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Circuits in the absence of cortical layers: increased callosal connectivity in reeler mice revealed by brain-wide input mapping of VIP neurons in barrel cortex

The neocortex is composed of layers. Whether layers constitute an essential framework for the formation of functional circuits is not well understood. We investigated if neurons require the layer organization to be embedded into brain-wide circuits using the reeler mouse. This mutant is characterized by a migration deficit of cortical neurons so that no layers are formed. Still, neurons retain their properties and reeler mice show little cognitive impairment. We focused on VIP neurons because they are known to receive strong long-range inputs and have a typical laminar bias towards upper layers. In reeler these neurons are more distributed across the cortex. We mapped the brain-wide inputs of VIP neurons in barrel cortex of wildtype and reeler mice with rabies virus tracing. Innervation by subcortical inputs was not altered in reeler, in contrast to the cortical circuitry. Numbers of long-range ipsilateral cortical inputs were reduced in reeler, while contralateral inputs were strongly increased. Reeler mice had more callosal projection neurons. Hence, the corpus callosum was larger in reeler as shown by structural imaging. We argue that in the absence of cortical layers, circuits with subcortical structures are maintained but cortical neurons establish a different network capable to preserve cognitive functions.

The neurons of the mammalian neocortex are stacked in layers (L). This organization 3 is considered as an essential scaffolding principle that evolution could expand to 4 generate increasingly sophisticated cognitive abilities (Shepherd and Rowe, 2017). 5 Layers are seen as units of processing and their input-output connectivity defines the 6 flow of information within the cortex (Feldmeyer, 2012;Miller, 2001). However, the 7 notion of layers structuring the flow of cortical processing might be misleading. 15 Therefore, we asked the question if correct laminar position is a necessary 16 prerequisite for a neuron to receive inputs and become properly embedded in brain-17 wide circuits. An excellent model system to study the significance of layers is the 18 reeler mouse (Guy and Staiger, 2017). Homozygous mice lack the protein reelin,  22 We first confirmed that VIP neurons lose their laminar bias in reeler mice, 23 establishing this mouse as a valid model system to study the circuitry of VIP cells in 24 the absence of layers. Then we generated maps of brain-wide long-range input to 25 VIP neurons in the barrel cortex of wildtype (WT) and reeler mice, using rabies virus 26 tracing. While we found that subcortical inputs innervated malpositioned VIP cells to 27 the same extent as in a layered cortex, the balance of cortical inputs was 28 fundamentally different. Ipsilateral cortical input was reduced, contralateral cortical 29 input was increased compared to WT. Reeler mice had more callosal projecting 30 neurons (CPNs) and hence a larger corpus callosum. We argue that the absence of 31 layers has little effect on innervation by subcortical structures but induces a different 32 circuit arrangement within the cortex. KpnI-HF and AgeI-HF to open the backbone and oG-EGFP fragment was reinserted 31 together with the TVA 66T fragment using infusion cloning with an In-Fusion HD 1 cloning kit (Takara bio). The plasmid was packaged into AAV8 by the SALK Viral 2 Vector Core, from where it is available.

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For sedation and analgesia, mice were injected intraperitoneally with 10 µg/g 16 xylazine (Xylariem, Ecuphar) and 0.065 µg/g buprenorphine (Temgesic, Individor UK 17 Limited) in sterile saline, respectively. Anesthesia was induced with 3% isoflurane 18 (vol/vol) and maintained between 0.5 and 1% throughout the entire surgical 19 procedure (Harvard Apparatus, USA). Mice were mounted on a custom-built frame 20 with rigid earbars. A mixture of 2µg/g bupivacaine/lidocaine (Astra Zeneca) was 21 injected subcutaneously under the scalp for local anesthesia. A heating pad was 22 used to maintain body temperature at 37 °C (ATC 1000, World Precision 23 Instruments, Florida). Subsequently, a small incision was made in the scalp to 24 expose the right hemisphere of the skull. The bone over the somatosensory area was 25 thinned to transparency with a dental drill (OS-40, Osada Electric Company, Japan).

