Structural development and dorsoventral maturation of the medial entorhinal cortex.

We investigated the structural development of superficial-layers of medial entorhinal cortex and parasubiculum in rats. The grid-layout and cholinergic-innervation of calbindin-positive pyramidal-cells in layer-2 emerged around birth while reelin-positive stellate-cells were scattered throughout development. Layer-3 and parasubiculum neurons had a transient calbindin-expression, which declined with age. Early postnatally, layer-2 pyramidal but not stellate-cells co-localized with doublecortin - a marker of immature neurons - suggesting delayed functional-maturation of pyramidal-cells. Three observations indicated a dorsal-to-ventral maturation of entorhinal cortex and parasubiculum: (i) calbindin-expression in layer-3 neurons decreased progressively from dorsal-to-ventral, (ii) doublecortin in layer-2 calbindin-positive-patches disappeared dorsally before ventrally, and (iii) wolframin-expression emerged earlier in dorsal than ventral parasubiculum. The early appearance of calbindin-pyramidal-grid-organization in layer-2 suggests that this pattern is instructed by genetic information rather than experience. Superficial-layer-microcircuits mature earlier in dorsal entorhinal cortex, where small spatial-scales are represented. Maturation of ventral-entorhinal-microcircuits - representing larger spatial-scales - follows later around the onset of exploratory behavior.

Introduction

The representation of space in the rodent brain has been investigated in detail. The functional development of spatial response properties has also been investigated in the cortico-hippocampal system (Ainge and Langston, 2012; Wills et al., 2014), with studies suggesting the early emergence of head-directional selectivity (Tan et al., 2015; Bjerknes et al., 2015), border representation (Bjerknes et al., 2014) and place cell firing, but a delayed maturation of grid cell discharges (Wills et al., 2010; Langston et al., 2010).

Even though there is information on the emergence of functional spatial properties in the hippocampal formation, remarkably little is known about the structural development of the microcircuits which bring about these properties. To understand this, we investigated the development of the architecture of the medial entorhinal cortex (MEC) and parasubiculum (PaS), two key structures in the cortico-hippocampal system.

In adult animals, layer 2 of MEC contains two types of principal cells, stellate and pyramidal cells (Alonso and Klink, 1993; Germroth et al., 1989). Stellate and pyramidal neurons are distinct in their intrinsic conductance (Alonso and Llinás, 1989; Klink and Alonso, 1997), immunoreactivity (Varga et al., 2010), projections (Lingenhöhl and Finch, 1991; Canto and Witter, 2012) and inhibitory inputs (Varga et al., 2010). Pyramidal neurons in layer 2 of MEC can be identified by calbindin-immuno-reactivity (Varga et al., 2010) and are clustered in patches across various mammalian species (Fujimaru and Kosaka, 1996; Ray et al., 2014; Naumann et al., 2016), while stellate cells can be identified by reelin-immuno-reactivity (Varga et al., 2010) and a lack of structural periodicity (Ray et al., 2014). In rodents, the grid-like arrangement of pyramidal cell patches is aligned to cholinergic inputs (Ray et al., 2014; Naumann et al., 2016). Functionally, about a third of all cells in layer 2 exhibit spatial tuning with grid, border, irregular and head-directional discharges being present (Tang et al., 2014).

Neurons in layer 3 of MEC are characterized by rather homogenous in vitro intrinsic and in vivo spatiotemporal properties (Tang et al., 2015). A majority of cells exhibit a lack of spatial modulation, and the remaining are mainly dominated by irregular spatial responses (Tang et al., 2015) with a fraction also exhibiting grid, border and head-directional responses (Boccara et al., 2010).

The parasubiculum is a long and narrow structure flanking the dorsal and medial extremities of MEC (Video 1). The superficial parasubiculum, corresponding to layer 1 of MEC is divided into large clusters, while the deeper part, corresponding to layers 2 and 3 of MEC, is rather homogenous (Tang et al., 2016). In terms of functional tuning of cells, a majority of the cells of PaS show spatially tuned responses, and include grid, border, head-directional and irregular spatial cells (Boccara et al., 2010; Tang et al., 2016).

Video 1.

Medial entorhinal cortex and parasubiculum in the rat brain.

The medial entorhinal cortex and parasubiculum are situated at the posterior extremity of the rat neocortex. This schematic video illustrates the location of the medial entorhinal cortex and parasubiculum in situ, the tangential sectioning process and the layout of parasubicular patches and calbindin-patches in the medial entorhinal cortex.

DOI: http://dx.doi.org/10.7554/eLife.13343.003

Here we investigate the emergence of the periodic pyramidal-cell patch pattern in layer 2 of MEC, as well as the development of cellular markers that characterize the architecture of adult MEC and PaS. The results indicate an early emergence of pyramidal cell organization, a delayed maturation of pyramidal but not stellate cells and a dorsal-to-ventral maturation of MEC circuits.

