth register to each case woman and matched for parity and same place and date of the index birth. According to register data mortality rate was 38-fold higher among cases than controls within 6-15 years after index birth. Especially, the risk for violent or accidental death was increased. The women with substance misuse problems had also elevated risk for viral hepatitis and psychiatric morbidity. They were more often reimbursed for psychopharmaceuticals. Disability pensions and rehabilitation allowances were more often granted to cases than controls. In total 626 children were born from these pregnancies. According to register data 38% of these children were placed in out-of-home care as part of child protection services by the age of two years, and half of them by the age of 12 years, the median follow-up time was 5.8 years. The risk for out-of-home care was associated with factors identifiable during the pre- and perinatal period. In 2002-2005 67 pregnant women with buprenorphine dependence were followed up at the Helsinki University Hospital, Department of Obstetrics and Gynecology. Their pregnancies were uneventful. The prematurity rate was similar and there were no more major anomalies compared to the national statistics. The neonates were lighter compared to the national statistics. They were also born in good condition, with no perinatal hypoxia as defined by standard clinical parameters or certain biochemical markers in the cord blood: erythropoietin, S100 and cardiac troponin-t. Almost 80% of newborns developed neonatal abstinence syndrome (NAS) and two third of them needed morphine medication for it. Maternal smoking over ten cigarettes per day aggravated and benzodiazepine use attenuated NAS. An infant s highest urinary norbuprenorphine concentration during their first 3 days of life correlated with the duration of morphine treatment. The average length of infant s hospital stay was 25 days.

Epoch extraction from linear prediction residual for identification of closed glottis interval

In voiced speech analysis epochal information is useful in accurate estimation of pitch periods and the frequency response of the vocal tract system. Ideally, linear prediction (LP) residual should give impulses at epochs. However, there are often ambiguities in the direct use of LP residual since samples of either polarity occur around epochs. Further, since the digital inverse filter does not compensate the phase response of the vocal tract system exactly, there is an uncertainty in the estimated epoch position. In this paper we present an interpretation of LP residual by considering the effect of the following factors: 1) the shape of glottal pulses, 2) inaccurate estimation of formants and bandwidths, 3) phase angles of formants at the instants of excitation, and 4) zeros in the vocal tract system. A method for the unambiguous identification of epochs from LP residual is then presented. The accuracy of the method is tested by comparing the results with the epochs obtained from the estimated glottal pulse shapes for several vowel segments. The method is used to identify the closed glottis interval for the estimation of the true frequency response of the vocal tract system.

Carnatic music analysis: Shadja, swara identification and rAga verification in AlApana using stochastic models

We analyze the AlApana of a Carnatic music piece without the prior knowledge of the singer or the rAga. AlApana is ameans to communicate to the audience, the flavor or the bhAva of the rAga through the permitted notes and its phrases. The input to our analysis is a recording of the vocal AlApana along with the accompanying instrument. The AdhAra shadja(base note) of the singer for that AlApana is estimated through a stochastic model of note frequencies. Based on the shadja, we identify the notes (swaras) used in the AlApana using a semi-continuous GMM. Using the probabilities of each note interval, we recognize swaras of the AlApana. For sampurNa rAgas, we can identify the possible rAga, based on the swaras. We have been able to achieve correct shadja identification, which is crucial to all further steps, in 88.8% of 55 AlApanas. Among them (48 AlApanas of 7 rAgas), we get 91.5% correct swara identification and 62.13% correct R (rAga) accuracy.

Zero-phase inverse filtering for extraction of source characteristics

The instants at which significant excitation of vocal tract take place during voicing are referred to as epochs. Epochs and strengths of excitation pulses at epochs are useful in characterizing voice source. Epoch filtering technique proposed by the authors determine epochs from speech waveform. In this paper we propose zero-phase inverse filtering to obtain strengths of excitation pulses at epochs. Zero-phase inverse filter compensates the gross spectral envelope of short-time spectrum of speech without affecting phase characteristics. Linear prediction analysis is used to realize the zero-phase inverse filter. Source characteristics that can be derived from speech using this technique are illustrated with examples.

A Scalable Architecture for VoIP Conferencing

Real-Time services are traditionally supported on circuit switched network. However, there is a need to port these services on packet switched network. Architecture for audio conferencing application over the Internet in the light of ITU-T H.323 recommendations is considered. In a conference, considering packets only from a set of selected clients can reduce speech quality degradation because mixing packets from all clients can lead to lack of speech clarity. A distributed algorithm and architecture for selecting clients for mixing is suggested here based on a new quantifier of the voice activity called “Loudness Number” (LN). The proposed system distributes the computation load and reduces the load on client terminals. The highlights of this architecture are scalability, bandwidth saving and speech quality enhancement. Client selection for playing out tries to mimic a physical conference where the most vocal participants attract more attention. The contributions of the paper are expected to aid H.323 recommendations implementations for Multipoint Processors (MP). A working prototype based on the proposed architecture is already functional.

