| 1. |
- Carinelli, S., et al.
(författare)
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Yoctomole electrochemical genosensing of Ebola virus cDNA by rolling circle and circle to circle amplification
- 2017
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 93, s. 65-71
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Tidskriftsartikel (refereegranskat)abstract
- This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries.
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| 2. |
- Chen, Wei-Ting, et al.
(författare)
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Spatial Transcriptomics and In Situ Sequencing to Study Alzheimer's Disease
- 2020
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 182:4, s. 976-
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Tidskriftsartikel (refereegranskat)abstract
- Although complex inflammatory-like alterations are observed around the amyloid plaques of Alzheimer's disease (AD), little is known about the molecular changes and cellular interactions that characterize this response, We investigate here, in an AD mouse model, the transcriptional changes occurring in tissue domains in a 100-mu m diameter around amyloid plaques using spatial transcriptomics. We demonstrate early alterations in a gene co-expression network enriched for myelin and oligodendrocyte genes (OLIGs), whereas a multicellular gene co-expression network of plaque-induced genes (PIGs) involving the complement system, oxidative stress, lysosomes, and inflammation is prominent in the later phase of the disease. We confirm the majority of the observed alterations at the cellular level using in situ sequencing on mouse and human brain sections. Genome-wide spatial transcriptomics analysis provides an unprecedented approach to untangle the dysregulated cellular network in the vicinity of pathogenic hallmarks of AD and other brain diseases.
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| 3. |
- Clausson, Carl-Magnus, 1985-, et al.
(författare)
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Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5, s. 12317:1-10
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Tidskriftsartikel (refereegranskat)abstract
- Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
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| 4. |
- Krzywkowski, Tomasz, et al.
(författare)
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Chimeric padlock and iLock probes for increased efficiency of targeted RNA detection
- 2019
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 25:1, s. 82-89
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Tidskriftsartikel (refereegranskat)abstract
- Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide poly-morphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.
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| 5. |
- Krzywkowski, Tomasz, et al.
(författare)
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Detection of miRNAs using chimeric DNA/RNA iLock probes utilizing novel activity of PBCV-1 DNA ligase : RNA-templated ligation of ssRNA
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Accurate detection of miRNAs with complementary probes is challenging due to the short target size, and often high sequence similarity between isoforms belonging to the same miRNA family. Ligation based methods can provide powerful discrimination of subtle sequence variation among target sequences, but they have been difficult to implement for direct RNA analysis due to the sloppiness and inefficiency of most DNA ligases on RNA substrates. In this work, we have studied if RNA substitutions in padlock probes can provide higher catalytic efficiencies for PBCV-1 DNA ligase on RNA substrates. We also characterise end-joining fidelity for Chlorella virus DNA ligase (PBCV DNA ligase 1) and T4RNA ligase 2 (T4Rnl2) in RNA-templated 3'-OH RNA/5’-pDNA chimeric probe ligation. Although we observed considerable ligation efficiency improvement towards short miRNA targets for PBCV-1 ligated chimeric probes, it showed no sequence specificity towards mismatches at the ligation junction. T4Rnl2 showed some base discrimination, but not satisfactory for robust RNA sequence analysis. To increase end-joining fidelity in PBCV-1 DNA ligase catalysed direct RNA detection assays (iLock probes), we have recently introduced an alternative ligation assay design in which ligation probes first undergo sequence- specific 5’ FLAP removal in order to create ligatable substrates. We have tested various chimeric iLock probe designs where RNA substitutions were introduced at different positions in the FLAP and at the ligation junction. We defined two particular nucleotide positions in the iLock probe sequence that when substituted with RNA, significantly increased iLock probe activation and ligation. We further characterized the end-joining fidelity of PBCV-1 and T4Rnl2 catalysed iLock reactions. Both enzymes showed high ligation fidelities for single nucleotide polymorphisms on RNA and miRNA. Finally, we demonstrate a multiplexed chimeric iLock probe miRNA profiling assay using sequencing-by-ligation as readout.
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| 6. |
- Krzywkowski, Tomasz, et al.