Results

We first investigated development of brain size and thickness of layers of the MEC (Figure 1) by observing rats at E18, P0, P4, P8, P12, P16, P20, P24 and adults (>P42). The majority of the brain development takes place within the first few weeks postnatally (Figure 1a), with the brain size increasing 1000% from 0.12 ± 0.00 g at E18 (mean ± SD; n=3) to 1.23 ± 0.07 g at P12 (n=5). Subsequently, the growth plateaus to ~25% with the brain weighing 1.71 ± 0.08 g at P24 (n=6) and having a weight of 2.11 ± 0.14 g in adults (n=9) (Figure 1b). The superficial layers (layers 1–3) of the MEC (Figure 1c) double in thickness during this early postnatal period from 243 ± 35 μm at P0 (mean ± SD; n=21, 4 rats) to 652 ± 50 μm at P12 (n=24, 4 rats). A similar increase is also observed in the deeper layers (layers 4–6) from 167 ± 21 μm at P0 (n=21, 4 rats) to 329 ± 54 μm at P12 (n=24, 4 rats).The overall thickness plateaus around this point to 981 ± 81 μm at P12 (n=24, 4 rats) and remains at 882 ± 78 μm in adults (n=24, 4 rats) (Figure 1d). Proportionally, the thickness of the layers remains similar during development, with layer 2 accounting for ~20% and layers 3 and 5/6 each accounting for ~30% of the MEC. Layers 1 and 4 are the thinnest at about 10% and 5% of the total thickness respectively (Figure 1d).

Figure 1.

Rat brain and medial entorhinal cortex laminar development.

(a) Growth in rat brain size from E18, P0, P4, P8, P12, P16, P20 to adult. Brains are overlaid on a 1 cm x 1 cm grid. (b) Mean weight (in grams) of E18 (n=3), P0 (n=6), P4 (n=5), P8 (n=5), P12 (n=5), P16 (n=5), P20 (n=5), P24 (n=6) and in adult (n=9) rat brains. Error bars indicate SD. (c) Parasagittal section double stained for calbindin-immunoreactivity (green) and Purkinje cell protein 4 immunoreactivity (pcp4; red), illustrating the superficial layers of the medial entorhinal cortex and parasubiculum. Calbindin+ neurons (green) are in layer 2, pcp4+ neurons (red) are in layer 3 MEC. (d) Development of mean layer width (in μm) of layer 1 (light-blue), layer 2 (green), layer 3 (red), layer 4 (gray-blue) and layer 5/6 (purple) from P0 to P24 and in adult rat medial entorhinal cortex. Scale bars 250 µm. PaS- Parasubiculum; L1- Layer 1; L2- Layer 2; L3- Layer 3; D- Dorsal; V-Ventral.

DOI: http://dx.doi.org/10.7554/eLife.13343.004

DOI: http://dx.doi.org/10.7554/eLife.13343.005

Laminar widths (in μm) of the medial entorhinal cortex for P0, P4, P8, P12, P16, P20, P24 and adult rats.

We next investigated the microcircuit organization of superficial layers of MEC. Calbindin, a calcium binding protein, is selectively expressed in layer 2 pyramidal cells (Varga et al., 2010; Fujimaru and Kosaka, 1996), which form a grid-like arrangement in adult animals (Ray et al., 2014). Concurrently, reelin, an extracellular matrix protein, is selectively expressed in stellate cells in layer 2 of MEC, which are scattered throughout (Ray et al., 2014) layer 2. To visualize the development of entorhinal microcircuits we first prepared tangential sections (see our video animation on preparing tangential sections, Video 1) through layer 2 of medial entorhinal cortex and stained for calbindin-immunoreactivity. From the earliest postnatal stages, calbindin+ neurons in the MEC exhibited clustering, forming patches at P0 (Figure 2a). The calbindin+ patches at P0 exhibited a grid-like (Figure 2a,b) regular arrangement (Figure 2c), determined by spatial autocorrelation analysis and grid scores, similar to that observed in adults (Ray et al., 2014; Naumann et al., 2016, Figure 2d–f). Similar preparations for visualizing stellate cells by reelin-immunoreactivity (Figure 2—figure supplement 1), exhibited the presence of stellate cells in early postnatal stages (Figure 2—figure supplement 1a,b) and a lack of periodicity (Figure 2—figure supplement 1c), similar to observations made in adults (Ray et al., 2014, Figure 2—figure supplement 1d–f). Calbindin+ pyramidal neurons in the MEC (Figure 2g) also received preferential cholinergic innervation early postnatally (Figure 2h–i), similar to adults (Ray et al., 2014; Naumann et al., 2016, Figure 2j–l).

