The granular retrosplenial cortex (RSG) is critical for both spatial and non-spatial behaviors, but the underlying neural codes remain poorly understood. Here, we use optogenetic circuit mapping in mice to reveal a double dissociation that allows parallel circuits in superficial RSG to process disparate inputs. The anterior thalamus and dorsal subiculum, sources of spatial information, strongly and selectively recruit small low-rheobase (LR) pyramidal cells in RSG. In contrast, neighboring regular-spiking (RS) cells are preferentially controlled by claustral and anterior cingulate inputs, sources of mostly non-spatial information. Precise sublaminar axonal and dendritic arborization within RSG layer 1, in particular, permits this parallel processing. Observed thalamocortical synaptic dynamics enable computational models of LR neurons to compute the speed of head rotation, despite receiving head direction inputs that do not explicitly encode speed. Thus, parallel input streams identify a distinct principal neuronal subtype ideally positioned to support spatial orientation computations in the RSG. Sitting in your car, about to drive home after a long day at work, you realize you have no idea which way to go: you recognize where you are right now, and you remember the name of the street your house is on, but you cannot figure out how to get there. This spatial disorientation happens to people with damage to a brain region called the retrosplenial cortex, whose role and inner workings remain poorly understood. Recent evidence has shown that this area contains ‘low-rheobase’ neurons which are not seen anywhere else in the brain, but what do these neurons do?Brennan, Jedrasiak-Cape, Kailasa et al. decided to explore the role of these neurons, focusing on the brain regions they are connected to. Experiments were conducted in mice using optogenetics, a technique that activates neurons using pulses of light. This revealed that brain areas involved in processing information about direction and position preferentially communicate with low-rheobase neurons rather than with nearby, more standard neurons in the retrosplenial cortex. The way these spatial signals are sent to the low-rheobase neurons allows these cells to ‘calculate’ how fast a mouse is turning its head using only information about which direction the mouse is facing. Essentially, this neuron can turn directional compass-like signals into a gyroscope signal that can track both direction and speed of head movement. These unique neurons may therefore be ideally suited to combine information about direction and space, suggesting that they may have evolved specifically to support spatial navigation.Individuals with Alzheimer’s disease show exactly the same type of spatial disorientation as individuals with direct damage to the retrosplenial cortex. This region is also one of the first to show altered activity in Alzheimer’s disease. Exploring whether these unique retrosplenial neurons and their communication patterns are altered in Alzheimer’s disease models could help to understand and potentially treat this debilitating condition. Activity in the granular retrosplenial cortex (RSG) is correlated with a wide variety of behaviors, including spatial navigation, learning, memory, fear conditioning, imagination, and planning for the future . The RSGCortical regions, including the RSG, receive inputs from one or more thalamic nuclei . ThalamiThe cortex, including the RSG, also receives widespread inputs from the densely connected claustrum . As withEach cortical region also receives inputs from select other cortical areas. The RSG receives direct innervation from the secondary motor cortex , contralThalamic, claustral, and cortical inputs all converge in the RSG, but whether they do so via parallel or integrative control of target excitatory neurons remains unknown. Within the RSG, the superficial layers contain two subtypes of principal excitatory pyramidal neurons that have strikingly distinct physiology, morphology, and computational capabilities: the low-rheobase (LR) neuron and the regular-spiking (RS) neuron . Here, wA antagonist, picrotoxin (50 µM), was added to the bath. Compared to their baseline activity, this stimulation evoked a significant increase in spiking in LR neurons  but not RS neurons  contains two types of principal pyramidal neurons: the low-rheobase (LR) neuron and the regular-spiking (RS) neuron . Using wLR neurons are morphologically distinct from their neighboring RS neurons . Given tTo further verify this, we examined the responses of L3 LR and RS neurons to ADAV input at L1a, where the highest projection density of ADAV inputs exist (see Methods). When delivering 1 ms high LED power light pulses, we indeed found that LR neurons had a significantly larger excitatory postsynaptic potential (EPSP) amplitude in response to the L1a LED stimulation compared to RS cell responses , while L5dp responses were significantly smaller than those at L1/2 , indicating that there is no significant lamination of functional responses to thalamic inputs in the RS population, likely due to their relative lack of response to these inputs in general  compared to those at L1a , which serves to transmit allocentric spatial information such as axis cell and boundary vector signals , also prWe next extended our physiological examination of superficial RSG circuits to include correlational analyses and dual injection experiments . We founWe also compared laminar expression of afferents from the four input regions and found that ADAV and DS inputs to RSG, which both at least in part target layer 3, were significantly positively correlated with one another . Similarly, CLA and ACC inputs, which both target the lower divisions of L1 as well as L5, were also significantly positively correlated . However, ADAV and DS inputs were significantly negatively correlated with both CLA and ACC inputs, confirming that these two streams target separate sublayers within RSG . DendritSupplemental examination of contralateral RSG (cRSG) inputs to RSG showed lamination and resulting cell-type-specific targeting resembling that of projections from CLA and ACC . SpecifiTo investigate the functional implications of these parallel circuits, we next examined the short-term dynamics of each input onto the RSG principal cells. LR short-term dynamics were examined in response to ADAV and DS inputs, while RS short-term dynamics were examined in response to CLA and ACC inputs . In contrast to previously documented thalamocortical matrix inputs to both superficial pyramidal cells and interneurons , 10 Hz aWe next used computational modeling to elucidate the functional role of synaptic depression in this circuit, especially with respect to the processing of head direction input . Up to 6We found that the firing rate of the postsynaptic LR cell receiving HD input via depressing synapses was strongly correlated with angular head speed, giving rise to symmetrical angular head velocity tuning . IdenticAnterior thalamic HD cells display anticipatory firing , a phenoIncreasing ATI from 0 ms (no anticipation by the HD cell) to higher values (HD cell firing prior to facing the preferred HD) improved the lag between LR firing rate and angular head speed, with larger ATIs resulting in shorter latency between the head movement and LR coding of that angular head speed . RemarkaAppendix). Moreover, the analysis showed that anticipatory firing compensates for the lag introduced by integration time, leading to a theoretical parameter regime for which postsynaptic speed coding can be essentially perfect . The observed improvement in quality of speed coding with increases in ATI corresponds to moving closer to this regime. Finally, our analysis showed that postsynaptic activity, at least at lower rotational speeds, is proportional to the square of head speed, thereby explaining the concave-up, parabolic shape of the speed-firing rate curve  neurons and regular spiking (RS) cells . Here, wThe small, excitable LR cells are unique among pyramidal cells in RSG, and there is no evidence, to our knowledge, for such cells in any other cortical regions . Indeed,The distinct electrophysiological properties of LR neurons, specifically, play an integral role in the RSG’s unique capacity to process anterior thalamic head direction inputs. We have previously reported that the low rheobase and lack of spike frequency adaptation of these neurons are their defining computational features . For thiPrevious work has reported TC-evoked responses in layer 5 RS neurons at their apical dendrites , with soThe dorsal subiculum, which serves to transmit allocentric spatial information such as axis and boundary vector signals , also prWe have shown that thalamocortical synapses onto LR cells are depressing . Our modOur results make specific predictions about the coding properties of LR cells that can be tested with in vivo recordings. First, based on their intrinsic properties , LR cellOur modeling work so far has focused on understanding the computational implications of thalamic inputs to LR cells. Future work, beyond the scope of the present study, will be needed to understand how these thalamic inputs interact precisely with dorsal subiculum inputs. Given the diversity of spatial and directional encoding seen in the dorsal subiculum , computaLR neurons do not synapse onto neighboring excitatory neurons within layers 2/3 , and no In contrast to thalamic and DS inputs to RSG, projections from both the claustrum and anterior cingulate cortex avoid laminar overlap with LR apical dendrites, instead projecting to the lower divisions of L1/2 and L5 to target RS apical and basal dendrites , and 6. Indeed, a recent study reported that selective ablation of putative retrosplenial RS cells located near the boundary and within upper layer 5 resulted in contextual fear amnesia , indicatAnother important consideration within the circuits examined here is the role of feedforward inhibition. Matrix TC inputs evoke feedforward inhibition to regulate cortical signaling through both disinhibition  and the In summary, our results highlight a superficial retrosplenial circuit enabled by the precise sublaminar organization of distinct principal neuronal subtypes and axonal afferents. LR neurons receive directional and spatial inputs from the anterior thalamus and dorsal subiculum. The synaptic dynamics of thalamic inputs to LR cells can give rise to rate coding of angular head speed in LR cells. In contrast, neighboring RS neurons in RSG respond very weakly to these directional inputs, as their apical and basal dendrites distinctly avoid laminar overlap with anterior thalamic and subicular afferents. Instead, RS neurons respond to claustrocortical and anterior cingulate inputs. Determining how these two parallel streams of information are integrated in downstream