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doctoral thesis
Morphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array
2024. urn:nbn:hr:171:302514
Radotić, Viktorija
University of Split
School of Medicine

Cite this document

Radotić, V. (2024). Morphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array (Doctoral thesis). Split: University of Split, School of Medicine. Retrieved from https://urn.nsk.hr/urn:nbn:hr:171:302514

Radotić, Viktorija. "Morphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array." Doctoral thesis, University of Split, School of Medicine, 2024. https://urn.nsk.hr/urn:nbn:hr:171:302514

Radotić, Viktorija. "Morphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array." Doctoral thesis, University of Split, School of Medicine, 2024. https://urn.nsk.hr/urn:nbn:hr:171:302514

Radotić, V. (2024). 'Morphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array', Doctoral thesis, University of Split, School of Medicine, accessed 19 November 2024, https://urn.nsk.hr/urn:nbn:hr:171:302514

Radotić V. Morphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array [Doctoral thesis]. Split: University of Split, School of Medicine; 2024 [cited 2024 November 19] Available at: https://urn.nsk.hr/urn:nbn:hr:171:302514

V. Radotić, "Morphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array", Doctoral thesis, University of Split, School of Medicine, Split, 2024. Available at: https://urn.nsk.hr/urn:nbn:hr:171:302514

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Metadata
TitleMorphological and electrophysiological characterization of spiral ganglion neurons cultured in vitro on high-density complementary semiconductor electrode array
Title (croatian)Morfološka i elektrofiziološka karakterizacija stanica spiralnog ganglija in vitro uzgojenih na komplementarnom poluvodiču sa elektrodnim sustavima visoke gustoće
AuthorViktorija Radotić
MentorDamir Kovačić (mentor)
Committee memberMarko Ljubković (predsjednik povjerenstva)
Committee memberZdravko Petanjek (član povjerenstva)
Committee memberMarija Raguž (član povjerenstva)
GranterUniversity of Split
School of Medicine
Split
Defense date and country2024-05-21, Croatia
Scientific / art field,
discipline and subdiscipline		BIOMEDICINE AND HEALTHCARE
Basic Medical Sciences
Universal decimal classification
(UDC)		61 - Medical sciences
Abstract
Objectives: This study investigated a micro-patterned, high-density complementary metal–oxide semiconductor (CMOS) electrode array to be used as a biologically permissive environment for organization, guidance, and simultaneous electrical stimulation and recording of spiral ganglion neurons (SGN). Methods: SGNs extracted and isolated from cochleae of P5-P7 rat pups and adult guinea pigs were cultured for 1, 4, and 7 days in vitro on glass coverslips (control) and CMOS electrode array. The cultures were analyzed visually and immunohistochemically for SGN presence, outgrowth, neurite alignment, neurite length, neurite asymmetry, and the contact of neuronal soma and neurites with the microelectrodes. Electrophysiological experiments were performed at 37° using a CMOS-like electrode array in two modes. The first, spontaneous, mode focuses on recording intrinsic spontaneous electrophysiological activity without external stimulation to determine whether cells can fire action potentials in the absence of a standard neurotransmitting mechanism via hair cells, while the second, stimulation mode recorded potential cell responses to an electrical biphasic stimulus (positive phase first) with a total duration of 60 µs and an amplitude of 500 nA or 800 nA. Results: Our findings indicate that the topographical environment of the CMOS chip with micro-patterned pillars enhanced growth, survival, morphology, neural orientation, and alignment of SGNs in vitro compared to control. Smaller spacing (0.8-1.6 µm) between protruding pillars on CMOS led SGNs to develop structured and guided neurites oriented along three topographical axes separated by 60°. These results constitute a morphological basis for positioning the microelectrodes on the chip appropriate for direct contact with SGNs. This configuration allowed the CMOS electrode array to electrically stimulate the SGN, whose responses were observed with live Fluo 4 calcium imaging and, at the same time, recorded electrophysiologically using the same CMOS electrode array. The average maximum spike amplitude