(författare)
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Limited reverse transcriptase activity of phi29 DNA polymerase
- 2018
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 46:7, s. 3625-3632
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Tidskriftsartikel (refereegranskat)abstract
- Phi29 (Phi 29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real time (SMRT) sequencing. Here, we report the ability of phi29 DNA polymerase to amplify RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions are amplified at a similar amplification rate as non-chimeric DNA substrates, and that consecutive RNA pyrimidines were generally preferred over purines. We observed RCA suppression with higher number of ribonucleotide substitutions, which was partially restored by interspacing RNA bases with DNA. We show that supplementing manganese ions as cofactor supports replication of RNAs during RCA. Sequencing of the RCA products demonstrated accurate base incorporation at the RNA base with both Mn2+ and Mg2+ as cofactors during replication, proving reverse transcriptase activity of the phi29 DNA polymerase. In summary, the ability of phi29 DNA polymerase to accept RNA-containing substrates broadens the spectrum of applications for phi29 DNA polymerase-mediated RCA. These include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.
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| 7. |
- Krzywkowski, Tomasz, et al.
(författare)
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Reverse-transcriptase activity of Phi29 DNA polymerase
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- F29 (Phi29) DNA polymerase is an enzyme commonly used in DNA amplification methods such as rolling circle amplification (RCA) and multiple strand displacement amplification (MDA), as well as in DNA sequencing methods such as single molecule real-time (SMRT) sequencing. Here we report the ability of F29 DNA polymerase to amplify partially RNA-containing circular substrates during RCA. We found that circular substrates with single RNA substitutions support a similar amplification rate as pure DNA substrates. We observed that increasing the number of consecutive RNA substitutions in the circular templates suppress replication, and cannot be recovered by addition of M-MuLV reverse-transcriptase. In summary, this novel ability of F29 to accept RNA-containing substrates broadens the spectrum of applications for F29 mediated RCA. Applications include amplification of chimeric circular probes, such as padlock probes and molecular inversion probes.
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| 8. |
- Kuhnemund, Malte, et al.
(författare)
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Circle-to-circle amplification on a digital microfluidic chip for amplified single molecule detection
- 2014
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0197 .- 1473-0189. ; 14:16, s. 2983-2992
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate a novel digital microfluidic nucleic acid amplification concept which is based on padlock probe mediated DNA detection and isothermal circle-to-circle amplification (C2CA). This assay platform combines two digital approaches. First, digital microfluidic manipulation of droplets which serve as micro-reaction chambers and shuttling magnetic particles between these droplets facilitates the integration of complex solid phase multistep assays. We demonstrate an optimized novel particle extraction and transfer protocol for superparamagnetic particles on a digital microfluidic chip that allows for nearly 100% extraction efficiencies securing high assay performance. Second, the compartmentalization required for digital single molecule detection is solved by simple molecular biological means, circumventing the need for complex microfabrication procedures necessary for most, if not all, other digital nucleic acid detection methods. For that purpose, padlock probes are circularized in a strictly target dependent ligation reaction and amplified through two rounds of rotting circle amplification, including an intermediate digestion step. The reaction results in hundreds of 500 nm sized individually countable DNA nanospheres per detected target molecule. We demonstrate that integrated miniaturized digital microfluidic C2CA results in equally high numbers of C2CA products mu L-1 as off-chip tube control experiments indicating high assay performance without signal loss. As low as 1 aM synthetic Pseudomonas aeruginosa DNA was detected with a linear dynamic range over 4 orders of magnitude up to 10 fM proving excellent suitability for infectious disease diagnostics.
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| 9. |
- Kuhnemund, Malte, et al.
(författare)
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Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore
- 2015
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Ingår i: Biosensors & bioelectronics. - : Elsevier BV. - 0956-5663 .- 1873-4235. ; 67, s. 11-17
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Tidskriftsartikel (refereegranskat)abstract
- Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches.
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| 10. |
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| 11. |
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| 12. |
- Kühnemund, Malte, et al.
(författare)
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Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules
- 2017
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 45:8
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Tidskriftsartikel (refereegranskat)abstract
- Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope.