neurons within both the ipsilateral and contralateral granular and dysgranular retrosplenial cortices is the next critical step toward a mechanistic understanding of retrosplenial computations.Camk2αCre , Grp-KH288Cre (RRID:MMRRC 037585-UCD), PvalbCre , and PvalbCre x Ai14 (crossed in house). A combined total of 70 mice of both sexes between the ages of P41–P184 were used in this study.All procedures and use of animals were approved by the University of Michigan Institutional Animal Care and Use Committee. The following mouse lines were used for the CRACM experiments: Ai14 , Ai32 , C57BL6 wildtypes , GrpCre mice and AAV2-hsyn-ChR2(H134R)-eYFP or AAV2-hsyn-ChR2(H134R)-mCherry for all other lines, UNC Gene Therapy Vector Core). For dual injection surgeries, the ChR2 viral construct was injected into the functional target, and a fluorescent tag (AAV2-hsyn-mCherry or AAV2-hsyn-eYFP) was injected into the other region for comparison of various afferent axonal and terminal arborizations within the same animal.Surgical anesthesia was induced via isoflurane inhalation at 4% and then maintained on a surgical anesthetic plane at 2–3% isoflurane. Upon induction, atropine was injected subcutaneously at 0.05 mg/kg. A Physitemp  controller monitored and maintained body temperature at 37°C. Ophthalmic ointment was placed on the eyes. The incision site was prepared using 1:40 Nolvasan followed with isopropyl alcohol before being subcutaneously injected with 1% lidocaine. The skull was then leveled, and bregma was identified. Using a digital stereotaxic coordinate system, the following injection target sites were identified: right anterodorsal thalamic nucleus , right claustrum , right dorsal subiculum , right anterior cingulate cortex , and contralateral RSG . They were visualized using an Olympus BX51WI microscope, Olympus 60x water immersion lens, and Andor Neo sCMOS camera . Patch pipettes had a diameter of 2–4 µm and resistances between 2 and 5 MΩ. The potassium gluconate internal solution used in these experiments contained 130 mM K-gluconate, 2 mM NaCl, 4 mM KCl, 10 mM HEPES, 0.2 mM EGTA, 0.3 mM GTP-Tris, 14 mM phosphocreatine-Tris, and 4 mM ATP-Mg and had a pH of 7.25 and osmolarity of 290 mOsm.Current-clamp recordings were conducted using the Multiclamp 700B and Digidata 1550B (Molecular Devices). Patched neurons were adjusted for series resistances and held at resting potentials around −65 mV. Recordings were not corrected post hoc for liquid junction potential. Resting membrane potential, defined as recorded potential within 30 s of break-in, was recorded, and cells with resting potentials more depolarized than −50 mV were not included in this study. Intrinsic neuronal properties were calculated using a set of protocols detailed below and measured with either Clampfit or simple custom MATLAB routines . PharmacChannelrhodopsin-assisted circuit mapping (CRACM) experiments  were consup: defined as 100 µm from the L3/5 boundary), and deep layer 5 . For LED-based stimulation over L1a, the objective was centered approximately 30 µm from the pia in L1. For LED-based stimulation over L1/2, the objective was centered over the border between L1 and L2. For LED-based stimulation over the cell body, the objective was centered on the patched cell body at its widest diameter. For L5sup, the objective was centered 100 µm into L5, and for L5dp, the objective was centered 200 µm into L5. All LED-based stimulations were aligned in the cortical column with the patched cell body. LED intensity was kept constant at each stimulation location and was set at the minimum intensity necessary to evoke the smallest visible response when stimulating at the minimum location (see below). For most cells, this minimal response was between 0.25 and 5 mV, while strongly activated cells had larger responses with the smallest possible LED intensity. The minimum location for ADAV experiments was L1/2, while the minimum location for CLA, ACC, and DS experiments was L1a, corresponding with the lowest projection density of inputs within the superficial layers of RSG. All layer test optogenetic stimulation protocols consisted of a 1 s 10 Hz train of 1 ms LED pulses. For short-term dynamics analyses, an additional optogenetic stimulation protocol was used that consisted of a 1 s 40 Hz train of 1 ms LED pulses.To examine the effect of afferent axonal input to retrosplenial neurons, two optogenetic stimulation protocols were used. First, a targeted optogenetic stimulation approach was used to examine the distribution of afferent axons and terminal arbors across the cortical layers in which the postsynaptic retrosplenial neurons resided. The main areas of interest included layer 1a , layer 1/2 boundary , the location of the patched cell body , upper layer 5  was added to the recording solution immediately before recording. Biocytin was allowed to diffuse into the cell for no less than 20 min. Shortly before removing the patch pipette from the neuron, ten current pulses (1-3 nA at 1 Hz) were applied to aid the diffusion process . After tZ-stacks of each filled cell were acquired using the Leica SP5 confocal microscope with a dry 40x lens or the Zeiss Axio Image M2 confocal microscope with 20x lens. Reconstructions from z-stacks were performed using NeuroLucida software in user-guided mode or NeuTube software . AdditioRepresentative images of axonal projection to RSG were obtained from mice injected with anterograde AAV2 into ADAV, CLA, ACC, DS, and cRSG. Slices were prepared as for electrophysiology recordings. After use, slices with ADAV, DS, and cRSG expression from AP range −1.7 to −2.06 mm were fixed overnight in 4% PFA and then stained with the fluorescent Nissl stain, NeuroTrace 435–455, to aid laminar demarcation. ACC and CLA slices underwent an additional signal amplification protocol. Those slices were first incubated overnight in PBS containing appropriate normal blocking serums with 0.2% TritonX. On the following day, anti-GFP (for eYFP) or anti-mCherry primary antibodies  were added and slices were incubated at 4°C on a shaker for up to 24 hr. Next, slices were washed in PBS and incubated on a shaker for 3 hr at room temperature in PBS containing 2% bovine serum albumin (BSA), 0.2% TritonX and biotin-conjugated secondary antibodies  then again washed in PBS. Finally, slices were incubated on a shaker in PBS with 0.2% TritonX and streptavidin-conjugated Alexa Fluor  and NeuroTrace 435–455 (dilution 1:200) for another 3 hr at room temperature, and then washed in PBS. All slices were then mounted on slides using Fluoromount-G and allowed to dry overnight at room temperature. Z-stack images (7 µm at 0.5 µm z-steps) of the slices were obtained the following day using confocal microscope Zeiss Axio Image M2 with 20x dry objective.in), input capacitance (Cin), and membrane time constant (τm) were measured. Spike amplitude, threshold, and width were calculated from the average of all spikes in a 600 ms current step that elicited a 5 Hz spike train. Amplitude was calculated as the voltage difference from threshold to the peak of the spike, threshold from the peak of the third derivative of membrane potential , input capacitance (Cin), and membrane time constant (τm) were all measured from a protocol that delivered a series of small negative current steps resulting in ~4 mV deflection in membrane potential. Rin was calculated using Ohm’s law, mean voltage change divided by mean current. τm was measured by fitting a single exponential to the average of the initial 60 ms of the negative voltage deflection, ignoring the first 20 ms. Lastly, Cin was calculated using the formula τm = Rin x Cin. Statistical significance of the differences in intrinsic properties between the retrosplenial neuronal subtypes was calculated using the Wilcoxon rank sum test.Multiple intrinsic neuronal properties were calculated as previously reported . Brieflyotential , and widTo quantify the effect of TC and ClaC input across the layers, the amplitude of the resulting postsynaptic responses from each laminar location of LED stimulation  were measured with Clampfit and Matlab. The average of the response to the first pulse in the 10 Hz train was calculated for each cell at each LED laminar location. Significant differences between cell types and/or laminar locations were calculated using the Wilcoxon rank sum test. From the current step protocol, raster plots and PSTHs were plotted for each cell in MATLAB. The percent increase in spike rate was calculated by subtracting the number of spikes in a 100 ms window after the LED pulse from the number of spikes in a 100 ms window before the LED pulse, then dividing this value by the number of spikes in the 100 ms window before the LED pulse. Significant differences between spike count pre- and post-stimulation for both LR and RS cells and LR and RS percent increase in spike rates were calculated using the Wilcoxon rank sum test.Short-term dynamics were calculated from 10 Hz or 40 Hz LED pulses protocols . To analyze 0.1 Hz short-term dynamics, the first response to the first pulse of each sweep from the 10 Hz protocols was used. Average response amplitudes of each pulse were measured for each cell. Amplitudes were then normalized using the equation stimulus n/stimulus 1, and normalized postsynaptic ratios were plotted. Cells with first pulse responses greater than 0.5 mV but not spiking were included. Significance of resulting short term synaptic depression of LR neuron responses to 10 Hz and 40 Hz thalamic stimulation were calculated using the Wilcoxon rank sum test.All z-stack images of axonal projections and their corresponding NeuroTrace channel were first collapsed in ImageJ (using Z project option with max intensity). A section of image spanning layers 1–6 from each image was chosen to serve as a representative sample of expression pattern in that image. In each sample, boundaries of retrosplenial layers 1, 2, 3, and 5 were measured as distance from pia based on NeuroTrace expression, and the information was saved. L1 was distinguished by a low cell density, L2 by a thin, densely packed band of small pyramidal cell bodies, L3 by a lower density (compared to L2) of small pyramidal neurons, L5 as having larger cell bodies relative to L3, and L6 as having smaller cell bodies. Images of fills of the reconstructed cells underwent a similar process . After all images (both cell reconstructions and sample projections into RSG) were measured and processed, they were turned into binary black and white thresholded versions using ImageJ. For the images of reconstructed cells, cell bodies were removed from the black and white images to allow the analysis of dendrites only.https://connectivity.brain-map.org; Dendrites and axons were analyzed as a function of cortical depth . White pOur model circuit consists of an RSG LR cell receiving input from an ensemble of Throughout, let Here, We draw these parameters from Beta distributions with statistics in accordance with the ADN HD cell statistics reported in Finally, the spike train of a single HD cell with PA anticipatory time interval, or ATI: for each cell, there exists some constant duration Ams such that the cell tends to spike Ams prior to when the animal’s heading equals the preferred direction of the cell. We define this notion more precisely following a previous study  model  of shortHere, Here, depression-only and facilitation-only dynamics, respectively. On the other hand, when In the regime of https://senselab.med.yale.edu/ModelDB/showmodel.cshtml?model=3815). For all simulations, we use the parameters:We utilized an implementation of the Tsodyks-Markram model as a NEURON mechanism, freely available for download from Model DB . Notably, this parallel processing identified by the authors provides a synaptic and circuit mechanism that likely supports the role of the RSG in spatial behaviors.