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Then, the location of the C2 whisker-related column was determined and mapped on 27 the blood vessel pattern as described below. The bone above the target area was 28 removed with a syringe tip. A glass pipette cut to 20 µm tip diameter (Drummond 29 Scientific Co, USA) was front-filled with AAV helper virus. The pipette holder was 30 attached to a micromanipulator (Luigs & Neumann, Germany). The pipette was 31 inserted at the target location into the brain in an approximately 45° angle, orthogonal 32 to the curvature of the cortex. AAV was pressure-injected with a syringe at three 1 depths (750 µm, 500 µm, and 250 µm below pia). To reduce backflow, the needle 2 was left in place at each depth for at least three minutes. The scalp was sutured and 3 the mouse received a subcutaneous injection of 5 µg/g Carprofen (Pfizer) for 4 prolonged pain relief. Fourteen days later, the mouse was injected with RV-mCherry 5 without prior imaging. The injection was guided based on the blood vessel pattern 6 and landmarks from the previous surgery." This procedure was performed as described earlier (Guy et al., 2015;Hafner et al., 10 2019). Briefly, Whisker C2 on the left muzzle was stimulated with a piezo actuator at 11 5Hz. Red light was shone on the cortex exposed, thinned cortex above the right 12 barrel field and its reflectance was measured with a CCD camera. Changes in 13 reflectance were average across 30 trials. The blood vessel pattern and the intrinsic 14 signal were overlaid to guide the subsequent injection.  Mice were sacrificed by injecting an overdose of ketamine (100 µg/g; Medistar) and 18 perfused transcardially with ice-cold 10% sucrose solution followed by 4% 19 paraformaldehyde (PFA) in 0.1M phosphate buffer. The brain was postfixed in 4% 20 PFA for 4h. A vibratome (VT 1200 S, Leica) was used to section the brain into 100 21 μm-thick coronal sections rostral and caudal to barrel cortex, while barrel cortex was  . The tdTomato signal was amplified after the in-situ hybridization using 19 the immunohistochemistry protocol described above but without Triton X-100.

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For overview images only one plane was imaged, for the injection sites stacks were 27 acquired. Tiles were stitched after imaging and stacks were deconvoluted in ZEN 28 Blue software (Zeiss) to reduce out-of-focus light. Confocal acquisitions were taken 29 on a LSM 880 (Zeiss) with a 25x objective operated by ZEN black software (Zeiss). The ex vivo brain samples (n=3 per group) were imaged in a high-field 9.4 Tesla MR 2 system, using a mouse brain 4-channel coil array (Bruker BioSpin MRI GmbH,  To measure the cross-sectional area of the CC at its midline crossing in a consistent 28 way across animals, we followed a multi-step process involving the computed maps 29 for MTsat and RGB FA. The MTsat maps, which exhibit good grey/white matter 30 contrast, were used to generate white matter masks through simple thresholding 31 (voxels with MTsat > 0.006 were classified as white matter). We subsequently 32 restricted the white matter masks to the five middle sagittal slices (left-to-right 1 thickness 0.625 mm) and further cropped them along the rostrocaudal (5 mm) and 2 the dorsoventral (3.125 mm) directions-thus confining them within a thin midline 3 slab that contained the entire CC crossing. This slab also included several other 4 white matter tracts, some of which (e.g. dorsal fornix) run adjacent to the CC and are 5 challenging to separate based on MTsat alone. We addressed this issue by 6 incorporating information about the orientation of white matter tracts. This was 7 available in the form of the RGB FA map, which is a color-coded representation of 8 the principal diffusion direction at each voxel (Pajevic and Pierpaoli, 1999). Using     For quantification of VIP neurons in VIP-Cre/tdTomato mice, neurons were manually 7 counted on six sections per mouse on a patch of barrel cortex that spanned from pia 8 to white matter and was 1000 µm wide. In the occasional case a section was lost 9 during the tissue preparation, we interpolated for this animal the number of cells to 10 the same volume.