Figure 2.

Adult-like grid layout and cholinergic innervation of calbindin+ pyramidal neurons in layer 2 of MEC at early postnatal stages.

(a) Tangential sections of the MEC processed for calbindin-immunoreactivity (green). Patches of calbindin+ neurons are evident already in the MEC, while the parasubicular patches at the right extremity also show calbindin-immunoreactivity in P0 rats. (b) Inset from (a), rotated 90 degrees clockwise, for presentation. (c) Two-dimensional spatial autocorrelation of the MEC region shown in (b) showing a periodic spatial organization of calbindin+ patches. The grid score is 0.59. (d) as (a) for adult animals. (e) Inset from (d). (f) Two-dimensional spatial autocorrelation of the MEC region shown in (e) showing a periodic spatial organization of calbindin+ patches. The grid score is 1.18. (g) Tangential section in a P4 animal processed for calbindin-immunoreactivity (green). Also note the calbindin-immunoreactive parasubicular patches present in a P4 rat. (h) Section from (g) co-stained for acetylcholinesterase activity (brown). (i) Overlay of inset regions from (g) and (h) shows overlap between calbindin and acetylcholinesterase in MEC in P4 rats. (j–l) as (g–i) for adult animals. (d–f, j–l) modified from Ray et al. (2014). Colour scale of spatial autocorrelation, -0.5 (blue) through 0 (green) to 0.5 (red). Scale bars 250 µm. D- Dorsal; V- Ventral; M- Medial; L- Lateral. Orientation in (d) applies to all sections apart from (b), where it’s rotated 90 degrees clockwise.

DOI: http://dx.doi.org/10.7554/eLife.13343.006

(a) Tangential sections of the MEC processed for reelin-immunoreactivity (red) in a P4 rat. (b) Inset from (a). (c) Two-dimensional spatial autocorrelation of the MEC region shown in (b) showing a lack of periodicity of reelin+ neurons. The grid score is -0.09. (d–f) as (a–c) for adult animals. The grid score in (f) is 0.03. Scale bars 250 µm. D- Dorsal; V- Ventral; M- Medial; L- Lateral. Orientation in (d) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.007

Figure 2—figure supplement 1.

Adult-like scattered distribution of reelin+ stellate cells in early postnatal stages.

(a) Tangential sections of the MEC processed for reelin-immunoreactivity (red) in a P4 rat. (b) Inset from (a). (c) Two-dimensional spatial autocorrelation of the MEC region shown in (b) showing a lack of periodicity of reelin+ neurons. The grid score is -0.09. (d–f) as (a–c) for adult animals. The grid score in (f) is 0.03. Scale bars 250 µm. D- Dorsal; V- Ventral; M- Medial; L- Lateral. Orientation in (d) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.007

In the parasubiculum, a transient presence of calbindin was observed with ~15 clusters of calbindin+ neurons at P0 (Figure 2a) and P4 (Figure 2g). This expression was curtailed in older animals, with only a calbindin+ stripe persisting in adults (Figure 2d).

To visualize the laminar development of MEC, we stained parasagittal sections for calbindin (Figure 3) and reelin (Figure 4) immunoreactivity. Indications of calbindin+ neuronal clusters were visible prenatally at E18 (Figure 3a). However, the calbindin+ patches in the MEC did not exhibit clustering of their dendrites, as previously described in adults (Ray et al., 2014) at E18 and P0 (Figure 3a,b). Some dendritic clustering could be observed at P4 (Figure 3c), while from P8 (Figure 3d–h) the dendritic clustering of calbindin+ pyramidal neurons was similar to that in adults. In layer 3 of the MEC, we observed a transient presence of calbindin expression. The number of calbindin+ neurons in layer 3 declined progressively from prenatal stages to P20 (Figure 3a–g), where it attained adult-like levels with rarely any calbindin+ neurons in layer 3 (Figure 3h). Quantitatively, calbindin+ neuronal density (calbindin+ neurons per mm2) decreased from 955 ± 315 (mean ± SD; count refers to n=3776 neurons in 8 rats) in P4-P8 rats to 333 ± 99 (n=2104 neurons, 8 rats) in P12-P16 rats to 141 ± 56 (n=828 neurons, 7 rats) in adults (Figure 3i).

Figure 3.

Transient presence of calbindin+ neurons in layer 3 of MEC in early postnatal stages reduces progressively to adult-like state by third postnatal week.

