Optogenetic Modulation and Multi-Electrode Analysis of Cerebellar Networks <i>In Vivo</i>

<div><p>The firing patterns of cerebellar Purkinje cells (PCs), as the sole output of the cerebellar cortex, determine and tune motor behavior. PC firing is modulated by various inputs from different brain regions and by cell-types including granule cells (GCs), climbing fibers and inhibitory interneurons. To understand how signal integration in PCs occurs and how subtle changes in the modulation of PC firing lead to adjustment of motor behaviors, it is important to precisely record PC firing <i>in</i><i>vivo</i> and to control modulatory pathways in a spatio-temporal manner. Combining optogenetic and multi-electrode approaches, we established a new method to integrate light-guides into a multi-electrode system. With this method we are able to variably position the light-guide in defined regions relative to the recording electrode with micrometer precision. We show that PC firing can be precisely monitored and modulated by light-activation of channelrhodopsin-2 (ChR2) expressed in PCs, GCs and interneurons. Thus, this method is ideally suited to investigate the spatio/temporal modulation of PCs in anesthetized and in behaving mice.</p></div

Light activation of PCs depends on the distance between the light source and the optogenetically targeted PC.

<p>(A) Schematic diagram of the relative distance of the light guide (red or blue) and the recording electrode (gray) around and along a PC. The light fiber was moved during the recording from responsive PCs in vertical direction relative to the recording electrode and 1000 ms pulses of light were delivered in various depths at 250 µm intervals. Laser power was varied between 0.5 and 5 mW, resulting in 0.125 to 1.25 mW in front of light fiber. The PC response to light was recorded, while the glass fiber was either in 330 µm horizontal distance relative to recording electrode (red fibers) or in 660 µm horizontal distance (blue fiber). (B) Diagram of the dependence of relative increase of PCs activity on the vertical distance of the light fiber tip. For each light guide position, activity during light application is expressed as multiples of spontaneous activity of the particular PC. Data points are aligned relative to the depth of the light fiber; negative distances correspond to positions of the light fiber above the electrodes. Activity increase elicited by light fiber positioned in neighboring guide tube (330 µm distance) are marked by red crosses (data from 10 PC recorded in 3 mice), rate increase caused by the light fiber positioned in the subsequent guide tube (660 µm horizontal distance) are marked by blue circles (data from 11 PC recorded in 3 mice). Averages are calculated for 200 µm intervals and plotted as horizontal bars, vertical lines indicate standard error of the mean. Note that strongest responses were elicited when the light fiber tip was up to 500 µm above the recorded PC.</p

Simultaneous recordings from multiple PCs in the vermis of mice.

<p>(A) Schematic representation of relative positions of three electrodes and a single light guide assembled in a linear array during a recording and stimulation experiment in the cerebellum. The guide-tubes (1 to 4) are part of the multi-electrode system, which allows for independent vertical movement of each fiber. Guide-tubes 1, 2 and 4 each house an electrode while guide-tube 3 contains a light fiber. The electrodes are placed to enable recordings from the Purkinje cell layer, whereas the tip of the light-guide resides in the molecular layer. (ML: molecular layer, PCL: Purkinje cell layer, GCL: granular cell layer). (B) Simultaneous recording from three PCs with electrodes e1, e2 and e4. Complex spikes can be identified by upward deflection of the action potential. (C) Superposition of 30 simple spikes (black) and 30 complex spikes (gray) recorded from electrode e2. (D) Complex spike triggered superposition of raw traces (n = 295) shows that simple spikes do not occur for 15 ms after complex spikes. The simple spike pause indicates that simple and complex spikes are recorded from the same cell.</p

Activation of PC simple spikes during light activation of ChR2 specifically expressed in PCs.

<p>(A) Raster plot (top) and peri-stimulus time histogram (PSTH, below) during light application to PCs. The raster plot exemplifies a single cell response during 20 light applications; the PSTH shows the averaged single cell response during these 20 repetitions (bin width 20 ms). (B) Average response profile of 14 cells, recorded from 6 tgPcp2-cre mice expressing ChR2 selectively in PCs. Illumination with 473 nm stably increases simple spike firing of PCs. Laser power was set between 1 and 5 mW, resulting in 0.25 to 1.25 mW in front of light fiber. (C) Same cell as in A, but PC responses to 60 light pulses plotted at faster timescale. In higher temporal resolution, rhythmic firing in single simple spike trains and in the PSTH becomes evident (bin width 1 ms). Note that the simple spikes are reliably triggered in less than 6 ms after light onset. Subsequent simple spikes appear with little temporal jitter, which diminishes the rhythmic modulation in the PSTH over time. (D) To analyze rhythmic firing during light driven and spontaneous activity, autocorrelation histograms (ACHs) were calculated on simple spike trains from periods without (gray) and with light application (blue), respectively. The rhythmic modulation is discernible in the autocorrelation of spontaneous activity (46.9 Hz), but becomes more prominent during light driven activity (93.8 Hz, modal frequency obtained from Fourier transformed of ACH). (E) Average firing rates increase during light application from 35 to 97 spikes/sec (left, n = 14). When spectra were computed from ACHs, modal frequencies were dominant in all 14 spectra obtained from light driven activity, but only in 10 spectra obtained from spontaneous activity. Modal frequencies shifted in average from 68.0 Hz (n = 10) to 116.6 Hz (n = 14, 40 to 100 light applications per cell). (F) The coefficient of variation (CV) is strongly diminished during light application, also indicating that simple spike firing becomes more regular. Similar effect is seen when CV₂ is calculated from adjacent inter spike intervals. (G) Parasagittal section of cerebellar cortex from Pcp2-cre mouse after virus injection. Red fluorescence indicates expression of hChR2(H134R)-mCherry specifically in PCs.</p

Thermal effect of light application on PC simple spike rates in tgGabra6-cre mice lacking ChR2 expression.