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For a layer-independent analysis of neuronal distribution in the cortex, we divided the 12 space between the pia and the white matter boarder in 20 bins of equal size. We   21 We used Cre-dependent rabies virus tracing, in order to map the monosynaptic long- surface. Furthermore, RV-mCherry is G-deleted, so that it needs the glycoprotein 32 13 provided in trans from the AAV to spread to presynaptic neurons. Its mCherry 1 expression labels these presynaptic neurons red. The starter cells appear yellow due 2 to the mixture of EGFP and mCherry ( Figure 2B). This two-virus system allows the 3 visualization of brain-wide monosynaptic inputs to unequivocally identifiable starter 4 cells.

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To check if our viruses are specific, we performed control experiments in BL6 6 animals without Cre. Injection of RV-mCherry alone did not result in any labeling, 7 showing that TVA is required for RV entry into cells ( Figure S1A). When we injected 8 AAV-TVA 66T -EGFP-oG and RV-mCherry as in tracing experiments, we did not detect 9 any RV-mCherry labeling, neither at the injection site nor in the thalamus (Figure 10 S1B). A few EGFP-positive cells most likely resulted from a minimal Cre-independent 11 expression of the AAV construct. While RV requires very little TVA to enter cells, 12 which can result in Cre-independent leak expression, TVA 66T has lower affinity to RV 13 and thus needs to be expressed in much higher levels (Miyamichi et al., 2013).
14 Therefore, this receptor abolishes leak expression and each mCherry-positive neuron 15 can be surely considered an input neuron.

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Injections were centered on the whisker C2 representation 18 In the barrel cortex, each whisker is represented by a cortical column. Although the 19 reeler barrel cortex has no laminar organization, it retains a somatotopic organization 20 such that adjacent whiskers are represented by adjacent modules (Guy et al., 2015). 21 We localized the cortical whisker C2 representation to guide our injections using 22 intrinsic signal optical imaging (Grinvald et al., 1986;Guy et al., 2015). Repetitive ). As a proof of principle, we injected AAV-TVA 66T -EGFP-oG and RV-mCherry 28 centered on C2 in a WT animal and sectioned the cortex tangentially. We could 29 confirm our target specificity as the highest density of inputs was present within C2 30 ( Figure S2E). In this way we could target VIP neurons that belong to the same 31 functional modules in WT and reeler mice to have optimal comparability between 32 genotypes.  When analyzing quantitative differences in the inputs between genotypes, we first 9 focused on more global categories of input ( Figure 4, Table S1). We first summed up 10 all long-range inputs and then separated inputs from ipsilateral and contralateral 11 cortical areas, from the thalamus and from all subcortical areas. We normalized the 12 inputs by dividing the cell count by the number of starter cells to calculate the input 13 magnitude, which can be seen as a proxy for the strength of an input. VIP neurons in 14 reeler received overall fewer inputs per cell. This reduction was exclusively due to 15 fewer inputs from the ipsilateral cortex in reeler, while they received more 16 contralateral inputs per cell. There was no difference for thalamic or total subcortical 17 input.

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The input magnitude might be influenced by the number of starter cells. One  In sum, VIP neurons in reeler mice are embedded in a different long-range cortical 1 circuitry that contains fewer inputs from the ipsilateral and more inputs from the 2 contralateral cortical hemisphere.

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Auditory and motor cortex showed the strongest reduction in ipsilateral input 5 Because each VIP neuron in reeler received on average less input, the subsequent 6 analyses for differences among individual areas using the input magnitude would be 7 inherently biased. Therefore, we employed another means of normalization: input 8 fraction. It is the number of inputs in an area divided by the number of total inputs in 9 the brain. It reflects how different inputs are balanced. We selected 41 consistently 10 labeled areas that constituted the majority of inputs (97.6% in WT; 98.8% in reeler).

