Разработка экспертно-распознающих изделий и методов в экономической диагностике

Автореферат диссертации на соискание ученой степени кандидата экономических наук

Neurotransmission and functional synaptic plasticity in the rat medial preoptic nucleus

Brain function implies complex information processing in neuronal circuits, critically dependent on the molecular machinery that enables signal transmission across synaptic contacts between neurons. The types of ion channels and receptors in the neuronal membranes vary with neuron types and brain regions and determine whether neuronal responses will be excitatory or inhibitory and often allow for functional synaptic plasticity which is thought to be the basis for much of the adaptability of the nervous system and for our ability to learn and store memories. The present thesis is a study of synaptic transmission in the medial preoptic nucleus (MPN), a regulatory center for several homeostatic functions but with most clearly established roles in reproductive behaviour. The latter behaviour typically shows several distinct phases with dramatically varying neuronal impulse activity and is also subject to experience-dependent modifications. It seems likely that the synapses in the MPN contribute to the behaviour by means of activity-dependent functional plasticity. Synaptic transmission in the MPN, however, has not been extensively studied and is not well understood. The present work was initiated to clarify the synaptic properties in the MPN. The aim was to achieve a better understanding of the functional properties of the MPN, but also to obtain information on the functional roles of ion channel types for neurotransmission and its plastic properties in general. The studies were carried out using a brain slice preparation from rat as well as acutely isolated neurons with adhering nerve terminals. Presynaptic nerve fibres were stimulated electrically or, in a few cases, by raised external K+ concentration, and postsynaptic responses were recorded by tight-seal perforated-patch techniques, often combined with voltage-clamp control of the post-synaptic membrane potential. Glutamate receptors of α-amino-3-hydroxy-5-methyl-4-izoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types were identified as mediating the main excitatory synaptic signals and γ-aminobutyric acid (GABA)A receptors as mediating the main inhibitory signals. Both types of signals were suppressed by serotonin. The efficacy of AMPA-receptor-mediated transmission displayed several types of short-term plasticity, including paired-pulse potentiation and paired-pulse depression, depending on the stimulus rate and pattern. The observed plasticity was attributed to mainly presynaptic mechanisms. To clarify some of the presynaptic factors controlling synaptic efficacy, the role of presynaptic L-type Ca2+ channels, usually assumed not to directly control transmitter release, was investigated. The analysis showed that (i) L-type channels are present in GABA-containing presynaptic terminals on MPN neurons, (ii) that these channels provide a means for differential control of spontaneous and impulse-evoked GABA release and (iii) that this differential control is prominent during short-term synaptic plasticity. A model where Ca2+ influx through L-type channels may lead to reduced GABA release via effects on Ca2+-activated K+ channels, membrane potential and other Ca2+-channel types explains the observed findings. In addition, massive Ca2+ influx through L-type channels during high-frequency stimulation may contribute to increased GABA release during post-tetanic potentiation. In conclusion, the findings obtained in the present study indicate that complex neurotransmission mechanisms and different forms of synaptic plasticity contribute to the specific functional properties of the MPN

Visual stimulation with blue wavelength light drives V1 effectively eliminating stray light contamination during two-photon calcium imaging

Background: Brain visual circuits are often studied in vivo by imaging Ca2+ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators´ emission spectra, resulting in significant contamination of calcium signals. New method: To overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicator´s emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials –VEPs) and 3D acousto-optic two-photon (2P) population Ca2+ imaging in mouse primary visual cortex (V1). Results: VEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca2+ imaging in a Thy1 GP4.3 line revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable. Comparison with existing methods: Masking or shielding the microscope are often used to minimize the contamination of Ca2+ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set-up. Blue stimuli not interfering with imaging allow to omit shielding. Conclusions: Together, our results show that the selected blue light stimuli evoke responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set-ups easier, and facilitate the combination of Ca2+ imaging with electrophysiology and optogenetics

Inactivation of K⁺ currents.