A finite-rate-of-innovation signal sampling perspective of the source-filter model of speech production

We consider the speech production mechanism and the asso- ciated linear source-filter model. For voiced speech sounds in particular, the source/glottal excitation is modeled as a stream of impulses and the filter as a cascade of second-order resonators. We show that the process of sampling speech signals can be modeled as filtering a stream of Dirac impulses (a model for the excitation) with a kernel function (the vocal tract response),and then sampling uniformly. We show that the problem of esti- mating the excitation is equivalent to the problem of recovering a stream of Dirac impulses from samples of a filtered version. We present associated algorithms based on the annihilating filter and also make a comparison with the classical linear prediction technique, which is well known in speech analysis. Results on synthesized as well as natural speech data are presented.

Microglia play a major role in direct viral-induced demyelination

Microglia are the resident macrophage-like populations in the central nervous system (CNS). Microglia remain quiescent, unable to perform effector and antigen presentation (APC) functions until activated by injury or infection, and have been suggested to represent the first line of defence for the CNS. Previous studies demonstrated that microglia can be persistently infected by neurotropic mouse hepatitis virus (MHV) which causes meningoencephalitis, myelitis with subsequent axonal loss, and demyelination and serve as a virus-induced model of human neurological disease multiple sclerosis (MS). Current studies revealed that MHV infection is associated with the pronounced activation of microglia during acute inflammation, as evidenced by characteristic changes in cellular morphology and increased expression of microglia-specific proteins, Iba1 (ionized calcium-binding adaptor molecule 1), which is a macrophage/microglia-specific novel calcium-binding protein and involved in membrane ruffling and phagocytosis. During chronic inflammation (day 30 postinfection), microglia were still present within areas of demyelination. Experiments performed in ex vivo spinal cord slice culture and in vitro neonatal microglial culture confirmed direct microglial infection. Our results suggest that MHV can directly infect and activate microglia during acute inflammation, which in turn during chronic inflammation stage causes phagocytosis of myelin sheath leading to chronic inflammatory demyelination.

Highly sensitive and rapid detection of acetylcholine using an ITO plate modified with platinum-graphene nanoparticles

Determining the concentrations of acetylcholine (ACh) and choline (Ch) is clinically important. ACh is a neurotransmitter that acts as a key link in the communication between neurons in the spinal cord and in nerve skeletal junctions in vertebrates, and plays an important role in transmitting signals in the brain. A bienzymatic sensor for the detection of ACh was prepared by co-immobilizing choline oxidase (ChO) and acetylcholinesterase (AChE) on graphene matrix/platinum nanoparticles, and then electrodepositing them on an ITO-coated glass plate. Graphene nanoparticles were decorated with platinum nanoparticles and were electrodeposited on a modified ITO-coated glass plate to form a modified electrode. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies. The optimum response of the enzyme electrode was obtained at pH 7.0 and 35 degrees C. The response time of this ACh-sensing system was shown to be 4 s. The linear range of responses to ACh was 0.005-700 mu M. This biosensor exhibits excellent anti-interferential abilities and good stability, retaining 50% of its original current even after 4 months. It has been applied for the detection of ACh levels in human serum samples.

Sensing of Stimulus Artifact Suppressed Signals From Electrode Interfaces

Stimulus artifacts inhibit reliable acquisition of biological evoked potentials for several milliseconds if an electrode contact is utilized for both electrical stimulation and recording purposes. This hinders the measurement of evoked short-latency biological responses, which is otherwise elicited by stimulation in implantable prosthetic devices. We present an improved stimulus artifact suppression scheme using two electrode simultaneous stimulation and differential readout using high-gain amplifiers. Substantial reduction of artifact duration has been shown possible through the common-mode rejection property of an instrumentation amplifier for electrode interfaces. The performance of this method depends on good matching of electrode-electrolyte interface properties of the chosen electrode pair. A novel calibration algorithm has been developed that helps in artificial matching of impedance and thereby achieves the required performance in artifact suppression. Stimulus artifact duration has been reduced down to 50 mu s from the stimulation-cum-recording electrodes, which is similar to 6x improvement over the present state of the art. The system is characterized with emulated resistor-capacitor loads and a variety of in-vitro metal electrodes dipped in saline environment. The proposed method is going to be useful for closed-loop electrical stimulation and recording studies, such as bidirectional neural prosthesis of retina, cochlea, brain, and spinal cord.