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We calculated the input fraction for each area and made pairwise comparisons 12 between genotypes ( Figure 5, Table S2). For motor, somatosensory body region, 13 auditory and visual cortex we also summed up counts in the individual sub-areas to 14 calculate a total input fraction. VIP neurons in reeler mice received a notably lower 15 fraction from almost all ipsilateral cortical areas. The strongest reduction was seen for 16 primary auditory cortex. The input fraction for motor cortex was also significantly 17 reduced. The contralateral barrel cortex as well as the contralateral secondary 18 somatosensory cortex contributed a significantly higher fraction of inputs in reeler.  Figure S4C). This increase 32 was the same as for contralateral inputs to VIP cells. Moreover, the distribution of 1 CPNs was very similar to the contralateral inputs' to VIP cells, being more biased 2 towards the lower part of the cortex ( Figure S4D, Figure 6F).

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If there are more CPNs in reeler, the corpus callosum should also contain more fibers 5 and hence be larger. To provide additional evidence for an increased callosal 6 connectivity, we performed magnetic resonance imaging (MRI) to measure the 7 dimensions of the corpus callosum. We performed a quantitative morphometric 8 analysis on three WT-reeler littermate pairs. We segmented the corpus callosum 9 based on structural information of magnetization transfer maps ( Figure S5A, A') and 10 directional information of fractional anisotropy maps ( Figure S5C, C'). We measured 11 the area of the corpus callosum on the mid-sagittal section, which was larger in reeler 12 in every littermate pair ( Figure S5D). This effect was not generated by differences in 13 cortical volume as the volume of the isocortex was fairly well matched between 14 littermates, although in one pair the volume was slightly larger in the reeler mouse 15 ( Figure S5E). Furthermore, we noticed on coronal sections that the corpus callosum 16 was shaped differently in reeler. It did not have a typical U-shape at the medial area looked notably different. In WT, most projection neurons were located around the LIII-32 IV border. This peak was smoothed out in reeler, indicating that the projection 1 neurons were rather dispersed than inverted in their arrangement ( Figure 6E). The 2 pattern of projection neurons in the contralateral barrel cortex, however, was 3 completely different ( Figure 6F). In WT, projection neurons were predominantly 4 located in the upper third of the cortex, corresponding to LII/III. In reeler, projection 5 neurons were predominantly located in the lower two-thirds of the cortex. The main aim of this study was to investigate if the distribution of cortical cells across 3 layers is linked to their capacity to integrate long-range inputs. We investigated the 4 long-range inputs to VIP neurons because they have a very typical laminar bias 5 towards the upper layers, which is lost in reeler mice. Using retrograde RV-tracing we 6 mapped the brain-wide inputs to VIP neurons in barrel cortex of WT and reeler mice.

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There was no difference in the innervation by subcortical inputs. Cortical inputs were 8 from the same sources, however, in reeler ipsilateral cortical inputs were reduced 9 and contralateral cortical inputs were much more numerous, revealing a different 10 cortical circuit architecture in the absence of layers.

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Input to VIP cells originated from the same sources in WT and reeler 13 We found that VIP cells in the barrel cortex of WT and reeler mice were innervated by 14 the same sources in both genotypes. This is not surprising because the existence of neuron classes in a cortical area receive input from the same sources. Therefore, the 21 feature, which is considered to distinguish cell types and relate to potential functional 22 implications, is the input's proportion a given neuron type receives. In consequence, 23 we expected to discover putative differences in the afferent connectivity to VIP 24 neurons between genotypes specifically on a quantitate level, which has not been 25 assessed for long-range projections in reeler yet.