Parasaggital sections of the MEC processed for calbindin-immunoreactivity (green). The sections show clustering of calbindin+ pyramidal cells in layer 2 and a transient presence of calbindin+ neurons in layer 3, which decrease with age in (a) E18 rat. (b) P0 rat. (c) P4 rat. (d) P8 rat. (e) P12 rat. (f) P16 rat. (g) P20 rat. (h) Adult rat. (i) Decreasing density of calbindin+ neurons in layer 3 of MEC from P4-P8 (n=3776 neurons, 8 rats); to P12-P16 (n=2104 neurons, 8 rats) to adults (n=828 neurons, 7 rats). Error bars denote SD. Scale bars 250 µm. D- Dorsal; V- Ventral. Orientation in (h) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.008

DOI: http://dx.doi.org/10.7554/eLife.13343.009

Figure 4.

Increase of reelin expression in layer 3 neurons of MEC through development.

Parasaggital sections of the MEC processed for reelin-immunoreactivity (red). The sections show reelin+ stellate cells in layer 2 and an increasing reelin expression in layer 3 neurons with development in (a) P4 rat. (b) P8 rat. (c) P12 rat. (d) P16 rat. (e) P20 rat. (f) Adult rat. (g) Increasing density of reelin+ neurons in layer 3 of MEC from P4-P8 (n=1405 neurons, 4 rats); to P12-P16 (n=3309 neurons, 3 rats) to adults (n=5039 neurons, 3 rats). Error bars denote SD. Scale bars 250 µm. D- Dorsal; V- Ventral. Orientation in (f) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.010

DOI: http://dx.doi.org/10.7554/eLife.13343.011

Calbindin+ neurons counted and areas (in µm2) in layer 3 for determining calbindin+ neuronal density in layer 3 in P4-P8, P12-P16 and adult rats.

Reelin+ neurons counted and areas (in µm2) in layer 3for determining reelin+ neuronal density in layer 3 in P4-P8, P12-P16 and adult rats.

Reelin was present in layer 2 from early postnatal stages (Figure 4a; Figure 2—figure supplement 1a,b), though the most prominent reelin-immunoreactive cells in the first two postnatal weeks were present in layer 1 (Figure 4a–c). Reelin expression increased in layer 3 of the MEC from early postnatal stages to P20 (Figure 4a–e), where it attained adult-like levels (Figure 4f). Quantitatively, reelin+ neuronal density in layer 3 increased from 729 ± 435 (n=1405 neurons, 4 rats) in P4-P8 rats to 1549 ± 115 (n=3309 neurons, 3 rats) in P12-P16 rats to 1996 ± 208 (n=5039 neurons, 3 rats) in adults.

Three observations indicated a dorsal-ventral developmental gradient in the superficial layers of medial entorhinal cortex and parasubiculum:

First, the transient calbindin expression in layer 3 disappeared from dorsal to ventral. Thus, most of layer 3 had calbindin+ neurons at P8 (Figure 5a), only the ventral half of layer 3 showed calbindin expression at P16 (Figure 5b), and in adults calbindin expression was largely absent from layer 3 of MEC (Figure 5c). This transient expression of calbindin in layer 3 followed a dorso-ventral developmental profile (Figure 5d). Early postnatally, in P4-P8 rats, we observed equitable densities of calbindin+ cells in dorsal, intermediate and ventral levels of MEC (n=3776 neurons, 8 rats). In contrast, around the end of the second postnatal week, in P12-P16 rats, we observed significantly lower densities (p=0.010, Mann-Whitney two tailed) in the dorsal (225 ± 96 cells / mm2), as opposed to the ventral (449 ± 161 cells / mm2) MEC (n=2104 neurons, 8 rats). In adults (n=828 neurons, 7 rats), calbindin+ neurons were largely absent in layer 3, but among the remaining population the density waxed from dorsal to intermediate and ventral MEC. The development of reelin expression in layer 3 neurons on the other hand (Figure 5—figure supplement 1a–c) occurred in equitable proportions in dorsal, intermediate and ventral levels of MEC (Figure 5—figure supplement 1d) with increasing age.

Figure 5.

Dorsal-to-ventral disappearance of layer 3 calbindin expression.

Parasaggital sections showing superficial layers of the MEC processed for calbindin-immunoreactivity (green). (a) Calbindin expression is seen throughout layer 3 in P8 rats. (b) Calbindin expression is seen only in ventral half of layer 3 in P16 rats. (c) Calbindin expression is largely absent in layer 3 in adult rats. (d) Proportion of layer 3 calbindin+ neurons in dorsal (white), intermediate (gray) and ventral (black) MEC in P4-P8 (n=3776 neurons, 8 rats); P12-P16 (n =2014 neurons, 8 rats); and adult (n=828 neurons, 7 rats) rats. The numbers represent layer 3 calbindin+ neuronal density and decay in a dorsal to ventral gradient with age as evident with the reduced proportions of the white (dorsal MEC) and gray (intermediate MEC) sections of the columns with increasing age. Scale bars 250 µm. L1- Layer 1; L2- Layer 2; L3- Layer 3; D- Dorsal; V-Ventral. Orientation in (c) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.012

