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1

Somatosensory responses in posteriol medial nucleus on non-anesthetized rats

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Borzymowska Z., Skladowska A., Wrobel A., Kublik E.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
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nr Supl.
BACKGROUND AND AIMS: Medial sector of posterior complex (PoM) of the thalamus receives two driving somatosensory inputs – from the periphery and from the cortical layer 5b and their functional significance was proposed to depend on arousal level. In anesthetized rats sensory evoked potentials in PoM revealed only late latency, cortex-dependent responses while in wakefulness they contained also fast latency components. In aroused animals this early activity is effectively transmitted to the sensory cortex. The current experiments were set up to record activity of single PoM neurons from conscious rats in order to confirm the field recording data and characterize the role of PoM in fast transmission of somatosensory information. METHODS: Rats were habituated to head fixation and body restrain, then implanted with chronic electrodes located in primary and higher order cortical somatosensory and motor areas. For extracellular recordings from PoM, microelectrodes were implanted on movable microdrives or the cranial window was opened for semichronic recording with silicon probe multichannel electrodes. Continuous signal containing field potentials and unitary activity was recorded for offline analysis. Single and multi-unit activity was extracted with template matching and clustering methods by Spike 2 software. The average evoked potentials and peristimulus time histograms were calculated to analyze the responses to whisker stimulations. RESULTS: Our preliminary results indicate that in awake rats PoM neurons respond to whisker stimulation with short-latency (5–6 ms) discharges followed by later, more dispersed activity CONCLUSIONS: Short-latency action potentials generated by PoM cells after vibrissae stimulation suggest that this nucleus participate in fast detection of tactile stimuli. Further research should elaborate the role of early response of this mixed-order somatosensory thalamic nucleus in more detail. Supported by National Science Centre grant DEC-2013/08/W/ NZ4/00691.
2

Influence of different optical stimulation parameters on strength of depolarization in ChR2-transfected neurons

88%
Skladowska A., Bekisz M., Majkowska M., Lempicka M., Kublik E.
Acta Neurobiologiae Experimentalis
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2017
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tom 77
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nr Suppl.1
INTRODUCTION: To use optogenetics in well control manner it is necessary to characterize the relation between the light power and resulting opening of light gated channels. AIM(S): To this end we tested the dependence of membrane depolarization on the parameters of light stimulation in channelrhodopsin-transfected neurones in rat’s central nucleus of amygdala (CeA). METHOD(S): Under general anesthesia rats were were injected with AAV-hSyn-ChR2-EYFP viral vector introducing ChR2 to CeA. During in vitro patch-clamp recording on brain slices, we measured the membrane depolarization evoked by a blue light emitted from LED source. Cells were stimulated with trains of light impulses with varying: 1) light power; 2) duration of light impulse; 3) frequency of light impulses. RESULTS: 1) Train of 2 ms light impulses delivered at 20 Hz, a driving current varying from 0.1 to 1 A: relation between light power and membrane depolarization can be approximated by a logarithmic function: 2.2 ln(x)+13 (at the resting potential kept at −50 mV) and 8 ln(x)+28 (at −60 mV). The dependency of the latency of first action potential on the light intensity can be approximated by a power function: 1.2 ×^(−1.3); 2) Train of light impulses of varying duration (range 2–20 ms) at 20 Hz, current 1 A and resting membrane potential kept at −50 mV: the relation between light impulse duration and resulting membrane depolarization can be approximated by a logarithmic function 3 ln(x)+4; 3) Train of 2 ms light impulses with a current set at 1 A and a frequency varying in a range 20–200 Hz (resting potential kept at −50 mV): relation between light impulse frequency and resulting membrane depolarization can be approximated by a logarithmic function: 2.5 ln(x)+3.5. CONCLUSIONS: Our results offers the guideline allowing to estimate expected depolarizing effects of light stimulation on the ChR2-transfected neuronal population in central nucleus of amygdala in rats. FINANCIAL SUPPORT: Supported by Polish National Science Centre grant 2013/08/W/NZ4/00691.
3

Spatial distribution of light evoked currents in ChR2-transfected rat’s cortex

88%
Kublik E., Skladowska A., Czerwinski M. B., Pasierski M., Wojcik D. K.
Acta Neurobiologiae Experimentalis
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2017
|
tom 77
|
nr Suppl.1
INTRODUCTION: Optogenetics allows to stimulate selected neuronal populations with high temporal resolution but the spatio‑temporal extent of resulting effects is not well characterized. AIM(S): Experiment were aimed to evaluate spatial distribution of the potentials and currents evoked by light impulses in the channelrhodopsin-transfected rat cortex. METHOD(S): Rats were injected with viral vector introducing ChR2 into large portion of somatosensory cortex. 2–3 weeks later we performed acute in vivo experiments recording multichannel local field potentials evoked (EP) by a blue light delivered either to the cortical surface (surf-stim) or into the cortex (deep-stim). We analyzed spatio-temporal patterns of EPs and their 2-D current source density (CSD) profiles (kernel CSD method, https://github. com/Neuroinflab/kCSD‑python). RESULTS: Our preliminary results indicated that light evoked potentials consisted of early waves, resulting from opening ChR2 channels, overlapping with later components related to the synaptic spread of activity within cortical network. As expected, largest EPs were recorded close to the fiber tip, in layer 2–3 with surf‑stim and layer 5 with deep-stim. Longer impulses (10 ver 1 ms) evoked around 20% stronger responses. Up to 600–800 µm from a light source EPs sustained ~50% of max amplitude. However, CSD analysis indicated that after surf-stim the early current sink (1–2 ms) was restricted to ~400 µm in layer 2–3. Later, postsynaptic sink developed at 5–8 ms in layer 5. Later components had wider lateral spread across few columns with clear reflection of cortical layering. After intra-cortical light delivery activity seemed to spread within, not across the cortical columns. CONCLUSIONS: For well controlled use of optogenetics it is not enough to ensure light beam of sufficient strength. The localization of the fiber tip can have specific impact on the activity developing within local neuronal network. FINANCIAL SUPPORT: Supported by Polish National Science Centre grant 2013/08/W/NZ4/00691.
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