Neurons in the dorsolateral geniculate nucleus (dLGN) of the thalamus receive a substantial proportion of modulatory inputs from corticothalamic (CT) feedback and brain stem nuclei. Hypothesizing that these modulatory influences might be differentially engaged depending on the visual stimulus and behavioral state, we performed in vivo extracellular recordings from mouse dLGN while optogenetically suppressing CT feedback and monitoring behavioral state by locomotion and pupil dilation. For naturalistic movie clips, we found CT feedback to consistently increase dLGN response gain and promote tonic firing. In contrast, for gratings, CT feedback effects on firing rates were mixed. For both stimulus types, the neural signatures of CT feedback closely resembled those of behavioral state, yet effects of behavioral state on responses to movies persisted even when CT feedback was suppressed. We conclude that CT feedback modulates visual information on its way to cortex in a stimulus-dependent manner, but largely independently of behavioral state. Mammalian vision is based on a hierarchy of processing stages that are connected by feedforward circuits projecting from lower to higher levels, and by feedback circuits projecting from higher to lower levels. Feedforward processing is thought to create feature selectivity  and invaFeedback in the mammalian visual system targets brain areas as early as the dorsolateral geniculate nucleus (dLGN) of the thalamus, where up to 30% of synaptic connections onto relay cells are established by corticothalamic (CT) feedback . Direct Most of the previous in vivo studies have probed the functional role of CT feedback with artificial stimuli, and often in anesthetized animals; CT feedback, however, might be most relevant for processing of dynamic naturalistic information and during wakefulness. From a conceptual perspective, if the role of feedback was to provide context based on an internal model built from the statistics of the world , naturalHere, we recorded spiking activity in dLGN of awake mice and investigated how CT feedback affected dLGN responses to naturalistic movie clips. Suppressing CT feedback either via photostimulation of V1 parvalbumin-positive (PV+) inhibitory interneurons or via direct photosuppression of L6CT neurons, we found that CT feedback had consistent modulatory effects on dLGN responses to movie clips, which could largely be captured by an increase in gain. Effects of CT feedback on dLGN responses to grating stimuli were more diverse, highlighting the stimulus-dependency of CT feedback effects. Finally, while geniculate responses to movies during V1 suppression resembled those during quiescence, we found effects of CT feedback and behavioral state to be largely independent. Overall, our results demonstrate that neural responses to naturalistic movies en route to cortex can be robustly modulated by extra-retinal influences such as cortical feedback and behavioral state, which seem to be largely conveyed via different modulatory pathways.To investigate the impact of CT feedback on visual processing of naturalistic stimuli, we presented to head-fixed mice full-screen movie clips and compared responses of dLGN neurons during optogenetic suppression of V1 activity to a control condition with CT feedback left intact , even duWe observed similar effects in the recorded population of dLGN neurons, where CT feedback enhanced overall responses and promoted tonic firing mode. Indeed, while mean firing rates varied almost 4 orders of magnitude across the population (∼ 0.1–100 spikes/s), they were higher in control conditions with CT feedback intact than during V1 suppression :  neurons . These e neurons .per se caused our findings. First, we repeated our experiments on an Ntsr1- control mouse, which was injected and underwent the same visual and photostimulation protocol. This negative control mouse did not show any effects of photostimulation on dLGN responses  to timepoint-by-timepoint responses in suppression vs. feedback conditions, and extracted the slope and threshold of the fit for each subsample  and the cycle-averaged response to the preferred orientation . Such diverse effects of CT feedback, as reported before for anesthetized mice . In contrast, for neuron 3, orientation selectivity was similar during control and V1 suppression conditions . These results were representative of the population, where CT feedback affected orientation selectivity in diverse ways, with virtually no difference in population means . To test whether V1 suppression affected the ability of dLGN to respond at the gratings’ temporal frequency, for each neuron we computed the amplitude of the response at the stimulus frequency (F1 component) relative to the mean response (F0 component) , we found that V1 suppression could advance response phase   and founse phase . This phzed cats , these azed cats . Also, dzed cats .Our analyses suggest that the impact of CT feedback on firing rates might be stronger overall for naturalistic movie stimuli than for gratings. To test this hypothesis, we focused on the subset of neurons recorded with both types of stimuli. Indeed, when we compared feedback modulation indices , we found that FMI was on average more positive for movies than for gratings  periods directly preceding each full-screen movie and grating trial . However, consistent with the observations shown in 1-4-4, ). In fact, the degree of running modulation was correlated between control conditions with feedback intact and V1 suppressed (firing rate: slope of 1-4). Interestingly, for firing rates and burst ratios, locomotion effects were slightly stronger, on average, with CT feedback intact compared to V1 suppression , indicating that these two modulatory influences likely interact.Finally, to further test the relationship between effects of behavioral state and CT feedback, we directly compared CT feedback and running-related modulations on a neuron-by-neuron basis. We focused on experiments with naturalistic movies, because this was the condition in which we observed robust effects of both CT feedback and behavioral state . This was clearly not the case, because CT feedback effects were correlated across behavioral states (firing rate: slope of 1-4). In addition, and similar to the slightly stronger run modulation with feedback left intact, we discovered a locomotion-dependent CT feedback effect for firing rates and burst ratios: CT feedback effects were slightly stronger, on average, during locomotion than during quiescence . This subtle interaction between behavioral state and CT feedback effects might relate to a previous finding, where careful dissection of brain states by depth of anesthesia had already suggested that the effects of transient cortical inactivation on dLGN responses were more evident during lighter anesthesia, that is, during desynchronized cortical activity . Contrary to this prediction, we found that effects of CT feedback (FMI) and behavioral state (RMI) were uncorrelated Arbr/J; #008069, Jackson Laboratory) and 3 adult Ntsr1-Cre mice -Tg(Ntsr1-cre)GN220Gsat/Mmcd; #030648-UCD, MMRRC) of either sex. Thirty minutes prior to the surgical procedure, mice were injected with an analgesic . To induce anesthesia, animals were placed in an induction chamber and exposed to isoflurane . After induction of anesthesia, mice were fixated in a stereotaxic frame  and the isoflurane level was lowered (0.5–2% in oxygen), such that a stable level of anesthesia could be achieved as judged by the absence of a pedal reflex. Throughout the procedure, the eyes were covered with an eye ointment  and a closed loop temperature control system  ensured that the animal’s body temperature was maintained at 37 ° C. At the beginning of the surgical procedure, an additional analgesic was administered  and the animal’s head was shaved and thoroughly disinfected using idodine solution . Before performing a scalp incision along the midline, a local analgesic was delivered . The skin covering the skull was partially removed and cleaned from tissue residues with a drop of H2O2 . Using four reference points , the animal’s head was positioned into a skull-flat configuration. The exposed skull was covered with OptiBond FL primer and adhesive  omitting three locations: V1 , dLGN , and a position roughly 1.5 mm anterior and 1 mm to the right of bregma, designated for a miniature reference screw  soldered to a custom-made connector pin. Two μL of the adeno-associated viral vector rAAV9/1.EF1a.DIO.hChR2(H134R)-eYFP.WPRE.hGH  was dyed with 0.3 μL fast green . After performing a small craniotomy over V1, in PV-Cre mice a total of ∼ 0.5 μL of this mixture was injected across the entire depth of cortex , using a glass pipette mounted on a Hamilton syringe . In V1 of Ntsr1-Cre mice, we injected 0.35 μL of stGtACR2 . A custom-made lightweight stainless steel head bar was positioned over the posterior part of the skull such that the round opening in the bar was centered on V1/dLGN. The head bar was attached with dental cement  to the primer/adhesive. The opening was later filled with the silicone elastomer sealant Kwik-Cast . At the end of the procedure, an antibiotic ointment  or iodine-based ointment  was applied to the edges of the wound and a long-term analgesic  was administered and for 3 consecutive days. For at least 5 days post-surgery, the animal’s health status was assessed via a score sheet. After at least 1 week of recovery, animals were gradually habituated to the experimental setup by first handling them and then simulating the experimental procedure. To allow for virus expression, neural recordings started no sooner than 3 weeks after injection. On the day prior to the first day of recording, mice were fully anesthetized using the same procedures as described for the initial surgery, and a craniotomy (ca. 1.5 mm2) was performed over dLGN and V1 and re-sealed with Kwik-Cast . As long as the animals did not show signs of discomfort, the long-term analgesic Metacam was administered only once at the end of surgery, to avoid any confounding effect on experimental results. Recordings were performed daily and continued for as long as the quality of the electrophysiological signals remained high.Experiments were carried out in 6 adult PV-Cre mice  for one of the final recording sessions.Head-fixed mice were placed on an air-cushioned Styrofoam ball, which allowed the animal to freely move. Two optical computer mice interfaced with a microcontroller (Arduino Duemilanove) sampled ball movements at 90 Hz. To record eye position and pupil size, the animal’s eye was illuminated with infrared light and monitored using a zoom lens (Navitar Zoom 6000) coupled with a camera . Extracellular signals were recorded at 30 kHz (Blackrock microsystems). For each recording session, the silicon plug sealing the craniotomy was removed. For V1 recordings, a 32- or 64 channel silicon probe  was lowered into the brain to a median depth of 1025 μm. For dLGN recordings, a 32-channel linear silicon probe (Neuronexus A1 × 32Edge-5mm-20–177 A32) was lowered to a depth of ∼2300–3611 μm below the brain surface. We judged recording sites to be located in dLGN based on the characteristic progression of RFs from upper to lower visual field along the electrode shank , the pre2 for activating ChR2 in PV-Cre mice, and 10.84 mW/mm2 for activating