Demodulation of Narrowband Speech Spectrograms Using the Riesz Transform

We propose a two-dimensional (2-D) multicomponent amplitude-modulation, frequency-modulation (AM-FM) model for a spectrogram patch corresponding to voiced speech, and develop a new demodulation algorithm to effectively separate the AM, which is related to the vocal tract response, and the carrier, which is related to the excitation. The demodulation algorithm is based on the Riesz transform and is developed along the lines of Hilbert-transform-based demodulation for 1-D AM-FM signals. We compare the performance of the Riesz transform technique with that of the sinusoidal demodulation technique on real speech data. Experimental results show that the Riesz-transform-based demodulation technique represents spectrogram patches accurately. The spectrograms reconstructed from the demodulated AM and carrier are inverted and the corresponding speech signal is synthesized. The signal-to-noise ratio (SNR) of the reconstructed speech signal, with respect to clean speech, was found to be 2 to 4 dB higher in case of the Riesz transform technique than the sinusoidal demodulation technique.

An Alternating l(p) - l(2) Projections Algorithm (ALPA) for Speech Modeling using Sparsity Constraints

We address the problem of separating a speech signal into its excitation and vocal-tract filter components, which falls within the framework of blind deconvolution. Typically, the excitation in case of voiced speech is assumed to be sparse and the vocal-tract filter stable. We develop an alternating l(p) - l(2) projections algorithm (ALPA) to perform deconvolution taking into account these constraints. The algorithm is iterative, and alternates between two solution spaces. The initialization is based on the standard linear prediction decomposition of a speech signal into an autoregressive filter and prediction residue. In every iteration, a sparse excitation is estimated by optimizing an l(p)-norm-based cost and the vocal-tract filter is derived as a solution to a standard least-squares minimization problem. We validate the algorithm on voiced segments of natural speech signals and show applications to epoch estimation. We also present comparisons with state-of-the-art techniques and show that ALPA gives a sparser impulse-like excitation, where the impulses directly denote the epochs or instants of significant excitation.

Ischaemic concentrations of lactate increase TREK1 channel activity by interacting with a single histidine residue in the carboxy terminal domain

Key points The physiological metabolite, lactate and the two-pore domain leak potassium channel, TREK1 are known neuroprotectants against cerebral ischaemia. However, it is not known whether lactate interacts with TREK1 channel to provide neuroprotection. In this study we show that lactate increases TREK1 channel activity and hyperpolarizes CA1 stratum radiatum astrocytes in hippocampal slices. Lactate increases open probability and decreases longer close time of the human (h)TREK1 channel in a concentration dependent manner. Lactate interacts with histidine 328 (H328) in the carboxy terminal domain of hTREK1 channel to decrease its dwell time in the longer closed state. This interaction was dependent on the charge on H328. Lactate-insensitive mutant H328A hTREK1 showed pH sensitivity similar to wild-type hTREK1, indicating that the effect of lactate on hTREK1 is independent of pH change. AbstractA rise in lactate concentration and the leak potassium channel TREK1 have been independently associated with cerebral ischaemia. Recent literature suggests lactate to be neuroprotective and TREK1 knockout mice show an increased sensitivity to brain and spinal cord ischaemia; however, the connecting link between the two is missing. Therefore we hypothesized that lactate might interact with TREK1 channels. In the present study, we show that lactate at ischaemic concentrations (15-30mm) at pH7.4 increases TREK1 current in CA1 stratum radiatum astrocytes and causes membrane hyperpolarization. We confirm the intracellular action of lactate on TREK1 in hippocampal slices using monocarboxylate transporter blockers and at single channel level in cell-free inside-out membrane patches. The intracellular effect of lactate on TREK1 is specific since other monocarboxylates such as pyruvate and acetate at pH7.4 failed to increase TREK1 current. Deletion and point mutation experiments suggest that lactate decreases the longer close dwell time incrementally with increase in lactate concentration by interacting with the histidine residue at position 328 (H328) in the carboxy terminal domain of the TREK1 channel. The interaction of lactate with H328 is dependent on the charge on the histidine residue since isosteric mutation of H328 to glutamine did not show an increase in TREK1 channel activity with lactate. This is the first demonstration of a direct effect of lactate on ion channel activity. The action of lactate on the TREK1 channel signifies a separate neuroprotective mechanism in ischaemia since it was found to be independent of the effect of acidic pH on channel activity. Key points The physiological metabolite, lactate and the two-pore domain leak potassium channel, TREK1 are known neuroprotectants against cerebral ischaemia. However, it is not known whether lactate interacts with TREK1 channel to provide neuroprotection. In this study we show that lactate increases TREK1 channel activity and hyperpolarizes CA1 stratum radiatum astrocytes in hippocampal slices. Lactate increases open probability and decreases longer close time of the human (h)TREK1 channel in a concentration dependent manner. Lactate interacts with histidine 328 (H328) in the carboxy terminal domain of hTREK1 channel to decrease its dwell time in the longer closed state. This interaction was dependent on the charge on H328. Lactate-insensitive mutant H328A hTREK1 showed pH sensitivity similar to wild-type hTREK1, indicating that the effect of lactate on hTREK1 is independent of pH change.

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.