was 135.4 ± 15.2 µV for recorded spontaneous activity and 130 ± 10 µV for recorded neural activity after stimulation. Conclusion: To the best of our knowledge, this study demonstrates for the first time that simultaneous stimulation and recording with the same electrode array is feasible for measuring auditory neuronal activity from dissociated SGNs. Immunostaining of SGNs cultures grown on a CMOS electrode array for the neuronal marker Tuj revealed both somata and processes of neurons on the electrode surface, in accordance with results that the position of neuronal soma and neurites affects recordings of electrical activity on the CMOS electrode array. The model presented in this study represents a novel in vitro tool to investigate strategies for simultaneous electrical stimulation and recording of auditory neurons to optimize CI technology.
Abstract (croatian)
Cilj: Ova studija istraživala je mogućnost korištenja elektrodnog sustava visoke gustoće temeljenog na CMOS tehnologiji, kao biološki prihvatljivog okruženja za organizaciju, navođenje te električnu stimulaciju živčanih stanica spiralnog ganlija (engl. SGNs). Metode: Stanice spiralnog ganglija, ekstrahirane i izolirane iz pužnica 5-7 dana starih (P5-P7) štakorskih mladunaca i odraslih zamoraca, uzgajane su 1, 4 i 7 dana in vitro na stakalcima (kontrola) te CMOS čipovima. Kulture su analizirane vizualno i imunohistokemijski na prisutnost živčanih stanica spiralnog ganglija, na rast, poravnanje neurita, duljinu neurita, asimetriju neurita te kontakt neuronske some i neurita sa elektrodama. Elektrofiziološki pokusi izvedeni su na 37°C u dva eksperimentalna modusa sa svrhom detekcije živčane aktivnosti koristeći elektrodni sustav koji se temelji na CMOSu. Prvi modus je usredotočen na snimanje intrizične, spontane elektrofiziološke aktivnosti bez vanjske stimulacije kako bi se utvrdilo mogu li neuroni generirati akcijske potencijale u odsutnosti standardnog mehanizma neurotransmisije putem osjetilnih slušnih stanica. Drugi modus je uključivao stimulaciju pomoću koje su bili zabilježeni potencijalni stanični odgovori na električni bifazični podražaj (s prvom pozitivnom fazom) s ukupnim trajanjem od 60 µs i amplitudom od 500 nA ili 800 nA. Rezultati: Rezultati pokazuju, da je topografsko okruženje CMOS čipa sa mikroiglicama poboljšalo rast, preživljavanje, morfologiju te neuralnu orientaciju in vitro uzgojenih živčanih stanica spiralnog ganglija u usporedbi sa kontrolom. Manji razmak (0.8-1.6 µm) između mikroiglica na CMOS čipu doveo je stanice spiralnog ganglija do razvoja strukturiranih i vođenih neurita usmjerenih duž tri topografske osi pomaknutih za 60°. Ti rezultati čine morfološku osnovu za pozicioniranje mikroelektroda na čipu prikladnih za izravan kontakt sa stanicama spiralnog ganglija. Ova konfiguracija je omogućila uspješnu električnu stimulaciju stanica spiralnog ganglija čiji su odgovori promatrani uživo pomoću Fluo 4 oslikavanja kalcija na CMOS čipu. Prosječna maksimalna vršna amplituda je bila 135.4 ± 15.2 µV za zabilježenu spontanu aktivnost te 130 ± 10 µV za zabilježenu živčanu aktivnost nastalu kao odgovor na stimulaciju. Ova studija pokazuje da je istovremena stimulacija te snimanje s istim elektrodnim sustavom elektroda visoke gustoće izvedivo za mjerenje elektrofiziološke aktivnosti živčanih stanica dobivenih iz in vitro kultura stanica spiralnog ganglija. Imunohistokemijsko bojanje kultura uzgojenih na CMOS čipu za neuronski marker Tuj otkrilo je tijela stanica (some) i procese (neurite) neurona na površini elektroda, te u skladu s rezultatima, pokazuje, da položaj neurona i neurita utječe na snimanje električne aktivnosti na CMOS čipu. Model predstavljen u ovoj studiji predstavlja pogodan in vitro alat za istraživanje stimulacijskih protokola za istovremenu električnu stimulaciju i snimanje živčanih stanica spiralnog ganglija u svrhu optimizacije tehnologije kohlearnih implantata (engl. CI).
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:171:302514
Study programmeTitle: Translational Research in Biomedicine - TRIBE
Study programme type: university
Study level: postgraduate
Academic / professional title: doktor/doktorica znanosti, interdisciplinarna područja znanosti (doktor/doktorica znanosti, interdisciplinarna područja znanosti)
Type of resourceText
File originBorn digital
Access conditionsOpen access
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Created on2024-05-17 06:33:18