stGtACR2 in Ntsr1-Cre mice, as measured at the tip of the optic fiber). Since the tip of the fiber never directly touched the surface of the brain, and since the clarity of the surface of the brain varied , the light intensity delivered even to superficial layers of V1 was inevitably lower. Importantly, changes in dLGN firing rates induced by V1 suppression  did not differ, on average, from those induced by behavioral state   was coupled to a light-emitting diode  and positioned with a micromanipulator less than 1 mm above the exposed surface of V1. A black metal foil surrounding the tip of the head bar holder prevented most of the photostimulation light from reaching the animal’s eyes. To ensure that the photostimulation was effective, the first recording session for each mouse was carried out in V1. Only if the exposure to light reliably induced suppression of V1 activity was the animal used for subsequent dLGN recordings. For gratings, photostimulation started either 0.1 s before stimulus onset and ended 0.1 s after stimulus offset (2 experiments), or photostimulation started 0.3 s before stimulus onset and ended 0.2 s after stimulus offset (11 experiments), or photostimulation started 0.3 s before stimulus onset and ended 0.45 s after stimulus offset (12 experiments). For movie clips, photostimulation started either 0.1 s before stimulus onset and ended 0.1 s after stimulus offset (2 experiments), or photostimulation started 0.3 s before stimulus onset and ended 0.45 s after stimulus offset (45 experiments). LED light intensity was adjusted on a daily basis to evoke reliable effects  and following a 30 min latency period were transcardially perfused under deep anesthesia using a cocktail of Medetomidin , Midazolam  and Fentanyl  (ip). A few animals, which were treated according to a different license, were anesthetized with sodium pentobarbital . Perfusion was first done with Ringer’s lactate solution followed by 4% paraformaldehyde (PFA) in 0.2 M sodium phosphate buffer (PBS).To verify recording site and virus expression, we performed histological analyses. Brains were removed, postfixed in PFA for 24 hr, and then rinsed with and stored in PBS at 4 °C. Slices (40 μm) were cut using a vibratome , stained with DAPI solution before  or after mounting on glass slides (Vectashield DAPI), and coverslipped. A fluorescent microscope  was used to inspect slices for the presence of yellow fluorescent protein (eYFP) and DiI. Recorded images were processed using FIJI .2) positioned at a distance of 25 cm from the animal’s right eye  using custom written software . The display was gamma-corrected for the presentation of artificial stimuli, but not for movies (see below).Visual stimuli were presented on a liquid crystal display (LCD) monitor , we mapped the ON and OFF subfields with a sparse noise stimulus. The stimulus consisted of nonoverlapping white and black squares on a square grid, each flashed for 200ms. For dLGN recordings, the square grid spanned 60° on a side, while individual squares spanned 5° on a side. For a single experiment, the vertical extent was reduced to 50°. For subsequent choices of stimuli, RF positions and other tuning preferences were determined online after each experiment based on multiunit activity, that is high-pass filtered signals crossing a threshold of 4.5–6.5 SD.We measured single unit orientation preference by presenting full-screen, full-contrast drifting sinusoidal gratings of either 12 (23 experiments) or 8 (2 experiments) different, pseudo-randomly interleaved orientations (30° or 45° steps). For dLGN recordings, spatial frequency was either 0.02 cyc/° (17 experiments) or 0.04 cyc/° (8 experiments) and temporal frequency was either 2 Hz (2 experiments) or 4 Hz (23 experiments). One blank condition (i.e. mean luminance gray screen) was included to allow measurements of spontaneous activity. The stimulus duration was either 2 s (23 experiments) or 5 s (2 experiments), with an interstimulus interval (ISI) of 2.4 s (21 experiments) or 1.25 s (2 experiments). For two Ntsr1-Cre experiments, ISIs varied and were either 0.58 s or 1.09 s.For laminar localization of neurons recorded in V1, we presented a full-screen, contrast-reversing checkerboard at 100% contrast, with a spatial frequency of either 0.01 cyc/° (2 experiments) or 0.02 cyc/° (5 experiments) and a temporal frequency of 0.5 cyc/s.Movies were acquired using a hand-held consumer-grade digital camera (Canon PowerShot SD200) at a resolution of 320 × 240 pixels and 60 frames/s. Movies were filmed close to the ground in a variety of wooded or grassy locations in Vancouver, BC, and contained little to no forward/backward optic flow, but did contain simulated gaze shifts (up to 275°/s), generated by manual camera movements  analysis for recordings in area V1 to determine the laminar position of electrode contacts. To obtain the LFP data we first down-sampled the signal to 1 kHz before applying a bandpass filter . We computed the CSD from the second spatial derivative of the local field potentials , and assIn recordings targeting dLGN, we used the envelope of multi-unit spiking activity (MUAe)  to deterEach unit’s peristimulus time histogram  was calculated by convolving a Gaussian of width We defined bursts according to To quantify the effect of V1 suppression on various response properties, we defined the feedback modulation index (FMI) asSignal to noise ratio (SNR) was calculated according to where The sparseness where Response reliability was quantified according to To detect response peaks in trial raster plots and measure their widths, clustering of spike times collapsed across trials was performed using the gradient ascent clustering (GAC) algorithm , with a To determine whether V1 suppression changes dLGN responses in a divisive or subtractive manner, we fit a threshold-linear model using repeated random subsampling cross-validation. To this end, we first selected a random set of 50% of the trials for each condition for fitting to the timepoint-by-timepoint responses a threshold linear model given by For display of spike rasters , trials R0, response at the preferred orientation In this expression, Orientation selectivity was quantified according to where We computed the first harmonic of the response tk are the times of the individual spikes. We excluded the first cycle to avoid contamination by the onset response. For  or not suppressed by contrast (non-SbC) by comparing their mean firing rates during full-screen drifting grating presentation to their mean firing rates during blank-screen presentation. Units were classified as SbC if they were visually responsive to gratings (see below) and had a median z-scored response across orientation conditions of  results , where STo identify electrode channels within the dLGN, and their relative depth, which could be useful to distinguish between shell and core, we concentrated on the RF progression as assessed with MUAe maps that were constructed using sparse noise experiments. Because RF progression is mainly along elevation, amplitudes of MUAe for each channel were collapsed across azimuth and then range normalized. Channels with normalized amplitudes higher than an empirically set threshold (0.4) were considered part of dLGN. Non-detected channels located between detected channels were also included.Direction selectivity index DSI,  was calcR0 is baseline firing rate independent of orientation.where The RF distance from the center of the screen was calculated for each unit by finding the position of the MUAe RF for the channel on which the unit’s mean spike waveform had the largest amplitude.lt0. For gratings, we converted firing rates in response to each orientation to z-scores relative to responses to the mean luminance gray screen. We only considered visually responsive neurons, with an absolute z-scored response 1/F0 ratio of at least 0.25.Neurons with mean evoked firing rates < 0.01 spikes/s were excluded from further analysis. For movie clips, only neurons with SNR We used the Euclidean norm of three perpendicular components of ball velocity  to compute animal running speed. For the analysis of neural responses as a function of behavioral state, locomotion trials were defined as those for which speed exceeded 1 cm/s for at least 50% of the stimulus presentation, and stationary trials as those for which speed fell below 0.25 cm/s for at least 50% of the stimulus presentation. To quantify the effect of running vs. sitting on various response properties, the run modulation index (RMI) was defined asThe stimulus viewing eye was filmed using an infrared camera under infrared LED illumination. Pupil position was extracted from the videos using a custom, semi-automated algorithm. Briefly, each video frame was equalized using an adaptive bi-histogram equalization procedure, and then smoothed using median and bilateral filters. The center of the pupil was detected by taking the darkest point in a convolution of the filtered image with a black square. Next, the peaks of the image gradient along lines extending radially from the center point were used to define the pupil contour. Lastly, an ellipse was fit to the contour, and the center of this ellipse was taken as the position of the pupil. A similar procedure was used to extract the position of the corneal reflection (CR) of the LED illumination. Eye blinks were automatically detected and the immediately adjacent data points were excluded. Adjustable algorithm parameters were set manually for each experiment. Output pupil position time-courses were lightly smoothed, and unreliable segments were automatically removed according to a priori criteria. Finally, the CR position was subtracted from the pupil position to eliminate translational eye movements, and pupil displacement in degrees relative to the baseline (median) position was determined bywhere  the eye . AngularEye position standard deviation was computed by first taking the standard deviation of the horizontal eye position at each time point across trials, and then averaging over the 5 s during which the visual stimulus was presented. We focused on horizontal eye position because horizontal and vertical eye movements tend to occur in tandem under head-fixed conditions, and the horizontal position variance is larger , thus seFollowing , changeslme4 package (lmertest package (Data and code availability).To assess statistical significance, we fitted and examined multilevel linear models . Such mo package . We esti package  implemen package . For eacThroughout the manuscript, uncertainty in estimated regression slopes is represented as  This paper will be of interest to neuroscientists interested in understanding the role of corticothalamic feedback in coding of sensory inputs. The authors show that feedback is stronger for natural stimuli compared to artificial stimuli. Surprisingly, the feedback from the cortex works in parallel with other modulatory influences reflecting changes in the arousal (measured here with pupil size) or changes in locomotion. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Tirin Moore as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Robust effects of corticothalamic feedback during naturalistic visual stimulation" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The authors present important data for the role of corticothalamic feedback in awake behaving animals. They find that cortical feedback serves as a gating mechanism for the transmission of signals from retina to cortex, specifically for natural scenes. Both Reviewers supported publication provided the manuscript is edited to improve clarity.Reviewer 1:Variability in pupil dilation can be examined even for repeated natural video presentations as in Figure 5 of Reimer et al. 2014.The visual presentation could be improved by a few small changes – this may seem nitpicky but I think it would make it easier to follow the argument from the figures:1. Using a more distinct color (i.e. not gray) for the feedback suppression period throughout with a legend for "Stimulus On" and "Light On" for these bars in each figure.2. Using more distinct  colors for feedback vs suppression .3. Rather than "Feedback" and "Suppression" something like "V1 Control" and "V1 inhibited" or "Feedback Inhibited" would be helpful.4. In most cases there seems to be plenty of room for each scatter plot to have it's own y axis label.5. Wherever there is room, having legends directly on the figures would be helpful, for example 3a3 and 3i there's no reason to make the reader go back and forth to the caption.2 and p values directly on the figures would be helpful, or at least indicating which ones are significant.6. There are a lot of scatter plots, and including RWould be good to indicate directly in the main text whether trials with running in figure 5 are completely overlapping with running or else what the criteria is.Figure 3 has neurons 1 and 3 but no neuron 2.In Figure 4a, some of the lines don't connect with dots on either side.Was there any difference between shell and core effects? If this was mentioned I missed it.Does silencing V1 cause the pupil to dilate? This is not essential to the paper, but the authors have the data in hand and I've wondered if becoming suddenly and temporarily cortically blind might drive a startle response, especially in earlier sessions.Reviewer 2:Spacek et al. study the corticothalamic feedback of different visual stimuli on visual thalamus. With optogenetic suppression of visual cortex feedback and simultaneous multi-channel recordings in visual thalamus, the authors succeeded to acquire important data about this essential feedback loop in awake, behaving animals. The paper is technically very impressive and the results are important for a wide range of readers.My concerns and suggestions:The Abstract is not mentioning any methods and therefore the reader has no clue where the results come from. Please change.The long Discussion should be structured by introducing headlines.The Introduction, Result, and Discussion is not up to date with the current literature. There were several papers published during the last 2 years which are not or only barely mentioned, but which are relevant for this manuscript. Please adapt.For example, several imaging papers of layer 6 were lately published . So, layer 6 is not as mysterious any more as claimed in the Introduction and Discussion.The physiology of dLGN TC neurons should be better described in the Introduction. The authors mention mGluR, but NMDA R and AMPA R are as important or even more important for the interpretation of the results. Especially, NMDA plateau potentials in the dendrites of TC neurons can easily explain the observed gain of CT feedback . Therefore, the statement in line 455-456 is not necessary – it can be easily replaced by a few sentences about TC neuron physiology.dLGN receives modulatory feedback from visual cortex L6 and modulatory input from brainstem. In this light, it is not surprising that locomotion and CT feedback are to some extent independent. This should be mentioned in the Discussion and the Abstract.The resting state has a wide range of attentive states which can be determined by the pupil diameter , p. 13289). The state of the animal is specifically important due to the spike/burst controversy  https://doi.org/10.1038/89434). The authors should comment if the pupil diameter, and thereby the state of the animal, was in any way taken into account during the analysis, especially for the extraction of bursts. Do the authors observe changes in pupil diameter?Related to the previous comment, the authors should discuss how reliably  busts can be separated from high frequency firing.When the video presentation ends, neuron #1 in Figure 1d shows a strong off response with the cortical inhibition still on. Is this a typical behavior? Can the authors explain? Reviewer 1:Variability in pupil dilation can be examined even for repeated natural video presentations as in Figure 5 of Reimer et al. 2014.We thank the reviewer for this remark. We have implemented the method proposed by Reimer et al. (2014) and performed the analyses for our dLGN data, comparing ring rates in the absence of locomotion, for more rapidly than usual dilating vs. constricting pupil sizes. We have done these analyses separately for trials in control conditions vs. V1 suppressed. We consistently observed that pupil dilation is associated with enhanced ring rates, and this effect persists even if CT feedback is suppressed. These new results strengthen the conclusions that were based on using locomotion as a marker of behavioral state.The visual presentation could be improved by a few small changes – this may seem nitpicky but I think it would make it easier to follow the argument from the figures:1. Using a more distinct color (i.e. not gray) for the feedback suppression period throughout with a legend for "Stimulus On" and "Light On" for these bars in each figure2. Using more distinct  colors for feedback vs suppression .We thank the reviewer for helping us to improve our visualization. We have revised the color palette for our plots and added the legends.3. Rather than "Feedback" and "Suppression" something like "V1 Control" and "V1 inhibited" or "Feedback Inhibited" would be helpful.Thank you for the suggestion. We followed the reviewer’s advice and now label the axes with V1 Control and V1 Suppressed.4. In most cases there seems to be plenty of room for each scatter plot to have it's own y axis labelWe have added y axis labels in all places where we felt that they would be helpful. In other places, we preferred to share y axis labels across panels to reduce unnecessary clutter.5. Wherever there is room, having legends directly on the figures would be helpful, for example 3a3 and 3i there's no reason to make the reader go back and forth to the caption.Agreed. We have added more fi gure legends.2 and p values directly on the figures would be helpful, or at least indicating which ones are significant.6. There are a lot of scatter plots, and including RWe have now added p values directly to the figures.Would be good to indicate directly in the main text whether trials with running in figure 5 are completely overlapping with running or else what the criteria is.We have added the requested information to the main text, as suggested by the reviewer.Figure 3 has neurons 1 and 3 but no neuron 2.Our intent was to use the same labels for the example neurons throughout the text. For Figure 3, we want to introduce another example neuron , because this neuron compellingly exempli es that effects of CT feedback can also enhance responses to gratings. We have clarified this in the respective figure captions.In Figure 4a, some of the lines don't connect with dots on either side.We apologize for the confusion. We randomly jittered the horizontal position of the points to avoid overlap, but the lines connecting the pairs still end at the central position to represent the actual values. To avoid future misunderstanding, we have added a brief explanation to the figure caption.Was there any difference between shell and core effects? If this was mentioned I missed it.We thank the reviewer for the interesting question. We analyzed the effects of V1 suppression more generally as a function of depth relative to the top-most channel recorded in dLGN, in order to avoid having to binarize into putative shell and core. These results are shown in panels Figure 1S3b and g, and Figure 3S1b and g. To increase the clarity of presentation, we have now stated in the methods and in the caption of Figure 1S3 that depth could serve as a proxy for dLGN shell and core.Does silencing V1 cause the pupil to dilate? This is not essential to the paper, but the authors have the data in hand and I've wondered if becoming suddenly and temporarily cortically blind might drive a startle response, especially in earlier sessions.2) than in PV-Cre mice (median intensity to activate ChR2: 13.66 mW/mm2).We thank the reviewer for the interesting question. We have now examined how pupil size changes during V1 suppression . Contrary to the hypothesis raised by the reviewer, we find either no change at all (in the case of direct L6CT suppression) or a slight pupil constriction (in the case of suppression via PV activation). We believe that the slight pupil constriction in the latter case is indicative of some light leaking through the shield that is attached to the well around the craniotomy, or perhaps through the brain itself to the retina, rather than an effect of arousal. Indeed, given the exquisite light sensitivity of stGtACR2, we used lower light intensities for direct photosuppression in Ntsr1-cre mice . Third, we analyzed additional experiments performed in a Crenegative Ntsr1 mouse that underwent the same surgical and injection procedures as the experimental animals. We found no effects of optogenetic light stimulation .Overall, we would like to thank the reviewer for the detailed read and thoughtful suggestions how to improve our manuscript.Reviewer 2:Spacek et al. study the corticothalamic feedback of different visual stimuli on visual thalamus. With optogenetic suppression of visual cortex feedback and simultaneous multi-channel recordings in visual thalamus, the authors succeeded to acquire important data about this essential feedback loop in awake, behaving animals. The paper is technically very impressive and the results are important for a wide range of readers.My concerns and suggestions:The Abstract is not mentioning any methods and therefore the reader has no clue where the results come from. Please change.We thank the reviewer for pointing this out. We have revised the abstract and modified the title.The long Discussion should be structured by introducing headlines.We have revised the Discussion to be more succinct and introduced headlines, as suggested by the reviewer.The Introduction, Result, and Discussion is not up to date with the current literature. There were several papers published during the last 2 years which are not or only barely mentioned, but which are relevant for this manuscript. Please adapt.For example, several imaging papers of layer 6 were lately published . So, layer 6 is not as mysterious any more as claimed in the Introduction and Discussion.We fully agree. We apologize for the lack of specificity and recency and have updated all parts of our manuscript with respect to the current literature. For instance, in the new version of the introduction, we now explicitly highlight the important work by Augustinaite and Kuhn (2020) and Liang et al. (2021) regarding the effects of behavioral state on V1 L6CT neurons.The physiology of dLGN TC neurons should be better described in the