Parasaggital sections showing superficial layers of the MEC processed for reelin-immunoreactivity (red). (a) Reelin expression is sporadic throughout layer 3 in P8 rats. (b) Reelin expression equitably increases in layer 3 in P16 rats. (c) Reelin expression is present throughout layer 3 in adult rats. (d) Proportion of layer 3 reelin+ neurons in dorsal (white), intermediate (gray) and ventral (black) MEC in P4-P8 (n=1405 neurons, 4 rats); P12-P16 (n =3309 neurons, 3 rats); and adult (n=5039 neurons, 3 rats) rats. The numbers represent layer 3 reelin+ neuronal density and increase equitably with age as evident with the similar proportions of the white (dorsal MEC), gray (intermediate MEC) and black (ventral MEC) sections of the columns with increasing age. Scale bars 250 µm. L1- Layer 1; L2- Layer 2; L3- Layer 3; D- Dorsal; V-Ventral. Orientation in (c) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.013

DOI: http://dx.doi.org/10.7554/eLife.13343.014

Figure 5—figure supplement 1.

Dorsal- ventral distribution of layer 3 reelin expression.

Parasaggital sections showing superficial layers of the MEC processed for reelin-immunoreactivity (red). (a) Reelin expression is sporadic throughout layer 3 in P8 rats. (b) Reelin expression equitably increases in layer 3 in P16 rats. (c) Reelin expression is present throughout layer 3 in adult rats. (d) Proportion of layer 3 reelin+ neurons in dorsal (white), intermediate (gray) and ventral (black) MEC in P4-P8 (n=1405 neurons, 4 rats); P12-P16 (n =3309 neurons, 3 rats); and adult (n=5039 neurons, 3 rats) rats. The numbers represent layer 3 reelin+ neuronal density and increase equitably with age as evident with the similar proportions of the white (dorsal MEC), gray (intermediate MEC) and black (ventral MEC) sections of the columns with increasing age. Scale bars 250 µm. L1- Layer 1; L2- Layer 2; L3- Layer 3; D- Dorsal; V-Ventral. Orientation in (c) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.013

DOI: http://dx.doi.org/10.7554/eLife.13343.014

Calbindin+ neurons (Figure 5) and reelin+ neurons (Figure 5—figure supplement 1) counted and areas (in μm2) in dorsal, intermediate and ventral parts of layer 3 for determining calbindin+ and reelin+ neuronal densities respectively in P4-P8, P12-P16 and adult rats.

Second, layer 2 calbindin+ patches in the MEC also exhibited a dorsal-to-ventral maturation profile. The calbindin+ patches (Figure 6a) co-localized with doublecortin (Figure 6b), a well-established marker for immature neurons (Brown et al., 2003) throughout layer 2 at P8 (Figure 6c,d). At P16, the dorsal calbindin+ patches (Figure 6e,g) did not express doublecortin (Figure 6f,g), while ventral calbindin+ patches still co-localized with doublecortin (Figure 6h). In adults, calbindin+ patches (Figure 6i) did not exhibit doublecortin (Figure 6j) in either dorsal (Figure 6k) or ventral (Figure 6l) parts. A similar dorsal-to-ventral development gradient was evident in the PaS, with doublecortin being present throughout the PaS in P8 (Figure 6b), only in the ventral part in P16 (Figure 6f) and not present in adults (Figure 6j). To quantify the overlap between calbindin and doublecortin we performed spatial cross-correlations (Figure 6m). P8-P12 rats exhibited a high degree of overlap between calbindin and doublecortin in both dorsal (0.74 ± 0.05; mean ± SD, Pearson’s cross-correlation coefficient) and ventral (0.61 ± 0.10) parts (n=9 regions, 5 rats). In P16-P20 rats (n=16 regions, 8 rats), the dorsal regions showed low correlations (0.14 ± 0.17), while the ventral regions still showed significantly higher overlap (0.60 ± 0.07; p=0.0008, Mann-Whitney two tailed). In adults (n=7 regions, 4 rats), both dorsal (0.19 ± 0.07) and ventral (0.20 ± 0.07) regions had low overlap. The difference in the Pearson’s cross correlation coefficient between overlapping regions (dorsal and ventral in P8-P12; ventral in P16-P20) and non-overlapping regions (dorsal in P16-P20; dorsal and ventral in adults) was significant at p=0.000001 (Mann-Whitney two tailed).

Figure 6.

Dorsal-to-ventral maturation of layer 2 calbindin+ patches and parasubiculum.