<p>A, K⁺ currents evoked by >60 s long voltage steps from −74 mV to +16 mV, in control solution (<i>upper trace</i>) and in 10 µM 17-β-estradiol (<i>lower trace</i>). Note the slow, but large inactivation in control solution as well as in estradiol. B, voltage dependence of steady-state inactivation. Peak current at +26 mV plotted <i>versus</i> the voltage of a 60 s long preceding interval. Control solution (<i>squares</i>) and 10 µM 17-β-estradiol (<i>circles</i>). Smooth lines are fitted Boltzmann relations (Equation 2) with voltage for half-maximal inactivation (U_½) and slope factor (U_S) indicated. A steady non-inactivating current component was subtracted. C, relative recovery from inactivation caused by a 16-s interval at +26 mV (ending at time 0): Superimposed curves represent the currents at +26 mV after varying recovery intervals at −74 mV with and without 17-β-estradiol, as indicated. D, relative recovery for peak currents as in C plotted <i>versus</i> the recovery interval at −74 mV. Smooth line is described by a monoexponential function with time constant 0.81 s. The differences between curves for control and estradiol are within the range of trial-to-trial variability for a single condition in these long-duration experiments.</p

Blocking effects of different steroids on K^<b>+</b> currents and lack of effect of ICI 182,780.

<p>All steroids were applied at a concentration of 10 µM and the estrogen-receptor blocker ICI 182,780 at a concentration of 50 µM. Currents were measured 590–600 ms after voltage steps from −74 mV to +6 mV. A–D, effects of the indicated substances at repetitive voltage steps given to individual neurons. E, summary of blocking effects plotted relative to the blocking effect of 10 µM 17-β-estradiol observed in the same neurons. The number of cells studied for each condition is given within parenthesis. The negative value for estrogen sulphate indicates that this substance caused a slight potentiation of the K⁺ currents.</p

Model of estradiol action on voltage-gated K^<b>+</b> currents.

<p>A, state diagram showing the model with estradiol (E) binding to open channels (O) to form the blocked state with estradiol bound (BE). Dashed box includes the voltage-dependent transitions between closed states (C1 and C2) and the open state. B, experimentally obtained raw data currents from one neuron in control conditions (<i>black curves</i>) and in the presence of 10 µM 17-β-estradiol (<i>grey curves</i>), for comparison with C. The currents were activated by voltage steps (600 ms) to indicated potentials from a holding potential of −74 mV. C, computed K⁺ currents for control conditions (corresponding to states enclosed by dashed box in A; <i>black curves</i>), and for the presence of 10 µM 17-β-estradiol according to the model (<i>grey curves</i>). Voltage steps as in B. D, computed currents at different concentrations of 17-β-estradiol, as indicated. Voltage step to +26 mV from −74 mV. E, concentration-response curve for computed currents 600 ms after a voltage step from −74 mV to +26 mV. EC₅₀ value and Hill coefficient (<i>n</i>) are given in the figure. F, voltage dependence of the block induced by 10 µM 17-β-estradiol (600 ms after a voltage step from −74 mV to indicated potentials) according to the model. The current in 17-β-estradiol is plotted relative to control. The line is a fitted exponential curve, with an <i>e</i>-fold change in relative current per 15 mV.</p

Sensitivity to blockers and concentration-dependent rate of current decline.