Subcortical fibers target VIP cells independent of laminar position 28
When we looked at the proportion of inputs to VIP cells, we found profound 29 differences between WT and reeler. VIP cells in reeler received less input per cell 30 than in WT, which was exclusively due to a reduction in cortical inputs, while the 31 subcortical inputs remained the same.  , 1976;Imai et al., 2012;Steindler and Colwell, 1976). At least, our observation 27 that the distribution of CPNs is biased towards deeper parts of the cortex is not 28 without precedent (Steindler and Colwell, 1976).             (A-F) The thickness of the cortex was divided into 20 equal-sized bins. We plotted the 7 fraction of inputs in a bin normalized against the total inputs of VIP cells from this area. In 8 ipsilateral areas, the distribution of projection neurons had one peak that was usually shifted 9 more superficially in reeler, except for visual cortex where the peak was just broader. The 10 distribution of contralateral projection neurons from the barrel cortex was very different, with 11 neurons in WT mostly in the upper third, while in reeler predominantly in the lower two thirds 12 of the cortex (Abbr.: S1, primary somatosensory cortex; BF, barrel field).     Orbital area  (A) Number of VIP neurons counted in a volume of cortex that spanned from pia to white matter and was 100 µm wide and 240 µm thick (n=5 WT mice; n=4 reeler mice; box plot: white line = median; white dot = mean). Counts were almost the same in WT and reeler.
(B) Distribution of VIP neurons across the cortical depth. In WT they showed a prominent peak in the upper layers. In reeler they were fairly uniformly distributed with few neurons close to pia and white matter.
(C) Section of barrel cortex of a VIP-Cre/reeler/tdTomato mouse stained against VIP-RNA (green) with fluorescent in-situ hybridization. Virtually all tdTomato cells overlapped with VIP signal (scale bar: 200 µm).  (B) AAV8-DIO-TVA66T-EGFP-oG and RV-mCherry were injected with the same titer as in experimental conditions but in a BL6 (wild type) animal. We did not observe any RV labeling at the injection site nor in the ventral posteromedial nucleus of the thalamus (insert), a structure with reliable input to the barrel cortex. Therefore, this AAV does not show leak expression in the absence of Cre that would confound the tracing experiments (scale bar: 200 µm).
(C) Input magnitude was plotted against starter cell count for WT and reeler. There was no correlation between the two variables. Therefore, the difference in input magnitude between genotypes cannot be explained by a difference in starter cell numbers. (A, A') Red light was shone on the exposed cortical surface and its reflectance was measured with a CCD-camera, while the contralateral C2 whisker was stimulated. Repetitive whisker stimulation over 30 trials led to a localized change in blood flow, which induced a change in light reflectance visible as a dark spot. WT and reeler mice had very similar signals in terms of dynamics and size (scale bar: 200 µm).
(B, B') Surface vasculature was overlaid with image in A and the location of the strongest change in reflectance was marked. The blood vessels were used as landmarks to guide the injection pipette to the dot (scale bar: 200 µm).
(C) Tangential section of barrel cortex after targeted injection of AAV-TVA66T-EGFP-oG and RV-mCherry into the C2 column. Staining thalamic terminals with vesicular glutamate transporter 2 (vG-LuT2) allowed to visualize the barrels. The density of input cells was highest in C2 indicating that the majority of VIP starter cells was located within this barrel-related column (scale bar: 200 µm). (D) Distribution of CPNs across the cortical depth. The cortex was divided into 20 bins of equal size and the relative fraction of CPNs in each bin was plotted. CPNs in WT were predominantly in the upper part corresponding to LII/III. In reeler they were more in the deeper parts of the cortex. The distribution of all CPNs wes similar to the distribution of CPNs targeting VIP cells in both genotypes. (B, B') Coronal MTsat map of WT versus reeler mouse. At the midline the CC showed a different geometry between genotypes. In reeler mice the characteristic curvature of WT mice was absent so that the top of the CC appeared flatter.
(C, C') Fractional anisotropy (FA) maps show the color-coded directionality of fibers. Because fibers of the CC run in the medio-lateral direction they could be distinguished from whiter matter bundles more dorsally or more ventrally that run in the rostro-caudal direction. (D) Mid-sagittal area of the CC of individual WT and reeler mice compared between littermate pairs. In each pair, the reeler mouse had a larger area, probably indicating that it is comprised of a higher number of callosal fibers.
(E) Total isocortical volume of individual WT and reeler mice compared between littermate pairs. The volume was almost the same except for one pair in which the reeler mouse showed a slightly higher volume.