Introduction. The authors mention mGluR, but NMDA R and AMPA R are as important or even more important for the interpretation of the results. Especially, NMDA plateau potentials in the dendrites of TC neurons can easily explain the observed gain of CT feedback . Therefore, the statement in line 455-456 is not necessary – it can be easily replaced by a few sentences about TC neuron physiology.Thank you very much for this very helpful comment and the pointer to the literature. We have modified the introduction to better introduce the cellular physiology of TC neurons and mention the NMDA plateau potentials as one potential source for depolarization associated with gain modulations in the introduction and discussion.dLGN receives modulatory feedback from visual cortex L6 and modulatory input from brainstem. In this light, it is not surprising that locomotion and CT feedback are to some extent independent. This should be mentioned in the Discussion and the Abstract.We thank the reviewer for this helpful recommendation. We have modi ed the abstract and added a brief note to the introduction that dLGN receives neuromodulatory inputs from the brain stem, which can also have depolarizing effects on dLGN relay cells. In addition, we have expanded this point in the discussion. Yet, we still think that our finding of rather independent effects of CT feedback and neuromodulation is interesting and relevant for a number of reasons. On the one hand, recently published results indicate that our finding is rather general, because signals in visual thalamic neurons related to whisker movements also seem to be independent from visual cortical activity . On the other hand, the emerging picture might not be as straightforward as suggested, as two recent studies report a relationship, rather than independence, between CT feedback and behavioral state modulations of dLGN neurons for spontaneous activity  or gratings . We have re-written the discussion to incorporate these new findings.The resting state has a wide range of attentive states which can be determined by the pupil diameter , p. 13289). The state of the animal is specifically important due to the spike/burst controversy . https://doi.org/10.1038/89434). The authors should comment if the pupil diameter, and thereby the state of the animal, was in any way taken into account during the analysis, especially for the extraction of bursts. Do the authors observe changes in pupil diameter?We thank the reviewer for the interesting question. So far, our manuscript had only taken locomotion as a proxy for behavioral state, as locomotion typically goes along with increased pupil size  and increased levels of arousal . To also study the effects of locomotion-independent arousal, we have now applied the analysis mentioned by the reviewer: following methods originally suggested by Reimer et al. (2014), we identified periods of the video presentation without locomotion that corresponded to the upper or lower quartile of pupil size change. Similar to the results that Reimer et al. (2014) found for primary visual cortex, we observed that ring rate in dLGN was enhanced during times when the pupil was dilating faster than usual vs. when it was constricting faster than usual. Like the effects of running, the modulations by pupil-indexed arousal persisted even with V1 suppression. We present these new results in Figure 5 – Supplement 2.Related to the previous comment, the authors should discuss how reliably  busts can be separated from high frequency firing.We thank the reviewer for this important technical comment. We fully agree that our assessment of bursting with extracellular recordings is indirect and hence less reliable than with intracellular recordings. We would like to point out, however, that our criteria for defining bursts have been shown to be a stringent criterion for selecting only those spikes originating from calcium-induced bursts . Accordingly, our spike trains show features that have been known from intracellular recordings of bursts riding on top of low-threshold calcium spikes, namely the lower spontaneous activity, phase advance in response to gratings, and the higher degree of non-linearity . We have added a cautionary note to the discussion and methods.When the video presentation ends, neuron #1 in Figure 1d shows a strong off response with the cortical inhibition still on. Is this a typical behavior? Can the authors explain?Based on visual inspection, we think that this behavior is fairly typical across the population of recorded neurons. At video offset, we have both an increase in luminance as well as a reduction in contrast. One might speculate that these changes at video offset might drive larger responses with V1 suppression, because ring rates in response to the video are overall lower and thus dLGN neurons might not be as exhausted as with CT feedback intact.


Strainflow, a supervised and causally predictive model using unsupervised latent space features of SARS-CoV-2 genome sequences. Strainflow was trained and validated on 0.9 million sequences for the period December, 2019 to June, 2021 and the frozen model was prospectively validated from July, 2021 to December, 2021. Strainflow captured the rise in cases 2 months ahead of the Delta and Omicron surges in most countries including the prediction of a surge in India as early as beginning of November, 2021. Entropy analysis of Strainflow unsupervised embeddings clearly reveals the explore-exploit cycles in genomic feature-space, thus adding interpretability to the deep learning based model. We also conducted codon-level analysis of our model for interpretability and biological validity of our unsupervised features. Strainflow application is openly available as an interactive web-application for prospective genomic surveillance of COVID-19 across the globe.The global efforts to control COVID-19 are threatened by the rapid emergence of novel SARS-CoV-2 variants that may display undesirable characteristics such as immune escape, increased transmissibility or pathogenicity. Early prediction for emergence of new strains with these features is critical for pandemic preparedness. We present  Nextstrain , Australia (green), and England (magenta) are highlighted in Strainflow captures the temporal emergence of strains and geographic information in a country-specific manner.The phylogenetic tree with globally collected sequences  demonstrEntropy of a latent dimension has biological significance as it intuitively captures the variation in codon level changes during a certain time window. Each latent dimension encodes a combination of codon weights and increase in entropy represents frequent changes to these weights. Temporal changes in entropy are therefore expected to uncover the explore-exploit cycles of SARS-CoV-2 spike gene changes, hence biologically indicative of future trends. To compare different geographical regions, the sum of sample entropy was computed for each latent dimension across all the months. This revealed that certain geospatial regions such as France and Germany  and UnitWe then attempted to decipher the relationship between monthly sample entropy and monthly new COVID-19 cases in different countries. Detrended cross-correlation coefficient was calculated at different lag values, which revealed that entropy dimensions have a leading relationship with new cases . This suOur model can be therefore used to predict the COVID-19 caseloads in several countries. Both United States  and Japa.We further assessed the potential link of the predictive features with SARS-COV-2 variants by extracting the top 10 contributing codons and their associated weights for each dimension . The intCodon weights  of a givFor investigating the potential of our predictive features to track the spread of SARS-CoV-2, we used the codon level information of the SARS-CoV-2 delta variant for the spike gene and extracted the weights of these codons specific to each feature. We selected Dimensions 3, 4, 12, 13, 15, 16, 25, 28, 30, 32 with high absolute weights for the codons related to the delta variants . The entOur case prediction model was frozen in June, 2021 and prospectively predicted the caseloads from July, 2021 to December, 2021. Our model predicted the case upsurge in India due to the Omicron variant in November, and December, 2021  2 monthsWe have implemented an approach for analyzing the emerging strains based on the latent space of spike protein coding nucleotide sequences. We chose the nucleotide sequences instead of proteins in order to capture and track the variations that may not have immediate functional consequences. Our approach has two main underlying tenets: 1) long-range interactions are known to modulate the functional interaction between receptor binding domain and ACE2 receptors, hence may be captured in the NLP models that capture 3-mer changes and context, and 2) latent dimensions may be differentially correlated with indicators of spread, thus providing a data-driven handle for tracking and predicting variants of concern and variants of interest . The pipWhile most research studies have attempted to predict the exact number of cases and have failed, our work is focussed on early prediction of trends from a non-obvious source of data. Unlike obvious data sources, the inter-relationships between codons in genome sequences are complex and less likely to be influenced or biased. Furthermore, sequencing data are made routinely available via various national and global consortia for genomic surveillance of SARS-CoV2. Our study also highlights the potential for triangulating insights from completely unrelated datasets, an approach that is expected to eliminate systematic biases in reporting by independent organizations. Further studies may triangulate insights from disparate, heterogeneous datasets such as mobility, genome surveillance, testing and case predictions to partially solve the problem of biases in individual datasets.Entropy is a measure of the disorder of a system. We hypothesized that mutations increase the chaotic dynamics in the latent space of spike genes. To calculate entropy, we used the accelerated versions of the Approximate Entropy and Sample Entropy algorithms, called Fast Approximate Entropy and Fast Sample Entropy . Both alTo predict new COVID-19 cases, a random forest regression model was trained on the monthly entropy data. With sample entropy, we achieved an R-squared value of 73% on the validation set, while with approximate entropy, the value was only 10%. Therefore the model trained on sample entropy was selected. The predictions from the model were found to be highly correlated with the actual cases, indicating that our model can be used for preemptive warning signals for the rise in cases in different countries. Further, the actual and the predicted difference in the number of cases in consecutive months was found to be correlated, which suggests that the relative change in the cases in consecutive months predicted by our model is linked to the relative change in the number of cases. Overall, we recommend that our model be used to predict dangerous trends and not the actual number of cases. Further, the mapping from latent dimensions to Variants of Concern (VOCs) and Variants of Interest (VOIs) may help us track the country-specific spread of different variants.The COVID-19 pandemic has