Tangential sections of the MEC double-stained for calbindin immunoreactivity (green) and doublecortin immunoreactivity (red). Doublecortin is a marker for immature neurons and disappears in a dorsal-ventral gradient. (a) Calbindin-expression (green) in P8 rats. (b) Doublecortin-expression (red) in P8 rats. Note the presence of doublecortin throughout the dorso-ventral extent of MEC and parasubiculum. (c) Overlay of the dorsal inset region (dashed) in (a) and (b), showing overlap of calbindin and doublecortin (hence the yellowish color). (d) Overlay of the ventral inset region (dashed) in (a) and (b), showing overlap of calbindin and doublecortin. (e–h) as (a–d) for P16 rats, respectively. However, note that dorsal inset region lacks doublecortin (g) while ventral inset region shows overlap of calbindin and doublecortin (h). Also, note the absence of doublecortin in the dorsal but not the ventral parasubiculum (f). (i–k) as (a–d) for adult rats. No doublecortin is present in either dorsal (k) or ventral (l) regions. (m) Spatial cross-correlations of calbindin and doublecortin in MEC showing high overlap in both dorsal and ventral regions in P8-P12 rats (dark green; n=9 regions, 5 rats); low correlation in dorsal but high overlap in ventral in P16-P20 rats (green; n=16 regions, 8 rats) and low correlations in both dorsal and ventral in adult rats (light green; n=7 regions, 4 rats). The Pearson’s cross-correlation coefficient can vary from -1 (anti-correlated) through 0 (un-correlated) to 1 (correlated). Scale bars 250 µm. D- Dorsal; V- Ventral; M- Medial; L- Lateral. Orientation in (i) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.015

A closer analysis of the co-localization of the immature neuronal marker doublecortin with calbindin+ pyramidal cells and reelin+ stellate cells (Figure 7a–c) revealed doublecortin to be mostly co-localized with calbindin+ rather than reelin+ neurons (Figure 7d). Spatial cross-correlations between doublecortin and either calbindin or reelin (Figure 7e; n=8 rats from ages P8 - P20) from triple-immunostained calbindin, reelin and doublecortin regions of layer 2 of the MEC revealed a greater overlap of doublecortin with calbindin (0.54 ± 0.10) than with reelin (0.08 ± 0.13). This difference in the Pearson’s cross correlation coefficient was significant at p=0.0009 (Mann-Whitney two tailed).

Figure 7.

Higher co-localization of doublecortin with calbindin+ pyramidal than reelin+ stellate cells in the developing medial entorhinal cortex.

Tangential sections of the MEC layer 2 triple-stained for calbindin immunoreactivity (CB; blue), doublecortin immunoreactivity (DCX; red) and reelin immunoreactivity (RL; green). Pyramidal but not stellate cells are structurally immature during early postnatal stages. (a) Calbindin-expression (blue) in layer 2 of MEC. (b) Doublecortin-expression (red) in layer 2 of MEC. (c) Reelin-expression (green) in layer 2 of MEC. (d) Overlay of the inset region (dashed) in (a), (b) and (c), showing a higher co-localization of doublecortin (red) with calbindin (blue), than reelin (green). (e) Spatial cross-correlations of doublecortin with calbindin and reelin showing high overlap of doublecortin with calbindin but not reelin (n=8 regions, 8 rats). Scale bars (a–c) 250 µm; (d) 100 µm. D- Dorsal; V- Ventral; M- Medial; L- Lateral. Orientation in (c) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.016

Third, wolframin expression, a marker which co-localizes with calbindin+ pyramidal neurons in layer 2 of MEC in adult rodents (Kitamura et al., 2014), develops from dorsal to ventral in layer 2 medial entorhinal cortex and parasubiculum (Figure 8). Specifically, wolframin expression starts to appear in the dorsal MEC and the dorsal PaS shortly after birth (Figure 8a) and is present only in the dorsal ~10% of the PaS. It extends progressively more ventrally (Figure 8b) and covers ~40% at P8 and ~75% at P12 of PaS. At P20 it is expressed throughout the full extent of medial entorhinal cortex and the parasubiculum (Figure 8c).

Figure 8.

Dorsal-to-ventral maturation of wolframin expression in the medial entorhinal cortex and parasubiculum.

(a) Tangential sections of the MEC and PaS (outlines dashed) double-stained for calbindin-immunoreactivity (green) and wolframin immunoreactivity (red) in a P4 rat. Shown is an overlay of red and green fluorescence. (b) as (a) for a P8 rat. (c) as (a) for a P20 rat. Wolframin is present in the dorsal ~10% of the parasubiculum at P4, ~40% at P8 and 100% at P20. Note that wolframin expression co-localizes with calbindin-expression in the MEC (hence the yellowish color) and increases from dorsal to ventral with age. Scale bars 250 µm. D- Dorsal; V- Ventral; M- Medial; L- Lateral. Orientation in (a) applies to all sections.