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| 13. |
- Kühnemund, Malte
(författare)
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Single Molecule Detection : Microfluidic Automation and Digital Quantification
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, which now permit the detection and analysis of single molecules with high sensitivity and specificity. Assay sensitivity is fundamentally limited by the efficiency of the detection method used for read-out. Inefficient detection systems are usually compensated for by molecular amplification at the cost of elevated assay complexity.This thesis presents microfluidic automation and digital quantification of targeted nucleic acid detection methods based on padlock and selector probes and rolling circle amplification (RCA). In paper I, the highly sensitive, yet complex circle-to-circle amplification assay was automated on a digital microfluidic chip. In paper II, a new RCA product (RCP) sensing principle was developed based on resistive pulse sensing that allows label free digital RCP quantification. In paper III, a microfluidic chip for spatial RCP enrichment was developed, which enables the detection of RCPs with an unprecedented efficiency and allows for deeper analysis of enriched RCPs through next generation sequencing chemistry. In paper IV, a smart phone was converted into a multiplex fluorescent imaging device that enables imaging and quantification of RCPs on slides as well as within cells and tissues. KRAS point mutations were detected (i) in situ, directly in tumor tissue, and (ii) by targeted sequencing of extracted tumor DNA, imaged with the smart phone RCP imager. This thesis describes the building blocks required for the development of highly sensitive low-cost RCA-based nucleic acid analysis devices for utilization in research and diagnostics.
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| 14. |
- Kühnemund, Malte, et al.
(författare)
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Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy
- 2017
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.
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| 15. |
- Mezger, Anja, et al.
(författare)
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Highly specific DNA detection employing ligation on suspension bead array readout
- 2015
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 32:5, s. 504-510
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Tidskriftsartikel (refereegranskat)abstract
- We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.
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| 16. |
- Saharil, Farizah, et al.
(författare)
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LIGATION-BASED MUTATION DETECTION AND RCA IN SURFACE UN-MODIFIED OSTE+ POLYMER MICROFLUIDIC CHAMBERS
- 2013
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Ingår i: 17th IEEE International Conference on Solid-State Sensors, Actuators and Microsystems & EUROSENSORS XXVII (IEEE TRANSDUCERS 2013). - : IEEE conference proceedings. - 9781467359818 ; , s. 357-360
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Konferensbidrag (refereegranskat)abstract
- For the first time, we demonstrate DNA mutation detection in surface un-modified polymeric microfluidic chambers without suffering from bubble trapping or bubble formation. Microfluidic devices were manufactured in off-stoichiometry thiol-ene epoxy (OSTE+) polymer using an uncomplicated and rapid fabrication scheme. The device performance was compared to that of PMMA and PDMS devices. In OSTE+ devices, we were able to perform ligation-based mutation detection and rolling circle amplification (RCA) assays directly on the un-modified surface without suffering from bubble formation or enzyme inhibition during bio-operation at elevated temperatures. In contrast, PMMA, PDMS and COP microfluidic devices required specific surface treatment.
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| 17. |
- Zelano, Johan, et al.
(författare)
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The synaptic protein encoded by the gene Slc10A4 suppresses epileptiform activity and regulates sensitivity to cholinergic chemoconvulsants
- 2013
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Ingår i: Experimental Neurology. - : Elsevier BV. - 0014-4886 .- 1090-2430. ; 239, s. 73-81
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Tidskriftsartikel (refereegranskat)abstract
- The expanding number of disease-causing dysfunctions of synaptic proteins illustrates the importance of investigating newly discovered proteins involved in neuronal transmission. The gene Slc10A4 encodes a recently described carrier protein present in pre-synaptic terminals of cholinergic and monoaminergic neurons. The biological significance of this recently described transporter protein is currently unknown. We here investigated whether absence of the Slc10a4 protein has any impact on function of the cholinergic system. We first investigated the sensitivity of Slc10a4 null mice to cholinergic stimulus in vitro. In contrast to wild type mice, gamma oscillations occurred spontaneously in hippocampal slices from Slc10a4 null mice. Furthermore, moderate treatment of Slc10a4 null slices with the cholinergic agonist carbachol induced epileptiform activity. In vivo, 3-channel EEG measurements in freely behaving mice revealed that Slc10a4 null mice had frequent epileptiform spike-activity before treatment, and developed epileptic seizures, detected by EEG and accompanied by observable behavioral components, more rapidly after injection of the cholinergic agonist pilocarpine. Similar results were obtained on non-operated mice, as evaluated by behavioral seizures and post mortem c-Fos immunohistochemistry. Importantly, Slc10a4 null mice and wild type control mice were equally sensitive to the glutamatergic chemoconvulsant kainic acid, demonstrating that absence of Slc10a4 led to a selective cholinergic hypersensitivity. In summary, we report that absence of the recently discovered synaptic vesicle protein Slc10a4 results in increased sensitivity to cholinergic stimulation.
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