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Laura Colgin as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Thalamus and claustrum control parallel layer 1 circuits in retrosplenial cortex" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at All three reviewers consider that the study addresses important questions in regard to the organization of the neuronal circuits of the granular retrosplenial cortex (RSG). Specifically, the observation that afferent input from the claustrum and the anterior thalamus synapse onto different classes of RSG neurons, is novel and well supported by the present data. However, the reviewers raised a series of concerns that must be addressed before we can reach a decision on the manuscript.While we consider that the points raised by the reviewers must be addressed in full, particular attention should be placed on addressing how the RSG can perform independent spatial and fear computations. Indeed, whereas the connectivity data presented here is suggestive of functional segregation within the proposed circuits, the behavioral and computational implications of these findings are at present highly speculative. The reviewers suggest that a connection between the present findings and circuit computation could be achieved via a model or further experimentation as described in detailed below.Reviewer #1:Brennan et al. present data concerning inputs to layer 1 of retrosplenial cortex arising from the anterior thalamus and claustrum. They provide strong evidence for non-overlap of inputs to retrosplenial cortex from these two structures that segregate both in terms of the specific layer 1 sub-regions and layers III/IV sub-regions that are targeted as well as two different groups of pyramidal cell types that are identified electrophysiologically. The two types are "low-rheobase" (LR) neurons and regular-spiking (RS) neurons. The anatomical and electrophysiological claims are well-evidenced and the data is presented at a time in which there is great interest in retrosplenial cortex and its lamina-specific inputs. There is a nice complement to work concerning inputs from subiculum and hippocampus. The authors also examine to some extent the differential tuning properties of LR and RS neurons in combined modeling/electrophysiology experiments.I find the work interesting, but some of the modeling work seems a bit derivative of work done in a prior publication by the same group (Cell Reports). In addition, the manuscript suffers from an over-interpretation based on the firing properties of anterior thalamic and claustrum neurons. The attribution of claustrum inputs as "higher-order" and "fear-related" goes much too far. Finally, the work lacks an experimental consideration of how the two groups of layer III pyramidal cells are integrated within retrosplenial cortex. For this reason, the emphasis on "parallel circuits" may be misleading. It is not clear if (at the extreme), there are independent circuits in retrosplenial cortex that happen to co-localize to the same region (perhaps to gain input from a third common source) or if the segregation at the level of layer III is followed by integration . The combination of anatomy, modeling and electrophysiology has merit, but some of the findings have precedent and, most importantly, the interpretation of the data with respect to the retrosplenial cortex as a whole needs improvement.1. While it is mostly reasonable to speak of anterior thalamic inputs as reflecting head direction inputs (with high input rates), the claustrum inputs cannot be described this way. Unfortunately, there is very little data concerning the response properties of claustrum inputs . In addition, a brain region with neurons responding in fear/avoidance based learning scenarios (or for which lesions impair such learning) is all too common. Neurons throughout the brain respond in some way in such conditions as they often do to reward. The authors should refrain from tagging the claustrum inputs in particular with psychology jargon. I realize that this leaves the meaning of the claustrum input as a bit of a mystery, but perhaps other more neuroanatomy or neurophysiology based arguments for the special properties of a claustrum input can be advanced. The authors might also find it much easier to advance arguments of function by putting more focus into the anterior cingulate inputs (cingulate responses are much better defined at this point).2. Much attention is paid to the layer 1 inputs, but the two input streams diverge equally with respect to layer 3-5. More discussion of the importance of this is desirable and might begin with why each input stream has two very different concentrations of lamina-specific input. What neurons and dendrites are targeted by the layer 3-5 inputs and how does this relate to the layer 1 inputs. In general, the manuscript does not go far enough to incorporate details of circuitry between neurons of retrosplenial cortex. The absence of this leaves the manuscript lacking some originality in terms of data presented and conceptual issues.3. The data presented in figure 4 is compelling with respect to latency to response (5ms is a long time in the brain), but I encourage the authors to spend more time working through the description in results. As written, I think many readers will need to re-read multiple times to understand what is going on (and probably won't re-read). Please make this section more accessible to the broader set of neuroscientists without special expertise in this kind of work.4. Can the authors provide greater context by speaking to the issue of axonal targets of retrosplenial LR and RS neurons?Reviewer #2:In this manuscript, Brennan et al. expand upon their previous observation of two different pyramidal neuron subtypes in RSG. They had recently shown that RS and LR neurons in superficial RSG exhibit distinct intrinsic excitability profiles and axo-dendritic arborization. Here, they provide evidence that this is paralleled by specific connectivity of long-range inputs and corresponding synaptic dynamics in the two cell types. The authors frame their work through the question of how RSG subserves both fear and spatial coding, concluding that these streams of information are processed by separate and largely independent circuits in RSG built around RS and LR neurons.eLife. How differential connectivity interacts with synaptic and/or intrinsic properties of distinct cell types to shape the computations that contribute to behavioral processes is a pressing problem in the field. And it is highly challenging to address. The specific observation at the core of the manuscript, that thalamic inputs preferentially target LR neurons while claustrum inputs preferentially target RS neurons, is quite interesting. However, the manuscript itself is preliminary and poorly organized, with a number of loose ends and an unclear impact. Substantial revision and expansion are necessary to do justice to the importance of the initial differential connectivity result.This is an exciting topic that is potentially a good fit for 1. The authors should finish the incomplete experiments shown in the supplement, which look potentially highly complementary to the data presented in the main figures. Specifically, the authors should complete the subiculum and ACC input anatomy, connectivity, and synaptic dynamics to LR and RS neurons. It is currently a serious missed opportunity. The resultant data will increase the impact of the paper, and it will be particularly useful for model building.2. The manuscript should be reorganized to present the comprehensive anatomy, connectivity, and synaptic dynamics of AT, CLA, ACC, and SUB in a principled way. Right now, the figures are all over the place, making comparisons challenging. For example, why is there no parallel figure to Figure 3 for CLA inputs? And why are some of the experiments repeated in L5 RS neurons? Is there a parallel system of RS and LR neurons in L5? Have the authors just not found the LR analogues in L5? I am not sure if the L5 data even adds anything – right now it seems like more of a distraction. Maybe it's best to stick to just L2/3.3. The authors assert  that they have identified and modeled how RSG can perform independent spatial and fear computations. They did not test this at all and should not push this agenda without specific, concrete experiments and data that support this direction. What the authors have shown is differential connectivity and synaptic dynamics for two inputs to two cell types. That is basically it. The biophysical model in Figure 4 really has nothing to do with spatial or HD coding. And Figure 7 is not a model at all, but a rudimentary cartoon schematic. In order for this manuscript to have broad impact in the field, the authors should perform a serious modeling effort that integrates what is known about the firing properties of the different neurons that output to RSG with the connectivity and synaptic dynamics they have discovered at RS and LR neurons to test how this system works in vivo. Receptive field properties of RSG neurons have previously been reported by multiple groups : the authors should develop a model that captures some of these in vivo properties and makes non-trivial, testable, and quantitative predictions about, for example, how parallel LR/RS circuitry is beneficial for some aspect of encoding.Reviewer #3:The authors distinguish between two previously characterized types of principal pyramidal neurons in Layer 3 of the granular retrosplenial cortex (RSG): low-rheobase (LR) and regular-spiking (RS) neurons, and identify them by their unique intrinsic properties using patch clamp.Viral injections into the anterodorsal and anteroventral thalamic nuclei (AD/AV) resulted in ChR2-eYFP being expressed in thalamocortical (TC) axons and terminals in layer 1a  