DOI: http://dx.doi.org/10.7554/eLife.13343.017

Discussion

Neurogenesis in the medial entorhinal cortex is completed prior to E18 (Bayer, 1980a; 1980b), and at this time the basic laminar organization of medial entorhinal cortex is already evident. While the basic structure of medial entorhinal cortex appears early, we observe massive developmental changes in the cortical structure, including a doubling of the thickness of the superficial layers during the first postnatal week.

The clustering of layer 2 MEC calbindin+ neurons into patches is also an early developmental event, and key aspects of the grid-layout of calbindin+ neurons are already present at birth. This observation indicates that the periodic structure of patches is a result of genetic signaling rather than spatial experience. Periodic patterns are ubiquitous in nature, and several chemical patterning systems have been explained on the basis of interaction between dynamical systems (Turing, 1952). Since it has been suggested that the grid layout of calbindin+ neurons is functionally relevant for grid cell activity (Brecht, 2014), it would be interesting to investigate, whether genetic manipulations would result in changes of layout periodicity and have functional effects. The dendritic clustering of calbindin+ pyramidal neurons is similar to dendritic development in the neocortex (Petit et al., 1988) and is established by the end of the first postnatal week. The cholinergic innervation of the calbindin+ patches was present by P4 in line with other long-range connectivity patterns in the MEC (O’Reilly et al., 2015), which are also established early in development.

Reelin is an important protein in cortical layer development (D'Arcangelo et al., 1995) and in the early stages of postnatal development we see the strongest reelin expression in layer 1, where reelin secreting Cajal-Retzius cells are involved in radial neuronal migration (Pesold et al., 1998). Stellate cells in layer 2 of MEC, which can be visualized by reelin-immunoreactivity (Varga et al., 2010), were scattered (Ray et al., 2014) throughout postnatal development.

Layer 3 of the MEC features a complementary transition of calbindin+ and reelin+ neurons during the first couple of postnatal weeks. While the density of reelin+ neurons increases, there is a concurrent decline in calbindin+ neuronal density in layer 3 of MEC, though part of the calbindin+ neuronal density decline can be attributed to the increasing brain size. Taken together with the presence of radial neuronal migration promoting Cajal-Retzius cells in layer 1 during this period, it would be interesting to investigate whether the transient calbindin+ neurons are migrating to layer 2 or changing their phenotype to reelin+ neurons, and what layer and cell-type specific functional differences are observed in this early postnatal development stage.

An interesting observation is the presence of clusters of neurons in the parasubiculum, which transiently express calbindin in early postnatal stages, and subsequently express wolframin. Transient expression of calbindin has been observed in early postnatal development in the neocortex (Hogan and Berman, 1993) and midbrain regions (Liu and Graybiel, 1992), but its functional significance remains largely unknown. Our data show, however, that at early developmental stages the parasubiculum and medial entorhinal cortex share a similar organization in calbindin+ patches. Additionally, the expression of wolframin in the parasubiculum persists in adults, while calbindin+ neurons in MEC layer 2 also exhibit wolframin (Kitamura et al., 2014) from the end of the first postnatal week. Current studies generally focus on cell-type specific investigations using proteins expressed by these cells. However, investigations to study the specific roles of these proteins (Li et al., 1995) might provide interesting insights towards understanding the finer differences in the functionalities exhibited by these cells. For instance, calbindin is a calcium buffer, and reduces the concentration of intracellular calcium (Mattson et al., 1991), while wolframin is implicated in increasing intracellular calcium levels (Osman et al., 2003). With the medial entorhinal cortex and parasubiculum having many similarities in their spatial discharge properties (Tang et al., 2014; Boccara et al., 2010; Tang et al., 2016), a structure-function comparison of the wolframin+/transiently-calbindin+ neurons in the parasubiculum and the wolframin+/ permanently-calbindin+ neurons in the medial entorhinal cortex would be worthwhile.

A dorsal-to-ventral development profile was observed in the superficial layers of the MEC and parasubiculum. This conclusion was suggested by the progressive disappearance of the calbindin expression in layer 3 from dorsal to ventral; the progressive disappearance of doublecortin expression in layer 2 and parasubiculum from dorsal to ventral; and the progressive appearance of the wolframin expression in superficial layer 2 of MEC and parasubiculum from dorsal to ventral. Homing behavior in rats, as well as spontaneous exploratory behavior develops around the end of second postnatal week (Wills et al., 2014; Bulut and Altman, 1974) while spontaneous exploration of larger environments outside the nest emerge towards the end of the third postnatal week (Wills et al., 2014). This is coincident with the timeline of maturation of calbindin+ patches in the dorsal and ventral MEC respectively. Since the dorsal MEC represents smaller spatial scales and the ventral MEC progressively larger scales (Hafting et al., 2005; Stensola et al., 2012), these data may indicate that the rat’s navigational system matures from small to large scales. Early eyelid opening experiments have indicated an accelerated development of spatial exploratory behaviour (Kenny and Turkewitz, 1986; Foreman and Altaha, 1991), and similar experiments might provide insights into whether early behavioral development is accompanied by an accelerated development of the microcircuit underlying spatial navigation.