been a dynamically evolving scenario, with new strains emerging and vaccines being developed. With the SARS-CoV-2 genome constantly mutating, we anticipate an underlying change in the grammar of the sequence, underpinning the need to update our language model every few months. Further, the regression model for caseloads needs to be periodically retrained too. An empirical analysis led us to discover that the Random forest model used for prospective validation from November, 2021 onwards performed better in terms of predicting the number of cases than the model used for prospective validation from July 2021 . HoweverFurther, models trained on genomic sequences can be used for predicting infection severity based on Co-associations between the SNPs of Co-morbid Diseases and COVID-19 . The macStrainflow pipeline for SARS-CoV-2. Furthermore, the present study does not consider the interaction between the spike gene and other genes in the SARS-CoV-2 genome. We have not considered the interaction between the ACE2 receptor sequence for the human and the spike gene sequences due to the unavailability of such large-scale paired data. However, we believe this is a strength of our study as we were able to extract relevant features as well as make valid predictions using the spike region of the SARS-CoV-2 gene alone.Our study has the following limitations. Our approach of codon embeddings does not indicate the position where the codon change may have happened in the spike gene. This is because low-dimensional embeddings do not preserve the positional encoding of words. However, we are investigating advanced approaches such as complex-valued word embeddings with positional encodings and transformer models such as BERT to overcome our current limitations . The latOur current approach does not explicitly capture specific positional mutations. Although the ad-hoc analysis for codon weights on significant dimensions allows us to rank the codon level changes, the predictive feature is a complex nonlinear combination of these changes which may eliminate strongly associated features. The E484Q mutation was not captured as the most important in our model. However, this may be because other codon level changes such as L452R and their combinations may be correlated and hence a proxy for E484Q. Importantly, the B.1.617 variant has both L452R and E484Q mutations and L452R change was predictive and captured in the top ten ranks for multiple latent dimensions .Finally, a relatively small number of samples were used to construct the supervised predictive model for case prediction. As more data becomes available in subsequent months, we can produce more confident case predictions. An empirical validation depicted that we require a minimum of 100 samples per month for calculating the sample entropy. This also underscores the need for a more reliable and agile approach to deposit country-level datasets on repositories such as GISAID. We make an appeal to the countries to facilitate the sharing of such data in order to be prepared for any future waves of the current pandemic and for preventing the new emergence of strains. We believe our study is an instance of the new paradigm of pathogen surveillance using a novel language modelling approach that is potentially scalable to infectious disease surveillance and antimicrobial resistance.Training dataset: The dataset was downloaded from GISAID EpiCoV  (release) . 0.36 mirelease) . AccessiEvaluation dataset: We downloaded around 0.6 million genome sequences submitted to GISAID from April 2021 to June 2021. We used our trained model to predict the latent representations for these sequences.In our Strainflow pipeline, we have adopted a word2vec model . Low dimVarious word2vec models were trained on the dataset with different vector sizes varying in multiples of three. Based on the PIP loss calculations, we found out that word embeddings with 36 dimensions showed a differential dent in the curve (change in straight line), due to which we selected this to be the dimension of the word embeddings .To evaluate the phylogenetic properties based on the latent dimensions of the spike gene, we computed the cosine distances among spike genes of SARS-CoV-2 with the 36 latent dimensions. The pairwise distance was further used for hierarchical clustering using the ‘hclust’ function in R statistical programming language. This analysis was performed using 400 random sequences of spike genes from 16 countries. The visualization of the phylogenetic tree derived based on the latent dimensions was done using “iTOL” software  Letunic.To quantify the properties of latent dimensions, we have used a well-known information theory based algorithm suitable for time series datasets, called “Fast Sample Entropy” . To compIn our case, “x” is the latent dimension of the spike genes of the SARS-CoV-2 strains per month for a given country with default values of “m” and “r.” Entropy was computed for each latent dimension on a monthly basis for each country. To compare geographies across months, we used average entropy derived from 36 latent dimensions, followed by normalization using all the monthly entropies for a given country . To comph and new cases at time t, where h = 0, ±1, ±2, ±3 … ±10.To investigate the information content (entropy) of the latent dimensions with the new cases observed for COVID-19, we used the Detrended Cross Correlation Analysis  Here, DCCountry-wise monthly total new cases data was taken at the end of each month. Total new cases data for each month was merged to monthly entropy dimensions data from March, 2020 to June, 2021. We used a regression based machine learning approach called “Boruta,” a wrapper algorithm around a random forest algorithm to select the most relevant entropy dimensions for the prediction of subsequent 2 months’ new cases. We usedTo predict the new cases in the next to next months, we used a regression based random forest model using the most relevant predictive features using the “Boruta” R package . The modTo find the top codons associated with the latent dimensions, we extracted the absolute weights of each codon for a given dimension. The top 10 codons having the highest absolute weights (contribution) were identified corresponding to each dimension to link these to SARS-CoV-2 variants. We collected the SARS-CoV-2 variants and their associated genetic variations at the codon level linked to the spike gene, and a list of codons associated with VOIs and VOCs was curated ; Lopez-. The cur1. We have collected the SARS-CoV-2 sequences from the GISAID EpiCoV database. High quality sequences with complete temporal information were filtered.2. We extracted the spike gene region of these sequences from FASTA files using the CoV-Seq tool. A CSV containing these sequences and other metadata such as country names and dates was created.3. The sequences were splitted into chunks of three characters (codons). A splitted sequence represents a document with three-letter words.4. We trained a word2Vec model on the spike gene sequences for learning 36-dimensional word embeddings. The average of all word embeddings in a given sequence was treated as the embedding of the sequence.5. We calculated the sample entropies of each dimension of our embeddings for each month and country.6. New COVID-19 cases for each country in each month were calculated using data from the JHU CSSE repository.7. A feature selection algorithm (Boruta) was used for selecting the entropy dimensions predictive of caseloads 2 months in advance.8. Random Forest regression algorithm was used for predicting new cases 2 months ahead of time. The inputs to the model are the country names and important features extracted from the Boruta algorithm. The predictor variable is the caseload 2 months ahead of time for each country.The algorithm for the Strainflow pipeline has been described below:


Virtual automatic bronchoscopic navigation (VBN) systems to determine the route to peripheral pulmonary lesions (PPLs) in lung cancer can improve diagnostic biopsy yields. However, compared with VBN, drawing manual routes using computed tomography images, especially with oblique methods, can identify more routes. The Ziostation2 VBN system combines the benefits of these 2 methods; we evaluated this performance by comparing 3 different route-determining methods.We retrospectively collected data from 50 patients with PPLs measuring <30 mm who underwent transbronchial biopsy with an ultrathin bronchoscope at the Osaka International Cancer Institute during January to December 2018. We compared automatic VBN (Ziostation2), manual route modification using an oblique method after automatic VBN, and manual navigation using a general application computed tomography viewer. Concordance between predicted and actual branching were determined. We also compared the predicted relationship between the terminal bronchi and the lesion by 2 of the methods with ultrasonographic images .P < .001). Expected route bifurcations were exact matches with actual branching in 45/48 of the patients using manual modification after automatic VBN. The predicted relationship between the terminal bronchi and the lesion using manual modification after VBN matched the radial-EBUS images in 35/50 of the patients.Manual modification after automatic VBN significantly increased the rate of determining routes to the target (66%) versus with the automatic VBN alone (32%) (Manual modification of routes to PPLs using an oblique method after automatic VBN predicted actual radial-EBUS route imaging and could help determine appropriate patients for bronchoscopy. In addition to surgical resection, the treatment options for early lung cancer, such as stereotactic radiotherapy and heavy particle radiotherapy, are varied, and the demand for a definitive diagnosis of early lung cancer without a surgical procedure is increasing. These techniques are recommended by the American College of Chest Physicians lung cancer guidelines. With VBN, the diagnostic yield is reportedly superior to that with manual bronchoscopic navigation (MBN) involving a manual analysis of 2-dimensional CT images. Bronchoscopic guidance lets physicians select the route to the target, and the importance of selecting a bronchus under bronchoscopic direct vision has increased compared with the importance of adjustment under fluoroscopic imaging.,4 Therefore, ultrathin bronchoscopy (UTB) is expected to be effective. Additionally, in one study, the diagnostic yield of the novel 3.0-mm ultrathin bronchoscope directed by radial-EBUS, VBN, and fluoroscopy was higher than that of conventional 4.0-mm thin bronchoscopes directed by the same methods. However, thinner bronchoscopes require more accurate navigation because these are able to reach more distal bronchi. In addition, the bronchoscopic instruments in UTB for sampling are smaller compared with the conventional procedure, and the sampling area acquired by UTB is narrower. Inadequate navigation is directly involved in biopsy failure.Various approaches are available for diagnosis when performing biopsies of these suspicious lesions. Recent modifications to bronchoscopy, such as the use of radial-probe endobronchial ultrasonography  with or without a guide sheath (GS), and virtual bronchoscopic navigation (VBN) have increased the diagnostic yield of bronchoscopy for small peripheral lung