<p>PC simple spike rate responds to light application in tissue after expression of the tdTomato specifically in granule cells using AAV2/1.CAG-Flex.tdTomato. (A) Average response profile of 20 PCs showing increased activity during the time course of prolonged light application. Since no ChR2 is present, the observed increase in PC firing must be related to warming of the tissue surrounding the glass fiber. Light was delivered at maximum output power of the laser (20 mW), corresponding to approximately 5 mW measured in front of the tip. Bold black line indicates mean time course of rate modulation averaged across 20 PCs, 20 light applications per cell. (B) Increase in PC simple spike rate during light activation of cerebellar tissue expressing tdTomato in GC. Average activity increased from 47.5 to 54.4 Hz during light application.</p

Multiple modes of modulation of PC simple spikes during light activation of ChR2 specifically expressed in GCs. Light activation of GCs results in inhibition and/or activation of PC simple spike activity.

<p>(A) Increase in PC spike rate during 5 sec light activation of GCs expressing ChR2. Raster plots and corresponding PSTH from 10 repetitions (bin size 100 ms). (B) Average response profile of 13 PCs showing increased activity during the time course of prolonged light application to GCs expressing ChR2 (12 to 20 light applications per cell). Bold black line indicates mean time course of rate modulation averaged across 13 PCs. Dotted red curve corresponds to example shown in (A) above. (C) Inhibition of PC simple spikes during 5 sec light activation of GCs expressing ChR2. Data from cells shown in A and C are recorded simultaneously with two different electrodes. (D) Average response profile of 12 cells showing decreased activity during 5 sec of light application to GCs. Data recorded from 3 mice, 12 to 20 light applications per cell. Dotted red curve corresponds to example shown in (C) above. Laser power was set to 10 mW, resulting in 2.5 mW in front of light fiber. (E) Mean increase of simple spike rate from 13 PCs showing excitation during light application and mean decrease of activity of 12 PCs showing inhibition during light application. (F) Regularity of simple spike firing was increased during light application. The increase in CV₂ values was similar in both subgroups and therefore independent of the overall change in activity during light application. (G) Parasagittal section of cerebellar cortex from Gabra6-cre mouse after virus injection. Red fluorescence indicates expression of hChR2(H134R)-mCherry specifically in GC somas and parallel fibers crossing the dendritic trees of PCs in the molecular layer.</p

Table_1_Rapid outpatient transient ischemic attack clinic and stroke service activity during the SARS-CoV-2 pandemic: a multicenter time series analysis.docx

Background and aimRapid outpatient evaluation and treatment of TIA in structured clinics have been shown to reduce stroke recurrence. It is unclear whether short-term downtrends in TIA incidence and admissions have had enduring impact on TIA clinic activity. This study aims to measure the impact of the pandemic on hospitals with rapid TIA clinics.MethodsRelevant services were identified by literature search and contacted. Three years of monthly data were requested – a baseline pre-COVID period (April 2018 to March 2020) and an intra-COVID period (April 2020 to March 2021). TIA presentations, ischemic stroke presentations, and reperfusion trends inclusive of IV thrombolysis (IVT) and endovascular thrombectomy (EVT) were recorded. Pandemic impact was measured with interrupted time series analysis, a segmented regression approach to test an effect of an intervention on a time-dependent outcome using a defined impact model.ResultsSix centers provided data for a total of 6,231 TIA and 13,191 ischemic stroke presentations from Australia (52.1%), Canada (35.0%), Italy (7.6%), and England (5.4%). TIA clinic volumes remained constant during the pandemic (2.9, 95% CI –1.8 to 7.6, p = 0.24), as did ischemic stroke (2.9, 95% CI –7.8 to 1.9, p = 0.25), IVT (−14.3, 95% CI −36.7, 6.1, p ConclusionThis suggests that the pandemic has not had an enduring effect on TIA clinic or stroke service activity for these centers. Furthermore, the disproportionate decrease in IVT suggests that patients may be presenting outside the IVT window during the pandemic – delays in seeking treatment in this group could be the target for public health intervention.</p

All consecutive patients with lower respiratory tract infection are potentially eligible for this trial

<p><b>Copyright information:</b></p><p>Taken from "Procalcitonin guided antibiotic therapy and hospitalization in patients with lower respiratory tract infections: a prospective, multicenter, randomized controlled trial"</p><p>http://www.biomedcentral.com/1472-6963/7/102</p><p>BMC Health Services Research 2007;7():102-102.</p><p>Published online 5 Jul 2007</p><p>PMCID:PMC1947969.</p><p></p> If all inclusion criteria are fulfilled and no exclusion criteria are present, the physician has to explain to the patient the trial, ask for participation and get informed consent. After inclusion, the patient is randomized by a web based computerized random allocation algorithm to either the guidelines group or the PCT group, respectively. denotes community-acquired pneumonia, acute exacerbation of chronic pulmonary disease, antibiotics, procalcitonin