and in layer 3 (L3) of superficial RSG. Optically stimulating the thalamic axons and terminals in RSC Layer 3 showedLR neurons to be strongly driven, but RS very little. Morophological studies show that LR and RS neurons' dendrites cluster with their own type.Interestingly the TC inputs to LR but not RS neurons show synaptic depression. The authors argue that this allows low latency responses, using some detailed modelling of the LR neuron.In a second experiment, ChR2 injected into claustrum (CLA) and an EYFP tag into AD/AV showed that TC and CLA axons and terminals have distinct and non-overlapping lamination within RSG, such that superficial TC arbors exist most strongly in layers 1a and 3 , while CLA arbors exist in layers 1b/c, 2, and 5. And RS, but not LR, apical dendrites anatomically overlap with CLA arbors in layers 1c and 2. Using the same targeted optogenetic stimulation approach RS neurons have significantly larger responses to CLA input compared to LR neurons (and similarly for inputs from anterior cingulate).The finding of apparently parallel circuits within the RSG with two different types of input area will be important for understanding the function of the RSG, which is currently receiving much attention.I have a few questions, mainly for clarification:1. They argue that facilitating synapses from subiculum to LR neurons would be better for rate coding – as better for overcoming hyperpolarisation from previous spiking. This is easily understood.It would be good to give an intuitive explanation of why depressing synapses would lead to lower latency responses to head direction inputs from AT – it is one thing to show that a complicated simulation shows the effect and another to explain why it does so.2. The anatomical demonstrations that TC inputs avoid RS neuron dendrites is somewhat confused by the evidence of such synaptic connections (showing weak facilitation) – maybe this could be explained (there are some but they are weak).3. Figure 7 implies that TC inputs are to Layer 1a and subicular inputs are to Layer 3, but I don't think this is intended? All three reviewers consider that the study addresses important questions in regard to the organization of the neuronal circuits of the granular retrosplenial cortex (RSG). Specifically, the observation that afferent input from the claustrum and the anterior thalamus synapse onto different classes of RSG neurons, is novel and well supported by the present data. However, the reviewers raised a series of concerns that must be addressed before we can reach a decision on the manuscript.While we consider that the points raised by the reviewers must be addressed in full, particular attention should be placed on addressing how the RSG can perform independent spatial and fear computations. Indeed, whereas the connectivity data presented here is suggestive of functional segregation within the proposed circuits, the behavioral and computational implications of these findings are at present highly speculative. The reviewers suggest that a connection between the present findings and circuit computation could be achieved via a model or further experimentation as described in detailed below.The manuscript has been revised in accordance with the constructive reviewer feedback. In particular, we have extended our circuit mapping experiments to reveal the cell-type specificity of anterior cingulate and dorsal subiculum inputs to superficial retrosplenial cortex . Comprehensive new computational and analytical modeling has also been added. We now show that the observed short-term depression of thalamocortical synapses  can allow low rheobase (LR) neurons in the retrosplenial cortex to encode both rotational head speed and direction, even when the thalamic inputs do not explicitly encode speed . We also predict the existence of a clear, explainable tradeoff in encoding of head speed vs direction across individual LR neurons . In addition to identifying the LR cell as the primary initial recipient of spatially-relevant inputs from the anterior thalamus and dorsal subiculum, we have also added more nuanced discussion of the possible functions of cingulate/claustral inputs to regular spiking retrosplenial cells as well as the possible convergence of spatial and non-spatial inputs downstream within the retrosplenial cortex. Detailed point by point responses follow.Reviewer #1:Brennan et al. present data concerning inputs to layer 1 of retrosplenial cortex arising from the anterior thalamus and claustrum. They provide strong evidence for non-overlap of inputs to retrosplenial cortex from these two structures that segregate both in terms of the specific layer 1 sub-regions and layers III/IV sub-regions that are targeted as well as two different groups of pyramidal cell types that are identified electrophysiologically. The two types are "low-rheobase" (LR) neurons and regular-spiking (RS) neurons. The anatomical and electrophysiological claims are well-evidenced and the data is presented at a time in which there is great interest in retrosplenial cortex and its lamina-specific inputs. There is a nice complement to work concerning inputs from subiculum and hippocampus. The authors also examine to some extent the differential tuning properties of LR and RS neurons in combined modeling/electrophysiology experiments.I find the work interesting, but some of the modeling work seems a bit derivative of work done in a prior publication by the same group (Cell Reports). In addition, the manuscript suffers from an over-interpretation based on the firing properties of anterior thalamic and claustrum neurons. The attribution of claustrum inputs as "higher-order" and "fear-related" goes much too far. Finally, the work lacks an experimental consideration of how the two groups of layer III pyramidal cells are integrated within retrosplenial cortex. For this reason, the emphasis on "parallel circuits" may be misleading. It is not clear if (at the extreme), there are independent circuits in retrosplenial cortex that happen to co-localize to the same region (perhaps to gain input from a third common source) or if the segregation at the level of layer III is followed by integration . The combination of anatomy, modeling and electrophysiology has merit, but some of the findings have precedent and, most importantly, the interpretation of the data with respect to the retrosplenial cortex as a whole needs improvement.1. While it is mostly reasonable to speak of anterior thalamic inputs as reflecting head direction inputs (with high input rates), the claustrum inputs cannot be described this way. Unfortunately, there is very little data concerning the response properties of claustrum inputs . In addition, a brain region with neurons responding in fear/avoidance based learning scenarios (or for which lesions impair such learning) is all too common. Neurons throughout the brain respond in some way in such conditions as they often do to reward. The authors should refrain from tagging the claustrum inputs in particular with psychology jargon. I realize that this leaves the meaning of the claustrum input as a bit of a mystery, but perhaps other more neuroanatomy or neurophysiology based arguments for the special properties of a claustrum input can be advanced. The authors might also find it much easier to advance arguments of function by putting more focus into the anterior cingulate inputs (cingulate responses are much better defined at this point).We now present a more nuanced view of the implications of the observed cingulate/claustral targeting of RS but not LR cells. Please see the Discussion sections titled “Downstream targets of LR cells and potential role of RS neurons in retrosplenial spatial computations” and “Potential role of claustral and anterior cingulate inputs” for details of the possible functional implications of the claustral and cingulate inputs to the RSG.2. Much attention is paid to the layer 1 inputs, but the two input streams diverge equally with respect to layer 3-5. More discussion of the importance of this is desirable and might begin with why each input stream has two very different concentrations of lamina-specific input. What neurons and dendrites are targeted by the layer 3-5 inputs and how does this relate to the layer 1 inputs. In general, the manuscript does not go far enough to incorporate details of circuitry between neurons of retrosplenial cortex. The absence of this leaves the manuscript lacking some originality in terms of data presented and conceptual issues.Our experiments now consider the deeper inputs using extended CRACM experiments with stimulation sites that range from the midline (layer 1a) through 200 μm into layer 5, encompassing the basal dendrites of the superficial principal neurons that are the focus of this study. We also present more experimental details and additional discussion of within-RSG connectivity. While we have previously reported that LR and RS cells in superficial layers of RSG are unconnected , we have now also extended our pair matrix to examine connectivity between layer 3 LR and layer 5 RS cells. Of the 18 connections tested , 0 showed connectivity between layer 3 LR cells and layer 5 RS cells. Furthermore, building on the axonal targets we had presented in the first submission, we have examined cross-hemispheric RSG-RSG connectivity using CRACM . These experiments show that inputs from contralateral RSG strongly drive RS cells while almost completely avoiding LR neurons. We discuss the potential implications of this connectivity in the Discussion section titled “Downstream targets of LR cells and potential role of RS neurons in retrosplenial spatial computations.”3. The data presented in figure 4 is compelling with respect to latency to response (5ms is a long time in the brain), but I encourage the authors to spend more time working through the description in results. As written, I think many readers will need to re-read multiple times to understand what is going on (and probably won't re-read). Please make this section more accessible to the broader set of neuroscientists without special expertise in this kind of work.Following reviewer 2’s suggestion, the entire modeling section has been substantially expanded to focus on head directional coding in LR cells. As such, these new sections have been written with careful focus on clarity, accessibility, and explicit functional implications. Please see response to reviewer 2 major point 3 for full details.4. Can the authors provide greater context by speaking to the issue of axonal targets of retrosplenial LR and RS neurons?Please see response to point 2 above and the new Discussion section titled “Downstream targets of LR cells and potential role of RS neurons in retrosplenial spatial computations.”Reviewer #2:In this manuscript, Brennan et al. expand upon their previous observation