Electrical Properties of Developing Sensory Neurons

Titas Petruša. „Electrical properties of developing sensory neurons“. Supervisor of master thesis - Doc. Artūras Grigaliūnas. Lithuanian University of Health Sciences, Academy of Medicine, Faculty of Medicine, department of Physics, mathematics and biophysics. Kaunas, 2020. Study goals – to analyze the electrical properties of trigeminal ganglia neurons of rat embryos at 13 days of age, and to compare this data with trigeminal ganglia neurons electrical properties at 16 days of age. Study tasks – 1) To describe the changes of electrical properties in developing trigeminal neurons 2) To evaluate properties of membrane ionic channels by means of ion channel blockers. 3) To compare differences in electrical properties of developing trigeminal neurons during 13th and 16th gestational days. To analyze origin of differences and possible applications in modern medicine. Methodology – timed-pregnancy Sprague Dawley rat embryos at gestational day 13 were extracted. Trigeminal ganglions were dissected and placed on a matrix culture system, they were cultivated for 5-8 days, changing the medium daily and performing measurements at 5th or later day. Measurements were done by means of a whole-cell current clamp method and data analyzed by pCLAMP software. RT-PCR was performed using RNeasy protocol, RT-PCR data was confirmed using DNA sequencing. Object of the research – embryos of 13-day timed-pregnant Sprague Dawley rats. Results – 48 trigeminal neurons were tested electrophysiologically, 15 were morphologically described. 2 of them were unipolar, 8 – bipolar, 5 – pseudounipolar. After 5 to 8 days of cultivation, the following electrical properties of trigeminal neurons have changed: electrical membrane capacity, afterhyperpolarization amplitude, threshold of excitation, measured in picoamperes (pA). Those properties increased in statistically relevant boundaries. After 5 to 8 days in culture, the following properties of trigeminal neurons did not change: resting membrane potential, input resistance, time constant, amplitude of action potential, duration at half-amplitude, maximal right and left slopes, threshold of excitation, measured in millivolts (mV), duration of afterhyperpolarization. In addition, during hyperpolarization, there was no depolarizing sag present until 6th day of cultivation. However, it was present in most trigeminal neurons since 7th cultivation day. After applying TTX, it was noticed that 13th day trigeminal neurons generate TTX-resistant action potentials. After confirming data with RT-PCR, it was found that NaV1.9 channels are not present in E13 neurons, but develop in E16, in comparison to NaV1.8 type, which is present in both E13 and E16 trigeminal neurons. Conclusions - 1) Neuron morphological and electrical properties change overtime. Neuron soma size increases, membrane electrical capacity becomes larger. Action potential changes also – slopes get more flat, and the action potential duration increases. There are also changes in depolarization curve during the afterhyperpolarization. 2) During cultivation for 5 to 8 days, action potentials of trigeminal ganglion neurons showed increased afterhyperpolarization amplitude, however resting membrane potential, input resistance, time constant did not change overtime. 3) After applying tetrodotoxin to trigeminal neurons, the measurement results showed that trigeminal ganglia in 13 days of gestation do not have as much TTX resistant ionic channels formed yet. 4) After comparing the differences in electrical properties of 13th and 16th gestational day trigeminal neurons, it was shown, that during this period, neuron soma increases in size, membrane capacity and time constant increase as well. Properties, related to action potential and hyperpolarization change as well. 16th gestational day neurons have more flat action potentials, action potential decay time increased. Action potential amplitude also increased