lesions. DOM supports precisely identifying airways without mental reconstruction, and the accuracy of identification and bronchi selection both in the drawing phase and in the bronchoscopic simulation phase is reportedly better than with automatic VBN.The direct oblique method (DOM) is a MBN technique that is not a software program but rather a manual procedure conducted using a general application CT viewer.It is important to develop a bronchoscopic guidance system that supports physicians by providing greater accuracy and visualization of images of the bronchial bifurcations than existing VBN systems. Some existing VBN systems, namely VINCENT-sim , DirectPath , and Bf-NAVI  can be used with manual CT analysis. With these systems, for example, DirectPath and Bf-Navi, it is possible to create a virtual image in the vicinity of the lesions by additional extraction if the automatic extraction of the bronchi is insufficient. Completing a bronchial tree by placing dots in the bronchi on the CT images from the target to the terminal of the automatic extraction can confirm a virtual bronchus. Although this option is especially useful, and the developers also recommend manual additional extraction, it is necessary to draw the bronchi three-dimensionally with CT, which requires time. Therefore, techniques for tracking branches three-dimensionally are needed because the CT images browsed for additional bronchi are not cross- or vertical sections of the bronchi.For these reasons, we considered it important for navigation to make manual modifications of bronchi easier and more accurate. Additional extraction using the oblique method makes it easy to confirm the continuity of the bronchi because it is possible to track the bronchi using cross- and vertical sections that rotate freely around the route as an axis. Because VB-like cross-section oblique CT images of the route can be used in the simulation phase, route addition is possible simply by adding dots on the CT images as a marker for each route branch. In this study, we investigated the usefulness of a method to perform additional extraction using the oblique method with automatic bronchial extraction obtained by VBN.2This study was approved by the institutional review board of our institute.This study was observation study designed to evaluate the accuracy of the manual modification by the oblique method with the Ziostation2 compare with automatic VBN to that of automatic VBN and that of direct oblique method.2.1We retrospectively collected data from all patients with small peripheral pulmonary lesions (PPLs)  who underwent bronchoscopic examination with a 3.0-mm ultrathin bronchoscope  at the Osaka International Cancer Institute, Japan, during January to December 2018. A total of 50 subjects were included in this study. Data comprised thin-section CT images and the calculated routes by automatic VBN  described below as automatic VBN ), with modification using the oblique method with the Ziostation2 automatic VBN ) and with DOM using a general application CT viewer .All patients underwent bronchoscopic examination directed by 2 navigation systems (Ziostation2 and DOM using Aquarius), radial-EBUS, fluoroscopy, and rapid onsite evaluation.2.2First, CT was performed using a helical CT scanner . The bronchus involved in the lesion was evaluated on images obtained by reconstruction at ≤0.65-mm intervals using the chest algorithm (window width: 1500 Hounsfield units (HU), window level: −600 HU) from helical CT data. Although reconstruction at ≤0.65 mm is recommended, this is unnecessary for analysis using DOM and VBN in Ziostation2.2.3DOM is not a software program, but a manual procedure conducted using general application CT viewers. DOM can be analyzed in general application CT viewers with availability of oblique CT images and manual placement of dots on the CT images. DOM can be conducted using several CT viewers, namely Vincent , the Aquarius thin-client viewer, Ziostation, and OsiriX.The concept of DOM is that adequately adjusted two-dimensional oblique CT images are VB-like regarding the shapes and angles of bronchial bifurcations, which is necessary and sufficient for performing bronchoscopy. Compared with traditional MBN, DOM does not require mental reconstruction in which the physician mentally visualizes a complex three-dimensional airway construction by scrolling through contiguous 2-dimentional CT images. In this study, the DOM procedure was implemented as follows using the Aquarius thin-client viewer: in the drawing phase, a bronchus leading to the target was manually identified and manually marked with dots on the CT images. The bronchus was confirmed using cross-sectional CT.2.4The concept of modification using DOM with automatic VBN in Ziostation2 is to connect the adequately adjusted two-dimensional oblique CT images to automatically extracted bronchi seamlessly both in the drawing phase and in the bronchoscopic simulation phase Fig. . In the In the bronchoscopic simulation phase, the drawn routes are shown by VB. The operator refers to the VB images up to the central bronchus (lobar bronchus to segmented bronchus in most cases), and on the peripheral side, then refers to the corresponding cross-sectional oblique CT images of the marked route. Adequately adjusted two-dimensional oblique CT images are VB-like regarding the shapes and angles of the bronchial bifurcations, which eliminates the need for mental reconstruction.After switching VB images to oblique CT, the oblique CT accurately provides the necessary and sufficient information, such as branch selection, angle, and the distance required for the bronchoscope. The signpost of the bronchi is also marked on the CT images, and the thumbnail of the VB images and oblique CT images at each bronchial bifurcation is displayed as a catalog.Routes were calculated for the 50 lesions using Ziostation2 and a general application CT viewer (Aquarius). The route was classified as follows . In 2 cases, airway identification for virtual navigation was difficult using Ziostation2 because the existing lung anatomy was abnormal owing to chronic inflammation and postoperative changes. In these cases, it was possible to confirm the bronchi using DOM.In order to assess inter-reliabilities coming from different operators, these and other classifications were independently confirmed by 2 respiratory specialists who had been trained in DOM for at least 1 year. At the time of bronchoscopy, both results were discussed, and the best route was chosen for the bronchoscopy.2.5The statistical analyses were performed with EZR, version 1.37 , which is a graphical user interface for R .P ≤ .05 was considered significant. Odds ratio and 95% confidence intervals were also calculated.The correlation of the locational relationship between the terminal bronchi and the lesion predicted by 3 navigation methods with ultrasonographic images acquired by radial-EBUS was tested by Spearman rank correlation coefficient, denoted by “rs.” For comparisons of the mean values for each group, we used the chi-square test. All statistical tests were 2-sided;  and applied the statistical power at 0.9 [2-sided]) and significance level at 0.05, a total of 40 patients would be sufficient.If we were to compare the airway detection rate by the Ziostation2 method (expected to be around 90%) versus that of the conventional automatic VBN (previously reported to be around 70%κ) coefficients were calculated. The κ coefficients were calculated using BellCurve for Excel version 3.21 .To assess inter coefficients for agreement in the route evaluation results between 2 observers, kappa , 0.73 , 0.86 , respectively.The best route provided by Zio(A) was classified “optimal route” in 16 cases 32%), “optimal halfway” in 13 cases (26%), “suboptimal route” in 6 cases (12%), “suboptimal halfway” in 7 cases (14%), and “not on the route” in 6 cases (12%). Manual modification by the oblique method increased the rate  to calculate the new “optimal route” to the target in addition to the automatic VBN . Ultrathin bronchoscopy confirmed directly that the radial-EBUS probe advanced through the final bifurcation to the target in 32 (64%) cases, and in 18 (36%) cases, the ultrathin bronchoscope advanced 1 or 2 branching before the bronchus, for which navigation could be analyzed. For 45 (90%) cases, the bronchial branching predicted by the navigations exactly corresponded to the actual branching. For 2 cases (4%), there was an overlooked fine bronchial branching by all navigation systems when analyzing the CT images. In 1 case (2%), there was more peripheral bronchial branching after the limit of the system's analysis. For 2 cases (2%), we could not confirm the actual branching, retrospectively.The relationship between the prediction by navigation and ultrasonographic imaging is shown in Table P < .01) than that of Zio(A) .The prediction by Zio(M) showed stronger correlation with the ultrasonographic image by radial-EBUS . Using the prediction by Zio(A), the diagnostic yields of the cases with calculated terminals “within the target” was significantly higher than the cases with calculated terminals “adjacent to the target” or “not on the route” .The diagnostic yields in histologic specimens and the factors affecting the diagnostic yields are shown in Table P = .004).Using the prediction by Zio(M), the diagnostic yields of the cases with calculated terminals “within the target” was significantly higher than for cases with calculated terminals “adjacent to the target” or “not on the route” .The position of the radial-EBUS probe affected the diagnostic yields. Cases in which the probe was confirmed “within the target” had significantly higher diagnostic yield than cases in which the probe was confirmed “adjacent to the target”  cases, which suggested that fully automatic analysis may be insufficient in nearly half of cases. Therefore, it is meaningful to predict the type of image with EBUS-GS, especially whether the probe is within the lesion. In this respect, the results with Zio(M) predicted 28 of 30 cases (93.3%) which were subsequently confirmed as “within the lesion” using EBUS-GS. Conversely, with Zio(A), it was possible to predict only 22 of 30 cases (73%) subsequently confirmed as “within the lesion” using EBUS-GS. These findings suggest that the probability of the bronchoscope being “within the target” is low by radial-EBUS when the evaluation of the route to the target calculated by Zio(M) is not “optimal” or “optimal halfway.” We believe that evaluating routes on oblique CT images may identify important criteria for selecting appropriate cases for bronchoscopy.We believe that manual modification is necessary for accurate bronchial evaluation. In addition, our results showed no significant difference between the results of fully manual analysis versus manual modification using the oblique method with automatic analysis. Manual modification based on the oblique method with automatic analysis could be useful because it can be used easily without experience or without special techniques. This approach is advantageous in theory; therefore, to verify the usefulness in clinical practice, we compared the navigation results with the results of actual bronchoscopy. Previous studies found that findings using EBUS-GS are causally linked to the diagnostic rate.There may be no need to analyze