of two different pyramidal neuron subtypes in RSG. They had recently shown that RS and LR neurons in superficial RSG exhibit distinct intrinsic excitability profiles and axo-dendritic arborization. Here, they provide evidence that this is paralleled by specific connectivity of long-range inputs and corresponding synaptic dynamics in the two cell types. The authors frame their work through the question of how RSG subserves both fear and spatial coding, concluding that these streams of information are processed by separate and largely independent circuits in RSG built around RS and LR neurons.This is an exciting topic that is potentially a good fit for eLife. How differential connectivity interacts with synaptic and/or intrinsic properties of distinct cell types to shape the computations that contribute to behavioral processes is a pressing problem in the field. And it is highly challenging to address. The specific observation at the core of the manuscript, that thalamic inputs preferentially target LR neurons while claustrum inputs preferentially target RS neurons, is quite interesting. However, the manuscript itself is preliminary and poorly organized, with a number of loose ends and an unclear impact. Substantial revision and expansion are necessary to do justice to the importance of the initial differential connectivity result.1. The authors should finish the incomplete experiments shown in the supplement, which look potentially highly complementary to the data presented in the main figures. Specifically, the authors should complete the subiculum and ACC input anatomy, connectivity, and synaptic dynamics to LR and RS neurons. It is currently a serious missed opportunity. The resultant data will increase the impact of the paper, and it will be particularly useful for model building.As suggested, we have completed a battery of additional experiments to include full analyses and figures for anterior cingulate and dorsal subiculum inputs to granular retrosplenial cortex. Please see the new Figure 4 (anterior cingulate), Figure 5 , Figure 6 , and Figure 7 (synaptic dynamics).2. The manuscript should be reorganized to present the comprehensive anatomy, connectivity, and synaptic dynamics of AT, CLA, ACC, and SUB in a principled way. Right now, the figures are all over the place, making comparisons challenging. For example, why is there no parallel figure to Figure 3 for CLA inputs? And why are some of the experiments repeated in L5 RS neurons? Is there a parallel system of RS and LR neurons in L5? Have the authors just not found the LR analogues in L5? I am not sure if the L5 data even adds anything – right now it seems like more of a distraction. Maybe it's best to stick to just L2/3.Each of the 4 input regions  examined are now presented using homogenous figure formats . We have also included an updated summary figure that directly compares incoming axon-axon, axon-local dendrite, and dendrite-dendrite lamination as Figure 6.As suggested, we have moved the previous layer 5 results to the supplements , in keeping with the reviewer’s suggestion to retain the central focus of the manuscript on the inputs and modeling of superficial RSG neurons.3. The authors assert  that they have identified and modeled how RSG can perform independent spatial and fear computations. They did not test this at all and should not push this agenda without specific, concrete experiments and data that support this direction. What the authors have shown is differential connectivity and synaptic dynamics for two inputs to two cell types. That is basically it. The biophysical model in Figure 4 really has nothing to do with spatial or HD coding. And Figure 7 is not a model at all, but a rudimentary cartoon schematic. In order for this manuscript to have broad impact in the field, the authors should perform a serious modeling effort that integrates what is known about the firing properties of the different neurons that output to RSG with the connectivity and synaptic dynamics they have discovered at RS and LR neurons to test how this system works in vivo. Receptive field properties of RSG neurons have previously been reported by multiple groups : the authors should develop a model that captures some of these in vivo properties and makes non-trivial, testable, and quantitative predictions about, for example, how parallel LR/RS circuitry is beneficial for some aspect of encoding.We have carried out extensive computational and analytical modeling of thalamic inputs to LR neurons to generate explicitly testable functional predictions about the directional coding capabilities of LR neurons based on the circuitry and dynamics reported in this manuscript. Please see Figures 8-10, and the corresponding new Results sections “Short-term depression of anterior thalamic inputs enables encoding of angular head speed by LR cells”, “Anticipatory firing of thalamic head direction cells improves postsynaptic speed tuning by LR neurons”, and “Non-uniform HD inputs can allow LR cells to encode both head direction and speed, with a tradeoff.” Implications and explicit predictions are then presented in the Discussion section titled “Thalamic inputs to LR cells show short-term depression: implications for angular head velocity and head direction coding in the RSG.”Reviewer #3:The authors distinguish between two previously characterized types of principal pyramidal neurons in Layer 3 of the granular retrosplenial cortex (RSG): low-rheobase (LR) and regular-spiking (RS) neurons, and identify them by their unique intrinsic properties using patch clamp.Viral injections into the anterodorsal and anteroventral thalamic nuclei (AD/AV) resulted in ChR2-eYFP being expressed in thalamocortical (TC) axons and terminals in layer 1a  and in layer 3 (L3) of superficial RSG. Optically stimulating the thalamic axons and terminals in RSC Layer 3 showedLR neurons to be strongly driven, but RS very little. Morophological studies show that LR and RS neurons' dendrites cluster with their own type.Interestingly the TC inputs to LR but not RS neurons show synaptic depression. The authors argue that this allows low latency responses, using some detailed modelling of the LR neuron.In a second experiment, ChR2 injected into claustrum (CLA) and an EYFP tag into AD/AV showed that TC and CLA axons and terminals have distinct and non-overlapping lamination within RSG, such that superficial TC arbors exist most strongly in layers 1a and 3 , while CLA arbors exist in layers 1b/c, 2, and 5. And RS, but not LR, apical dendrites anatomically overlap with CLA arbors in layers 1c and 2. Using the same targeted optogenetic stimulation approach RS neurons have significantly larger responses to CLA input compared to LR neurons (and similarly for inputs from anterior cingulate).The finding of apparently parallel circuits within the RSG with two different types of input area will be important for understanding the function of the RSG, which is currently receiving much attention.I have a few questions, mainly for clarification:1. They argue that facilitating synapses from subiculum to LR neurons would be better for rate coding – as better for overcoming hyperpolarisation from previous spiking. This is easily understood.It would be good to give an intuitive explanation of why depressing synapses would lead to lower latency responses to head direction inputs from AT – it is one thing to show that a complicated simulation shows the effect and another to explain why it does so.The modelling work has been substantially expanded with new insights and predictions about LR function. As such, these new sections have been written with careful focus on clarity, accessibility, and explicit functional implications. Please see response to reviewer 2 major point 3 for full details.2. The anatomical demonstrations that TC inputs avoid RS neuron dendrites is somewhat confused by the evidence of such synaptic connections (showing weak facilitation) – maybe this could be explained (there are some but they are weak).We agree with the reviewer that it is best to focus on the short-term dynamics of the synapses that our findings show are prominent and robust . This is especially true with the expanded focus that now includes cingulate and dorsal subiculum and thus increases the number of synapses being examined. Please see the new, hopefully clearer, version of the short-term dynamics figure .3. Figure 7 implies that TC inputs are to Layer 1a and subicular inputs are to Layer 3, but I don't think this is intended?The new version of this figure  has been updated to comprehensively represent laminar inputs into RSG through both the modification of the summary panel and inclusion of incoming axon-axon, axon-local dendrite, and dendrite-dendrite laminar correlations. The data-generated projection density plot shown in panel B (left) now clearly presents the lamination of inputs from all 4 main input regions examined in this manuscript, and this is simplified in the summary circuit cartoon in panel D.


To investigate the association of the myelomeningocele (MMC) volume with prenatal and postnatal motor function (MF) in cases who underwent a prenatal repair. Retrospective cohort study (11/2011 to 03/2019) of 63 patients who underwent a prenatal MMC repair . At referral, measurements of the volume of MMC was performed based on ultrasound scans. A large MMC was defined as greater than the optimal volume threshold  for the prediction of intact MF at referral (2.7 cc). Prenatal or postnatal intact motor function (S1) was defined as the observation of plantar flexion of the ankle based on ultrasound scan or postnatal examination. 