<p>A, currents (averages of 10 traces) evoked by a voltage step from −74 mV to +6 mV in solutions as indicated. Note that 30 mM TEA blocked a major fraction of the voltage-activated K⁺ current without leaving any significant transient current remaining. Note also that the blocking effects of TEA and 17-β-estradiol were overlapping. B, currents evoked by voltage steps from −74mV to +26 mV, in control solution and in the indicated concentrations of 17-β-estradiol. Note the faster current decline and the larger block with increasing concentration of 17-β-estradiol. C, ratio of current (<i>grey</i>) in 17-β-estradiol to that in control solution, to show the time course of current inhibition. The traces were well fitted by exponential functions with time constants 134ms (<i>top</i>), 75ms (<i>middle</i>) and 49 ms (<i>bottom</i>), shown as superimposed black lines. Computed from the traces shown in B. D, dependence of time constant of relative current, as in C, on concentration of 17-β-estradiol. Mean±S.E.M. for the number of neurons indicated. Voltage steps to +26mV from −74 mV. E-F, overlapping block by estradiol and r-stromatoxin-1. E, time-course of block of the K⁺ current (mean current 590–600 ms after a voltage step from −74 to +26 mV) by estradiol, r-stromatoxin-1 and a combination of these substances, as indicated. F, K⁺ currents (averages of 10 traces) evoked by voltage steps from −74 to +26 mV. Concentration of blockers as in E. Note the lack of effect of estradiol in the presence of r-stromatoxin-1. Data in E and F from the same neuron.</p

17-β-estradiol rapidly reduces K^<b>+</b> currents in MPN neurons.

<p>A, K⁺ currents evoked by a voltage step from −74 mV to +36 mV, in control solution, after the addition of 10 µM 17-β-estradiol and after wash-out of 17-β-estradiol, as indicated. B, time course of estradiol-induced depression, from the neuron in A. Mean current 190–200 ms after voltage steps to +26 mV from −74 mV. (No leak current subtraction.) 17-β-estradiol was applied as indicated. Superimposed lines show fitted exponentials. C, concentration-response relation for 17-β-estradiol-induced depression of K⁺ currents. Mean currents 590–600 ms after a voltage step to +26 mV from −74 mV. Smooth line is described by Equation 1 with EC₅₀ = 9.7 µM, n = 1.2, <i>Inh</i>_max = 58%. Data from 7 neurons. D, I–V relations for mean current 590–600 ms after a voltage step from −74 mV to the potentials indicated, for one MPN neuron. Current in solutions as indicated. E, relation between the effect of 10 µM 17-β-estradiol (ratio of current in estradiol to that in control solution; mean current 590–600 ms after voltage step from −74 mV) and membrane voltage for 12–13 neurons. Mean±S.E.M. The superimposed line is an exponential function, with <i>e</i>-fold change per 14 mV, fitted to the data. F–G, Ca²⁺ independence of estradiol-sensitive current. F, currents evoked by a voltage step from −74 mV to +6 mV, with extracellular solution modified as indicated. Note that estradiol (10 µM) induced a similar depression in the presence and absence of Ca²⁺. G, current depressed (mean current 590–600 ms after a voltage step to +6 mV) by 10 µM 17-β-estradiol added to standard extracellular solution (left bar) and to a solution with Co²⁺ substituted for Ca²⁺ (right bar). The same 9 neurons were used for both conditions. The difference was not significant.</p

Effects of altered [K^<b>+</b>]_o on block by 17-β-estradiol.

<p>A, currents recorded at 600-ms voltage steps to −24 mV from −74 mV. At −24 mV currents in standard [K⁺]_o of 5 mM are outward (<i>top traces, control black</i>) with some reduction caused by 10 µM 17-β-estradiol (<i>grey</i>). With a [K⁺]_o of 140 mM, currents (<i>lower traces, control black</i>) are inward and 10 µM 17-β-estradiol (<i>grey</i>) also reduces the current. B, relative current (mean±S.E.M. from 6 cells) in 10 µM 17-β-estradiol, measured 590–600 ms after a voltage step as in A. C, currents as in A but with voltage step to +16 mV. D, relative current (mean±S.E.M. from 5 cells) in 10 µM 17-β-estradiol, measured 590–600 ms after a voltage step as in C. E, currents recorded as in C, but with comparison between standard [K⁺]_o of 5 mM and [K⁺]_o = 0 mM. F, relative current (mean±S.E.M. from 7 cells) in 10 µM 17-β-estradiol, measured 590–600 ms after a voltage step as in E. Note the slight, but significant difference in blocking effect recorded from the same cells when [K⁺]_o = 5 mM and when [K⁺]_o = 0 mM.</p