the bronchi in high detail because the range of the bronchoscope reach is limited. Indeed, the rate of arrival at the final bifurcation was 64%, in this study. However, in most of the other cases, the bronchoscope could be advanced one additional branching before reaching the final bifurcation. In some cases, reaching the final bifurcation is critical to collect samples. In addition, evaluating the relationship between the target and the final bronchus is useful to select the bronchoscope and the sampling device. If the device can be advanced to within the target, forceps are the appropriate sampling device, and if the device can be advanced adjacent to the target, transbronchial aspiration cytology (TBAC) and curettage using thin bronchoscopy are options to consider.In this study, the navigation modified by the Ziostation2 could not completely predict the radial-EBUS image. We have 2 hypotheses for this limitation; 1 is the limit of the bronchoscope's advancement. As mentioned above, in only 64% of the cases could we insert the radial-EBUS to the final bronchus under direct view. The radial-EBUS probe is a delicate device; physicians tend to avoid applying strong pressure, and it is sometimes difficult to bend this equipment at a sharp angle. In some cases, the proper material was sampled by forceps despite having an “adjacent to radial-EBUS” image. The other hypothesis is that during bronchoscopy, we sometimes inject a small amount of saline into the peripheral bronchus to clear the view while the bronchoscope is wedged in the bronchus, and the obstructed bronchus under CT scan may dilate when the sampling device is advanced to the target.In conclusion, modification using the oblique method with VBN systems could predict the ultrasonographic image by radial-EBUS, which could lead to the identification of important criteria for selecting appropriate cases for bronchoscopy.Conceptualization: Takako Inoue.Data curation: Takako Inoue.Formal analysis: Takako Inoue, Satomi Odani.Funding acquisition: Takako Inoue.Investigation: Takako Inoue, Takahisa Kawamura, Kei Kunimasa, Motohiro Tamiya, Hanako Kuhara, Kazumi Nishino.Methodology: Kotaro Miyake.Project administration: Kotaro Miyake.Supervision: Fumio Imamura, Toru Kumagai, Kotaro Miyake.Writing – original draft: Takako Inoue.Writing – review & editing: Toru Kumagai, Kotaro Miyake.


The postnatal period is a critical period for the health of both mother and infant. Studies show that postnatal care reduces neonatal mortality and other adverse mother and child health outcomes. While the World Health Organization recommends four postnatal care contacts, South African guidelines only specify three, excluding a 7-14-day post-birth contact. This study aimed to assess whether a telephonic contact at 7-14 days following delivery had any effect on use of additional postnatal services.A randomized controlled trial design was used to address the study objectives. Two groups of new mothers were randomly allocated to either receive the 7-14-day telephonic contact or not from a research nurse. Data for this study was collected at Maphutha L Malatjie Hospital (MLMH). Descriptive analysis was performed first, then a multivariable logistic regression analysis was conducted to assess the factors associated with access to other health care services.p-value < 0.05). We found statistically insignificant associations between receiving the 7-14-day call and accessing other health care services.A total of 882 mothers were recruited, 854 (97%) were classified as high risk, 28 (3%) were classified as low risk. 417 (49%) of the high risk received the 7-14-day call (intervention group) whilst the remainder of 437 (51%) from the high risk plus all mothers classified as low risk (28) did not receive the call (control group). 686 (78%) of all mothers received the 3 month follow up call. The call showed that 17 mothers from the control group and 10 mothers from the intervention group accessed other healthcare services. We find that hypertension , mental health risk , PV bleeding during pregnancy , problem during labor  were positively associated with access to other health services, with statistically significant associations (The 7-14-day call had no statistically significant impact on access to other health services, however, high levels of satisfaction with the call may point to an unmet need for care at this time. It is important to investigate other innovative solutions to postnatal care improvement in South Africa. In South Africa, maternal mortality rate (MMR) was estimated to be 119 per 100 000 live births in 2017, and neonatal morality rate (NMR) was estimated at 12 per 1,000 live births in 2019/2020 . As a noPrevious studies have assessed the effects of postnatal care on newborn outcomes –11. A stGiven how critical the postnatal phase is, the WHO provides guidelines on timelines of postnatal contacts and the content of these postnatal contacts . The WHOThe study methods and results section adhere to the Consolidated Standards of Reporting Trials (CONSORT) guidelines. The guidelines provide a minimum set of reporting items aimed at improving the quality of randomized controlled trial (RCT) reporting .This study was conducted in Mopani District, which is one of five districts within Limpopo province, in South Africa. Among the five district municipalities, Mopani has the third largest population at approximately 1.2 million . The disData for this study was collected at Maphutha L Malatjie Hospital (MLMH). It is a district level hospital, and is the only hospital located in the sub-district of Ba-Phalaborwa see . As per The study formed part of the Mphatlalatsane Project, which is a collaboration of the South African Medical Research Council, National Department of Health, and the Clinton Health Access Initiative (CHAI) and aims to improve the quality of sexual and reproductive health in the Mopani District, Limpopo.A randomized controlled trial design was used to address the study objectives. The study made use of the following tools: a risk assessment tool, 7-to-14-day check tool, a follow up tool, 3 months follow up tool and a file review.The research nurse made use of this tool to conduct risk assessments of MIPs. Face to face interviews were conducted at the time of discharge from MLMH, after delivery. The research nurse made use of a private space close to the maternity ward to conduct the face-to-face interviews with the mothers. As there is no known validated tool for identifying postnatal risk of complication following discharge the RA tool was developed by combining known risk factors of antenatal, intrapartum and postnatal complications. A risk assessment tool for perinatal depression and anxiety developed by the Perinatal Mental Health Project was also included. Those with a single identified risk factor were classified as high risk and randomly allocated into two groups (1) those that received the additional postnatal contact at between 7 to 14 days (intervention) and (2) those that did not receive the call (non-intervention). Those that were classified as low risk did not receive the 7-to-14-day call and were included in the non-intervention group during the analysis. Those who did not receive the call received the South African standard of postnatal care which includes a 3-6 day and 6-week postnatal facility visit and were able to seek additional care through standard pathways.The research nurse contacted mothers telephonically between days 7 to 14 after delivery. This took place in addition to the South African standard of postnatal care, including the 3–6-day postnatal visit. During the call the research nurse administered a checklist that included a review of the care that the MIPS received at their 3–6 days facility visits, an assessment of maternal postnatal complications and neonatal danger signs, and a validated tool to screen for postpartum depression in pregnancy. Any MIP with problems or complications identified during this call was referred to the appropriate level of care by the research nurse.A week after the 7-to-14-day call, the research nurse would follow up on all MIPs with postnatal problems or complications identified at the 7-to-14-day call. The objective of this call was to ensure that the MIP received services for the issues identified during the 7-to-14-day call and that the issues were resolved.A telephonic follow up was conducted by the research nurse with all study participants (high risk and low risk) enrolled at the beginning of the study. The purpose of the call was to determine whether MIPs experienced any complications, had any additional interactions with health services   and their self-reported resolution of postnatal problems.A file review was then conducted at MLMH for all MIPs who accessed further care outside of the standard South African postnatal care. This was done to gather information on the complaint, diagnosis and treatment prescribed. In cases were MIPs accessed additional care from private hospitals such records were not available in the government sector hospital files and could therefore not be included in the analysis.Ethical approval for data collection was obtained from the Human Sciences Research Council Research Ethics Committee REC 5/21/08/19 and the Limpopo Provincial Health Research Committee (ref: LP-201909-012). Each mother also provided written informed consent to participate in the study.There are an estimated 1000 deliveries in a 6-month period at MLMH. We determined the appropriate sample size for detecting a difference between the proportion of mothers who accessed other healthcare services in the intervention vs. the control group. The sample size was estimated at 683, assuming a sample proportion in the intervention group of 15% and 10% in the control group, a confidence interval of 95% and power of 80%. We added another 10% to this number to account for the possibility of refusals to participate. Postnatal women were excluded from the study if (1) they refused to participate (2) lived outside the Ba-Phalaborwa sub-district (3) were <18 years old and not accompanied by their parent or legal guardian.Data collection was conducted from 3 August 2020 to 29 January 2021 during working hours (Monday to Friday). All mothers discharged from the postnatal ward of MLMH during the study period were invited to participate. No incentives or inducement was offered to the mothers to participate in the study. Face to face interviews and telephonic interviews were conducted with consenting mothers by an experienced research nurse based at the hospital during the study period. Interviews were conducted in the respondents preferred language to overcome language barriers and ensure participants were comfortable and confident with their response. Quality checks for all data collected by the research nurse was done once a week during the study.To keep similar numbers of mothers in the intervention and control groups, block randomization was applied. High risk MIP's study identifiers (IDs) were aggregated in blocks of ten, according to when they were recruited, and then randomized into two: (1) MIPs receiving the additional postnatal contact at 7 to 14 days intervention (2) MIPs receiving the standard South African postnatal care with no additional postnatal contact at 7 to 14 days. Mothers were then informed whether to expect a 7-14-day