23/63 participants presented a large MMC. Large MMC lesions was associated with an increased risk of having clubfeet by 9.5 times , and reduces the chances of having an intact MF at referral by 0.19 times . At birth, a large MMC reduces the chance of having an intact MF by 0.09 times , and increases the risk of having clubfeet by 3.7 times . A lower proportion of intact MF and a higher proportion of clubfeet pre- or postnatally were observed in cases with a large MMC sac who underwent a prenatal repair.Trial registration: Clinicaltrials.gov NCT02230072 and NCT03794011 registered on September 3rd, 2014 and January 4th, 2019. Myelomeningocele occurs when the neural placode extends through the spinal defect with accompanying herniation of the meninges. Exposure of the neural elements to amniotic fluid causes progressive injury, which leads to changes in the cytoarchitecture of the spinal cord from irritation of the primary lesion7. Because of this, motor and sensory deficits accompany the diagnosis of MMC8. The Management of Myelomeningocele Study (MOMS), a multicenter randomized clinical trial, reported improved motor function and a decrease in the need for ventriculo-peritoneal shunting during the first 12 months of life in those patients who underwent prenatal MMC repair as compared to the postnatal repair patients9.In the United States, myelomeningocele (MMC), a type of open neural tube defect (NTD), is seen in 1 in 3000 live-births10. They hypothesized that additional neurologic injury may result from cerebrospinal fluid hydrostatic forces within the MMC sac with secondary stretching of the neural placode and spinal nerves, which would represent another acquired mechanism of injury in cases of open spinal dysraphism.Oliver et al. demonstrated that MMCs prenatally or postnatally repaired were associated with a higher rate of clubfeet and lower extremity impairment when compared to myeloschisis lesions, another type of open NTD where the spinal cord remains within the spinal canalIn large MMCs, the excessive posterior herniation of the neural placode outside of the level of the wide open osseous spinal canal results in more significant stretching and thinning of the neural placode as well as the spinal nerves that are located along the anterior surface of the neural placode. We hypothesized that the overstretching of the neural placode in cases with large MMC volumes will result in higher motor function impairment.The objectives of our study were: (1) to compare brain findings between large and non-large MMC sac volumes at the time of referral and 6 weeks after the prenatal surgery; (2) to compare prenatal motor function from mid-gestation to delivery between large and non-large MMC sac volume; and (3) to determine the relationship between the volume of the lesions and postnatal neurological short-term outcomes.Sixty-three MMC cases who underwent prenatal NTD repair were included in this study (37 fetoscopic and 26 open-hysterotomy). The median volume of the sac at the time of referral was 1.6 (0.05–44.5) cc. Twenty-three cases presented with a “large” sac and 40 with a “non-large” sac.At the time of referral, the anatomical level of the lesion and ventricle size were similar between large and non-large MMC lesions Table . Six weeComparisons between large and non-large MMC lesions in the fetoscopic or the open-repair groups are presented in Supplementary Table At referral, there was no correlation between the volume of the lesion and ventricular size , or anatomical level of the lesion .At six post-operative weeks, there was no correlation between the volume of the lesion and hindbrain herniation (HBH) grade  or ventricular size .Fetuses with large MMC sac volumes showed a higher rate of clubfeet and a lower proportion of intact motor function at the time of referral Table . At the During the last US scan before delivery, the proportion of cases with an intact motor function was higher in cases of non-large lesions compared to large lesions Table .In the fetoscopic subgroup, there was no difference between large and non-large MMC lesions for the proportion of clubfeet or intact motor function at the time of referral, 6 weeks after the surgery or during the last US before delivery  after adjusting for the presence of an intact motor function at initial evaluation, anatomical level of the lesion, type of surgery, gestational age at delivery, and learning curve.Dehiscence or leakage of CSF, need for postnatal repair of the defect at birth, need for hydrocephalus treatment in the first year of life, and independent ambulation at 30 months of age was similar between large and non-large MMC Table .In the fetoscopic subgroup, there were significantly fewer cases with an intact motor function at birth in the large MMC group compared to the non-large MMC group, and a trend for a higher proportion of cases with clubfeet in the large MMC group  and are less likely to have an intact motor function  at birth (0.09-fold) after a prenatal repair compared to non-large MMC lesions.10. Their findings are consistent with previous reports of improved postnatal lower extremity function in prenatally repaired cases of myeloschisis compared to MMC13. A recent study exploring prenatal neurologic findings in cases of open spinal dysraphism found no association between MMC sac volume and prenatal clubfeet14. However, comparison with our cohort may be difficult because they did not report the anatomical level of the lesion, which is a well-known predictive factor for clubfeet12.Oliver et al. reported their experience with 182 NTD cases prenatally repaired (142 MMC and 40 MS), using the same methodology we used in this study to assess the volume of the lesion with similar volumes (4.7 cc in cases of MMC with clubfeet or 3 cc in cases of MMC without clubfeet)14. We only included cases that underwent prenatal repair who had HBH, so we did not compare cases with or without HBH.An inverse relationship between sac volume and degree of HBH severity was reported by Nagaraj et al., and it was hypothesized that MMC sac volume may have a protective effect on the degree of HBH by maintaining some hydrostatic pressure within the spinal canal16. Our study confirms previous reports that concluded that larger MMC sizes are associated with lower extremity impairment10, an observation that could be described as a “third-hit” hypothesis.Experimental and clinical studies suggest that the neurological deficits associated with NTD occur in a step-wise fashion6. The leak of cerebro spinal fluid (CSF) through the defect exerts a siphoning effect on the cerebellum and medulla. As the gestation proceeds, HBH impairs the path and dynamics of CSF flow resulting in hydrocephalus, which exacerbates the herniation through the foramen magnum, worsening the problem.First, NTD results in a closure defect in the posterior portion of the vertebral spine in the third week of gestation18.Second, the segmental neurological damage at the NTD site can be secondary to both chemical toxicity and recurrent physical trauma of the neural placode in the intra-uterine environment, as reported in animal studiesThird, the lower extremity impairment that we described in cases of large MMC, may be due to increased traction of the neural placode and nerve roots due to mechanical expansion of the larger sacs. It is possible that the additive effects of all these mechanisms may lead to a more permanent damage of the placode nerve root even after prenatal repair or large MMC.Our results indicate that the fetal spinal cord and nerves are sensitive to stretch injury which could impact fetal surgical planning for in-utero treatment. When deciding technique and closure strategies for repair, additional care should be shown to minimize traction to the MMC lesion and nerves. Based on our results, measuring the volume of the lesion prior to surgery would be worth considering to assist in intraoperative decision making.This study included fetuses from two different operative approaches undergoing prenatal repair from a single center. Standard protocols for evaluation and management of care were used for all cases. We acknowledge that the retrospective design is a major limitation of our study. Due to the outcomes seen in NTD cases being multifactorial in nature, it is possible the assumed effect secondary to nerve root overstretching in large lesions could be due to accumulating effects of the volume of the lesion or intrauterine insults that are unknown.In our cohort, fetuses who present with a large MMC (> 2.7 cc in our cohort) at referral for the evaluation of a prenatal MMC repair are less likely to have an intact motor function at birth and more likely to present clubfeet at birth. These results suggest that spinal cord and nerves are sensitive to stretch injury, a “third-hit” hypothesis.9.This is a retrospective study of 103 fetuses who underwent a prenatal MMC repair (fetoscopic or open-hysterotomy technique) performed at a single institution between November 2011 and March 2019. All patients who underwent prenatal NTD repair during this period were included in this study. Myeloschisis cases (n = 34), cases where surgery was aborted due to fetal intolerance (n = 3), the lesion was not confirmed to be a myelomeningocele (MMC) by direct visualization (n = 2), or when the mother had a contraindication for an MRI study (n = 1) were excluded from the analysis. All fetuses who underwent prenatal repair and satisfied all MOMS trial inclusion criteria and none of the exclusion criteria were includedAt the time of referral, all patients underwent fetal MRI on a 1.5-T Philips Ingenia superconducting magnet . Details of imaging protocol are presented in 9.At the time of referral, detailed transabdominal fetal US examinations  were performed in all patients referred to our Fetal Center in order to evaluate patient eligibility for prenatal NTD repair. The anatomical level of the lesion was defined as the upper bony spinal defect on US scans. A high level of the lesion was defined as an upper bony spinal defect at or above the second vertebral body (L2)10 ROC curve analysis and Youden index were performed to determine the best cut-off to predict an intact motor function at referral based on the volume of the lesion. A 2.7 cc cut-off was found to have the best sensitivity (0.63) and best specificity (0.75) to predict an intact motor function at referral . Therefo9. From 2014, fetoscopic repair was offered under our fetoscopic repair protocol provided by the U.S. Food and Drug Administration (FDA) IDE #G140201, the Baylor College of Medicine Institutional Review Board (IRB) protocols H-34680 (7/1/2014) and H-43359 (11/15/2018), our Fetal Therapy Board, and a Data Safety Monitoring Board. These protocols are registered on ClinicalTrials.gov (NCT02230072 and NCT03794011)19. The fetoscopic technique was performed as described elsewhere20. A single layer closure was performed in the first 23 cases of our MMC cohort. In all subsequent cases, a three layer closure was performed using a collagen patch on top of the dura , with muscle flaps and skin closure21. During both fetoscopic and open-hysterotomy surgeries, if a complete skin closure was not expected to be possible due to excessive tension on the skin edges, relaxing incisions were performed. These are bilateral vertically oriented incisions performed 2–3 cm from the edge of the defect to facilitate a tension-free midline closure.The surgical approach for NTD repair was determined based on maternal choice. During the pre-surgical evaluation, patients were not informed specifically about the volume of the lesion; however, the anatomical level of the lesion was discussed with them. Patients who underwent a prenatal NTD repair were managed per our standard protocol of steroid administration, prophylactic tocolysis , and prophylactic antibiotics . Open repair was considered the standard of care for prenatal NTD repair and was performed through an open-hysterotomy approach using the same methodology as reported by the MOMS trial20.Data was retrospectively obtained under an IRB (Baylor College of Medicine) approved protocol H-38479 (2/11/2016). All patients signed an informed consent prior to inclusion in the study. All methods were performed in accordance with the relevant guidelines and regulations. The first 12 cases in both the fetoscopic and the open-hysterotomy techniques, including prenatal repair of myeloschisis, were considered to be part of our learning curve22  walking independently (with or without orthotics), (2) walking with an assistive device , or (3) non-ambulatory.Data analysis was performed using the Statistical Package for Social Sciences (SPSS), version 24.0 . Quantitative variables were expressed as mean ± standard deviation if they had a normal distribution (by Kolmorov-Smirnov test) and compared with Student’s t-test. Quantitatives variables with non-normal distribution were expressed as median [range] and compared with Mann–Whitney U test. Pearson’s Chi square or Fisher’s exact tests were used to compare qualitative data. Correlations between the volume of the lesion and other quantitative variables such as the anatomical level of the lesion , ventricular size, and the HBH grading classification, were performed using Spearman correlation analyses. Linear and logistic regression analyses were performed, controlling for potential confounding variables  when indicated. Predictive values of large NTD for neurosurgical, post-operative and neonatal outcomes were expressed as odds ratios (OR) with a 95% confidence interval (95% CI).Supplementary Information.


Photosynthetic active radiation (PAR) favors vegetable growth with a specific blue and red light ratio. Thus, increasing the amount of red light improves chlorophyll absorption and photosynthetic efficiency. In this article, we present a hybrid system that combines luminescent materials and photonic crystals for better management of the light reaching the greenhouse. The luminescent dyes considered herein are combined ensuring a Förster resonance energy transfer (FRET) nonradiative mechanism to enhance the absorption range. The designed photonic crystal maximizes reflections in the Near-Infrared (NIR) range, and therefore, thermal losses are minimized. Thus, by converting harmful or ineffective radiation for plant growth to the PAR region, we aim to demonstrate growth-condition enhancement for the different vegetables that have been used as a model.The world population is growing by 1 billion people every 10 years. There will come a time when there will be more people to feed but less land to grow food. Greenhouses can be the solution to this problem because they provide the highest production yield per m The manufacturing of any product requires energy to transform raw materials into useful goods. The use of renewable energy sources and sustainable conversion processes, aligned with environmental protection, are key parameters for the development of society. Industrial companies have to integrate energy-efficient performance in production management . The humIn the last decades, the idea of using closed greenhouses was assumed in order to save water and energy. Thus, greenhouse agriculture has been considered as an intensive production structure creating aligned boundary conditions of climate control and increasing efficiency of resources . HoweverThe cost balance of a greenhouse depends on many parameters, but the most important ones are primary energy cost and total production yield . To achiThe temperature profile is the most studied parameter in the literature. A greenhouse gets very hot during the day and remains warm enough at night compared with the environmental conditions. To keep temperature distribution constant at the optimum range is essential for optimal crop growth ,6,7,8,9.The second parameter, external irradiation, is critical because it influences the photosynthesis crop process, and also the temperature distribution inside the greenhouse. Photon energy of the irradiation spectrum is absorbed by chlorophyll molecules in chloroplasts, where the solar energy is converted into chemical energy. Hemming et al.  reportedIncoming irradiation out of this spectral range only generates undesired effects that reduce the greenhouse performance: near ultraviolet (NUV) radiation affects film degradation, plant damage, and pollinator behavior, and near-infrared (NIR) radiation is a direct source of heat. Much of NIR radiation penetrates into greenhouses and causes heat loads .There are some previous research studies analyzing the effect of using photoselective plastic films to modify the light spectrum that enters the greenhouse, so the photosynthesis of the plants can be affected. Fletcher et al.  demonstrIn sum, two phenomena have to be considered to improve greenhouse performance: (1) avoid transmission of NIR inside of the greenhouse in order to minimize overheating during the day and cooling during the night; and (2) convert green light into red light to enhance the chemical energy transformed by chlorophylls. Both objectives can be achieved with the combination of photonic crystals and luminescent materials, as thin-film coatings or dye-doped polymers on the greenhouse structure .Photonic crystals (PCs) are periodic dielectric structures that are designed to form the energy band structure for photons, and either allow or forbid the propagation of electromagnetic waves of certain frequency ranges, making them ideal for light-harvesting applications . PhotoniThus, the appropriate combination of the PC concept for greenhouse energy-management purposes might be suitable to remove heat radiation (infrared radiation) from the walls, and then to minimize internal temperatures during the day. Moreover, this wavelength does not affect the crop-development mechanism: 1000 nm is the critical wavelength; up to this value radiation is transmitted, and it is reflected onward.Additionally, photoluminescent materials absorb light at a certain wavelength, and the light is re-emitted at another wavelength with lower energy. This means that they are able to tune the incoming solar spectrum; for example, by transforming the green spectral range to the red part of the spectrum. There is an available wide portfolio of organic luminescent molecules, so downshifting the described process is easy to adjust to different applications, such as sensors , energy So, luminescent materials can be directly applied on greenhouses to improve the useful energy requested for crop development (PAR), or even Luminescent Solar Concentratos (LSC) can be integrated into agricultural structures to benefit the photosynthesis processes at the same time that electricity is produced, either for self-energy consumption or for selling energy to the electrical market. The scope is to seize the opportunity that PAR is not influenced by green solar spectrum, so thanks to this family of materials, this energy can be easily transformed into the red part of the spectrum for a better crop exploitation.During the 1990s, Novoplansky et al.  analyzedFragaria × ananassa “Elsanta”. In order to optimize light quality and quantity growth, several photoselective greenhouse-covering materials were developed, with most of them based on red dyes (with photoluminescence between 610 and 690 nm) in different concentrations.Gonzalez et al.  investigCorrado et al.  investig(1−x)ZnxSe, but they concluded that their film had a low quantum yield, so another chemical compound approach was needed.Simakin et al.  and WangThe majority of these experiments considered the commercial luminescent material from BASF, called Lumogen 305 Red. However, the main disadvantages observed in this type of organic dye were: (1) narrow absorption spectrum; (2) high reabsorption due to the overlapping of the absorption and emission peaks (Stokes losses); and (3) limited tuneability to provide this service for a wider range of PAR specifications.An interesting alternative strategy is to consider a mix of dyes embedded in the host material, ensuring they transfer the energy between them in a nonradiative mechanism. This is called Förster resonance energy transfer (FRET) ,30. It hThe aim of this research study is to provide a new novel design based on the combination of photonic crystals and multiple luminescent dyes transferring energy between them with a FRET nonradiative mechanism. Thanks to this original approach, the flexible film will ensure: (1) reflection of the light in the infrared spectrum, responsible for increasing the internal temperature of the greenhouse; (2) wide absorption spectra range for those wavelengths that are not critical for photosynthesis; (3) light photoemitted in the proper energy compared to PAR; (4) minimization of Stokes losses; and (5) a high degree of tuneability of the system, upon the dyes selected, to modify the absorption and emission peaks, upon the desired PAR and the boundary conditions for each specific location.Luminescent films were manufactured with a combination of three dyes to cover the green absorption spectrum range from 450 to 600 nm c.a., and to improve photoemission around 650 nm c.a., according to PAR crops. The selected dyes were Lumogen Red (LR305), 4-(Dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM) and 2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-1,3,3-trimethyl-3H-indolium perchlorate (LDS), which were all used as received; the first one from BASF and the others from Exciton. The host material selected was a low-density polyethylene (LDPE) from LyondelBasell, as it is currently the most commonly used material for the manufacturing of conventional greenhouse films.This research study comprised two stages: laboratory and pilot scale . The samCompounding at pilot scale was carried out at 140 °C by gradually adding LDPE and the luminescent dye to a solution of p-Xilen (Sigma Aldrich). The solution remained for 2 h under stirring conditions to ensure the optimal mixing of the LDPE and dye molecules. After this time, p-Xileno was removed from the mixture by means of a distillation procedure to obtain a homogeneous solid. Different dye concentrations were tested from 0 to 5% w/w. One masterbatch of luminescent dye and LDPE was prepared from the previous solid and raw LDPE by employing a corotating twin-screw extrusion line with a pelletizing system (SIEPLA model SHJ-20). All resulting samples were processed in a mill machine with stainless steel blades (J Purchades model 250) in order to ensure homogeneity with an average particle size of 3 μm. The extrusion process was carried out by using a specific thermal profile of 90 °C/150 °C/180 °C/190 °C. During the entire process, the screws were rotating at 300 rpm. Finally, three different regrinds for each luminescent combination were fabricated: 100, 200, and 300 ppm. To end the procedure, luminescent film was obtained  from a b2 (1) and TiO2 (2), resulting in the sequence 1 2 1 2 1 