call prior to being discharged from the hospital.The main outcome variable in this study is access to other healthcare services. At three months mothers were asked if the MIP accessed any other health services during the three months post-delivery. This was coded into a binary variable. Secondary measures used to describe the postnatal period after hospital discharge were post-discharge infant deaths following discharge and maternal experiences during the prescribed 3-6-day visit.The 7-to-14-day call was the main independent variable for this study. Those who received the call were part of the intervention group and those who did not receive it were within the non-intervention group. Covariate variables included in our analysis were taken from the risk assessment and 3 months follow up tool. Demographic variables, health related variables, pregnancy factors, delivery and baby factors considered for the analyses were as follows: For the demographic variables we included age, employment, and education. Age was measured in years and categorized as 18 years and below, 19 to 24 years, 25 to 34 years, and 35 years and older. Employment was included as a binary variable. Education was categorized as follows: –did not complete or completed grade 12Health related factors included HIV status, hypertension during pregnancy , maternal pre-existing chronic conditions, and mental health risk factors. These were included as binary variables. Pregnancy related factors included having four or more pregnancies, PV bleeding during pregnancy and no antenatal care (not booked). These were also included as binary variables in the analysis. Delivery related factors included delivery out-of-facility, problems during labor, preterm labor, cesarean delivery, perineal problems. Mothers were asked if they experienced any complications, and these were also checked in the discharge summary. All delivery related factors were included as binary variables. All baby factors, which included low birthweight (<2500 g), high birthweight (4000 g or over) and resuscitation at birth were included as binary variables.p-value of ≤ 0.3 were included for the final multivariable logistic regression model.The analysis was restricted to mothers who received the 3 months follow up call. Statistical analysis was conducted in STATA 14. Descriptive analysis and multivariable logistic regression analysis were conducted to assess the factors associated with access to other health care services amongst the MIPs. Variables were evaluated as potential determinants of access to health care through bivariate logistic regression analysis. Collinear variables were omitted from the analysis and explanatory variables that had an association A total of 906 mothers were invited to take part in the study of which 24 mothers did not meet the inclusion criteria see . A totalThe characteristics of the study sample are shown in As shown in For the pregnancy factors 22% had more than four pregnancies, 1% experienced PV bleeding during pregnancy and 3% did not have an antenatal booking. Amongst the delivery factors we find five (2%) delivered out of facility, 20 (6%) reported experiencing a problem during labor, 74 (24%) had a cesarean delivery, 109 (35%) experienced puerperium problems. For the baby factors 38 (13%) babies were low birth weight, 8 (3%) babies were macrosomic, and six (2%) babies needed resuscitation at delivery. Amongst those who received the 7-14-day call, 10 (3%) accessed other health services. Our study recorded 31 stillbirths, 14 early neonatal deaths and four post discharge deaths.p-value < 0.05) associated with access to other health services. Although not statistically significant, the odds of accessing other health services were lower amongst those who received the 7-14-day call from the research nurse .p-value < 0.05) with access to other health services. Four or more pregnancies  and having a cesarean delivery  were negatively, significantly associated with access to other health services.In the adjusted model we find that hypertension , mental health risk , PV bleeding during pregnancy , problem in labor  were positively, significantly associated . All mothers for the post discharge deaths were discharged in <24 h and none of the mothers were referred for additional care or follow up at discharge. Baby A was born a twin and died a day after discharge. No problems were identified in either baby. Baby B was born with a low birthweight and died after the 3-6-day visit. During the 7-14-day call the mother reported that the baby experienced convulsions and breathing problems after their return from the 3-6-day checkup. The mother reported that baby died soon afterward, without receiving healthcare. Baby C died between 7 and 14 days after discharge. The hospital file review showed that the baby was referred to the hospital twice for respiratory distress and grunting noise. The first referral was during the 3-6-day visit and the second referral was during an additional non-routine visit that took place 4 days after the 3-6-day visit. During the 7-14-day call the mother reported that baby C was transferred to the hospital were the condition worsened and the baby died. Baby D was born with a low birthweight and died 2-6-weeks after discharge. The mother reported having attended the 3-6-day visit at their health facility and being satisfied with this visit. No problems were identified during the 7–14-day call. During the 3 months follow-up the mother reported that the baby had experienced feeding problems. Three of the post discharge deaths were before the 7-14-day call and the 7-14-day call was not able to identify high risk in the last baby death.During the 7-14-day call the research nurse administered a checklist that included a review of the care that the MIPS received at their 3-6 days facility visits. Out of the 417 mothers who received the call 390 reported attending their 3-6-day visit. Overall, the baby checks were well completed, although 10% were not weighed. The mother only reported being asked about her mood in 5% of cases, and 18% of those with a wound report no wound check. Three of the four post-discharge deaths were after the 3-6-day visit. All three mothers attended their 3-6-day visit, two out of the three mothers reported being satisfied with the visit and all baby checks were performed see . All thrOur study found that that the 7-14-day call did not have a statistically significant impact on additional service utilization. There was, however, non-significant lower odds of accessing additional services, which may point to needs being met during the 7-14 days call. Important factors we found to impact additional service utilization were hypertension, mental health risk, PV bleeding during pregnancy and complications during labor. We also found that the call was not able to identify risk in the one post-discharge infant death that occurred after 7-14-days. Whilst three of the four post discharge deaths were before the 7-14 day-call, three of the deaths were after the 3-6-day visit. It would also appear the 3-6-day visit was able to identify risk in one of the three post-discharge deaths. Our study was able to capture the content of these visits, however, we were unable to assess the quality.Various studies have shown the effectiveness of postnatal care interventions on mother and baby outcomes , 22. It The study also finds various other factors that are associated with the use of additional postnatal care. Various studies have assessed the determinants of postnatal care service utilization  but studHealth related factors found to be statistically associated with access to other health services in our study were hypertension and mental health risk. Previous research has shown that hypertension is one of the top three leading causes of maternal mortality in sub-Saharan Africa . TherefoOur study recorded 14 early neonatal deaths and four post discharge deaths (20 per 1000). This is below the inpatient neonatal mortality rate in Ba-Phalaborwa sub-district which was estimated to be 23 per 1,000 live births in 2016/17 .As previous literature indicates, some mothers do not attend their recommended routine postnatal visits. The District Health Barometer indicates that in 2016/2017 approximately 82% of women in Ba-Phalaborwa sub-district had their postnatal visit within 6 days, . In our Care of the newborn at the 3-6-day visit should include weighing, examination of the cord and a feeding check to ensure the baby is well. In our study we find that during the 3-6-day postnatal visit most postnatal checks were adhered to except mental health. Although studies have shown that postnatal depression is linked with poor child growth , 30 the Despite the importance and novelty of our study it is subject to some limitations. Our study is not nationally representative as data used was collected in rural district in Mopani. Our participants are therefore not from diverse backgrounds and mostly of low socioeconomic status. Despite this limitation our study does provide useful insights for interventions targeting at improving postnatal care and health outcomes. It is also important to note that although the study took place during the COVID-19 pandemic, antenatal care and data collection was not impacted by the restrictions in place at the time .The postnatal period has often been described as a critical opportunity to save mothers and newborns through the provision of effective health care. The study provides insights into some of the outcomes within the postnatal period and the factors associated with accessing additional health services. Although we find that the 7-14-day call did not have a statistically significant impact on additional service utilization, our study shows that hypertension, mental health risk, labor problems and delivery problems were associated with additional postnatal health care access and mothers who received the call do express gratitude at having received this check-up. There is likely a need to mandate additional postnatal care services for women and infants with certain pregnancy and labor-associated issues, however further work is needed to refine these conditions and the risk factors that cause them. This would allow for these women and their infants to be screened prior to discharge and classified into those requiring additional care and those who can have the traditional standard of care. Additional postnatal care can and should be provided in facility as well as in the home to ensure mothers receive sufficient support in the first 6 weeks following delivery.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethical approval for data collection was obtained from the Human Sciences Research Council Research Ethics Committee REC 5/21/08/19 and the Limpopo Provincial Health Research Committee (ref: LP-201909-012). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CM: data analysis and manuscript drafting. JD: review of the data analysis and critical review of the manuscript. HS and JM: critical review of analysis and manuscript. KR: review of study design, data analysis and critical review of the manuscript. All authors provided approval for the version to be published.This study has been funded by the South African Medical Research Council, linked to the Mphatlalatsane Project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.