2 1 2 1 2 1 2 1  structures were designed to reflect the maximum energy at the near-infrared spectral range. For that purpose, periodic dielectric structure was defined as 13 layers alternating SiO 2 1 2 1 . Refract2. Low-density polyethylene film was used as a substrate, with a dimension of 300 × 300 mm2. To avoid any contamination from the substrate, it was cleaned using a glow discharge in the load-lock chamber. The process chamber was evacuated below 5 × 10−7 mbar prior to any deposition, and all depositions were carried out in an Ar/O2 mixture under constant pressure of 2 × 10−3 mbar. The O2 flow rate was selected in order to operate beyond the transition region of the cathode voltage versus oxygen flow curve, where the deposition fully works in the oxide mode. The process was performed at plasma temperature.Dielectric layers were deposited by DC magnetron sputtering (44 Solar) using ceramic targets (99.999%) with a dimension of 500 × 100 mmThickness was measured using a mechanical profilometer . Optical properties were characterized using a UV-VIS spectrometer setup including a Xe-lamp (Newport model 71228), monochromator (Oriel Cornerstone 260), integer sphere (Labsphere 3P-GPS-030-SF), and UV-VIS detector (Newport 71610). The signal was recorded using a dual lock-in amplifier (Stanford Research SRS-830).The modification of light for plant growth was based on the principle that not all energies are equal, with red wavelengths having been proven as the most photosynthetically efficient energies. By converting a fraction of the incoming blue light and green light to red light, this film installed in the greenhouse increased the photosynthetic efficiency of light reaching the canopy, allowing growers to favor root development and flowering. These principles were demonstrated in this research study.3 at laboratory scale in order to identify whether their photoemission peaks were aligned with the PAR spectrum requested. It was observed that all dyes had a maximum luminescent emission in the range of approximately 600 nm . Moreond LR305 c: the acIt is remarkable that the absorption spectra were dramatically increased by increasing the concentration of the dyes used, and the absorption range was enhanced when DCM was combined with LDS or LR305. This increase was accompanied by a rich vibrational profile as the spectra became broader around the main absorption peaks. This behavior can be explained by the formation of dimers and higher molecular weight aggregates recognized by large spectral broadening, and obvious deviation from the Lambert–Beer law. Two main types of molecular aggregates can be created by high concentrations of fluorescent dye molecules: J-aggregates, which are face-to-face molecular arrangements that cause a bathochromic (red) shift; and H-aggregates, which are face-to-face molecular arrangements that yield a hypochromic (blue) shift .These lab designs were scaled up to a production scale to obtain large LDPE films doped with luminescent dyes, following the procedure described in The effect of the different dyes at lab scale was investigated at pilot scale. Industrial LDPE doped films were manufactured, and their normalized transmittance spectra to the reference sample (LDPE undoped) are illustrated in 01) of the DCM molecule. The second band lay in the green region, corresponding to the electronic transitions of the LR305 perylene molecule. The absorption contribution from LR305 remained, but the DCM extended the absorption spectra to a high-energy region. Both dyes, DCM and LR305, were transferring energy in a nonradiative mechanism (FRET); this phenomenon explains why the fluorescence spectra were similar to the single LR305 dye, but with the additional benefit of amplifying the intensity of the light photoemitted, in good agreement with the previous results achieved at the laboratory scale. Moreover, this photoemitted light was well aligned with PAR requirements to improve the photosynthesis process.Absorption was significantly increased when the LR305 molecule was combined with the DCM one (blue dots) with respect to single LR305 dye doping (red dots). Therefore, photoemission intensity increased, but the spectral profile remained the same, as it was expected in the FRET nonradiative process. When both dyes were mixed, two major absorption bands were observed; the first one was located in the UV region around characterizing the electronic transition of the first excited singlet state (S2) and titania (TiO2) layers.Photonic crystals are periodic dielectric structures that are designed to form the energy band structure for photons, which either allows or forbids the propagation of electromagnetic waves of certain frequency ranges, making them ideal for light-harvesting applications , such as2 (layer 1) and TiO2 (layer 2) according to this pattern: 1 2 1 2 1 2 1 2 1 2 1 2 1. The thickness of the first layer  was optimized to 229 nm, while the thickness of the second layer  was selected to be 130 nm. These refractive indices and the thickness of the layers were very large, and directly controlled the strip space of the photonic crystal. These parameters were used to model the reflectance, and the Bragg peak obtained was limited from 1000 to 1600 nm, so the NIR region was not transmitted inside of the greenhouse. Furthermore, this optical response showed that there was not any optical incompatibility between the luminescent material and the photonic crystal, but both of them were well coupled to ensure maximum performance of the films.In this work, a multilayer structure consisted of a periodic structure of SiO2/TiO2. The measured high reflection bands exactly matched with the simulated high-reflection bands. The interface fringes outside of the high-reflection bands almost matched with the theoretically generated fringes. We observed that most of the power irradiation was reflected from 1000 nm, which corresponded to the NIR region. The Bragg peak was centered at approximately λ0 = 1300 nm. However, the parameter λL represented the opening wavelength of the photonic band, and the parameter λH the wavelength of the closing. ∆λ = 600 nm was the photonic bandwidth, which represented a wider spectra range to reflect the low-energy photons corresponding to the NIR region, minimizing the internal temperature of the greenhouse. The index contrast between layers with high and low refractive index had a significant influence on the Bragg peak’s characteristics: central position and width. For instance, another theoretical model, replacing the TiO2 layer with Si3N4 (120 nm), and using the same periodic patterns (N = 13), offered a wider Bragg peak (900 nm) centered at 1450 nm.The simulation and experimental characterization are given in 2/TiO2 1DPC manufactured had infrared spectrally selective high reflectivity, thus meeting the requirements for our greenhouse design. In addition, the dielectric materials covered the entire 1000–1600 nm spectral band. This range of energy reflected might be increased by replacing SiO2 with Si3N4 and keeping the same pattern. The achieved high-reflection band was much wider than the values reported by Chiasera [2/SiO2 periodic photonic crystal.The above results indicate that the SiOChiasera , ScotognChiasera , and JenChiasera , in the A side-by-side comparison was conducted using two identical research greenhouses, one covered with the doped luminescent and photonic crystals using LDPE host materials, and the other one with the conventional LDPE films. To investigate the impact of the solar spectrum modification, the following indicators were analyzed: (a) tomato canopy performance (productivity and physiology), and (b) greenhouse internal microclimate (uniformity and crop-microclimate interaction). In addition, a survey study was conducted in the same greenhouses to find the performance of lettuce, herbs, and tomato seedlings.Three cropping zones were established in each greenhouse: (1) tomato crop production zone with Rockwool-based growing media and a high-wire system ; (2) lettuce (Rex and Magenta varieties) production zone with a deep-flow hydroponics system; and (3) an ebb-and-flow bench system to produce seedlings and herbs on a bench as part of the survey study.The following observations were obtained after one week of monitoring : compareTwo lettuce varieties, Rex and Magenta, were grown; the lettuce was seeded, and seedlings were transplanted one week later. A modified Hoagland nutrient solution was used, and pH was maintained at 6.64 during the production period. The results obtained in the first crop cycle are shown in The light quality was measured using a spectroradiometer on a clear sunny day, after the prototype installation. The light quality is shown in This research study demonstrated that the combination of an LDPE film doped with luminescent dyes and a photonic crystal improved the greenhouse’s performance. The radiation transmitted by the film was modified to match the PAR response of the crops: green light was re-emitted into red light, and the NIR spectra responsible for the thermal losses was minimized.The right combination of two dyes (DCM and LR305) to ensure a FRET nonradiative mechanism resulted in a system with a wider absorption range when compared with results reported previously by other authors (LR305). The wider the absorption spectrum was, the higher the photoemission measured, with obvious better optical performance. The 1DPC design showed a reflectivity over 97% in the NIR spectrum (from 1020 to 1680 nm), with excellent matching between theoretical and experimental values. Thus, thermal conditions inside of the greenhouse remained isolated, as the low-energy photons could not be transmitted by the film.2 and SiO2 thicknesses and refractive indexes of the 1DPC could be tuned to adapt the film’s reflectivity to a different spectrum range.In addition, it was demonstrated that the optical properties of the film  could be easily modified to adapt the greenhouse materials to any specific location . Additional dyes could be added to increase or modify the absorption and emission spectra. Moreover, the TiOTwo vegetables were selected to evaluate the benefit of this greenhouse film: lettuce and tomato crops. Crop growth under these conditions exhibited faster development of leaves and extension of stems (elongation). The enhanced leaf expansion increased overall photosynthetic capacity, and thereby the growth.The technology described in this paper can manipulate sunlight in a similar way to LEDs, without any use of electricity at all. Future work will concern deeper analysis of this design in different climate locations; an exhaustive energy model will be elaborated, and the photostability of the films over long-term periods will be evaluated. This information will be checked by the market to determine a better economic and feasibility model.