The higher co-localization of doublecortin with calbindin+ pyramidal cells than reelin+ stellate cells, supports further the dichotomy of structure-function relationships exhibited by these two cell types (Ray et al., 2014; Tang et al., 2014). Grid and border cells have been implicated to be largely specific to pyramidal and stellate cells respectively (Tang et al., 2014)and the delayed structural maturation of pyramidal cells might reflect the delayed functional maturation of grid cells (Wills et al., 2010; Langston et al., 2010), with the converse being applicable to stellate and border cells (Bjerknes et al., 2014). The divergent projection patterns of pyramidal and stellate cells, with the former projecting to CA1 (Kitamura et al., 2014) and contralateral MEC (Varga et al., 2010) and the latter to dentate gyrus (Varga et al., 2010; Ray et al., 2014) and deep layers of MEC (Sürmeli et al., 2015), have differing theoretical interpretations in spatial information processing.

The same sets of neurons, which correspond to grid and border cells (Tang et al., 2014), have also been implicated to be differentially involved in temporal association memory (Kitamura et al., 2014) and contextual memory (Kitamura et al., 2015) respectively. An underlying differential structural maturation timeline of the microcircuit governing these processes may also translate into a differential functional maturation profile of these memories.

We conclude that the structural maturation of medial entorhinal cortex can be coarsely divided into an early appearance of the calbindin+ neuron patches and a progressive cell-type specific refinement of the cellular structure, which proceeds along the dorsal to ventral axis.

Materials and methods

All experimental procedures were performed according to the German guidelines on animal welfare under the supervision of local ethics committees (LaGeSo) under the permit T0106-14.

Brain tissue preparation

Male and female Wistar rats (n=83) from E18 to P24 and adults (>P42) were used in the study. The ages were accurate to ± 1 day. Animals were anaesthetized by isoflurane, and then euthanized by an intraperitoneal injection of 20% urethane. They were then perfused transcardially with first 0.9% phosphate buffered saline solution, followed by 4% formaldehyde, from paraformaldehyde, in 0.1 M phosphate buffer (PFA). For prenatal animals, pregnant rats at E18 were perfused in the aforesaid manner and the E18 animals were then extracted from the uterus. Subsequently, brains were removed from the skull and postfixed in PFA overnight. Brains were then transferred to 10% sucrose solution for one night and subsequently immersed in 30% sucrose solution for at least one night for cryoprotection. The brains were embedded in Jung Tissue Freezing Medium (Leica Microsystems Nussloch, Germany), and subsequently mounted on the freezing microtome (Leica 2035 Biocut) to obtain 60 μm thick sagittal sections or tangential sections parallel to the pia.

Tangential sections of the medial entorhinal cortex were obtained by separating the entorhinal cortex from the remaining hemisphere by a cut parallel to the surface of the medial entorhinal cortex (Video 1). For subsequent sectioning the surface of the entorhinal cortex was attached to the block face of the microtome.

Histochemistry and immunohistochemistry

Acetylcholinesterase (AChE) activity was visualized according to previously published procedures (Ichinohe et al., 2008; Tsuji, 1998). After washing brain sections in a solution containing 1 ml of 0.1 M citrate buffer (pH 6.2) and 9 ml 0.9% NaCl saline solution (CS), sections were incubated with CS containing 3 mM CuSO4, 0.5 mMK3Fe(CN)6, and 1.8 mM acetylthiocholine iodide for 30 min. After rinsing in PB, reaction products were visualized by incubating the sections in PB containing 0.05% 3,3’- Diaminobenzidine (DAB) and 0.03% nickel ammonium sulfate.

Immunohistochemical stainings were performed according to standard procedures. Briefly, brain sections were pre-incubated in a blocking solution containing 0.1 M PBS, 2% Bovine Serum Albumin (BSA) and 0.5% Triton X-100 (PBS-X) for an hour at room temperature (RT). Following this, primary antibodies were diluted in a solution containing PBS-X and 1% BSA. Primary antibodies against the calcium binding protein Calbindin (Swant: CB300, CB 38; 1:5000), the extracellular matrix protein Reelin (Millipore: MAB5364; 1:1000), the transmembrane protein Wolframin (Proteintech: 11558-1-AP; 1:200), the microtubule associated protein Doublecortin (Santa Cruz Biotechnology: sc-8086; 1:200) and the calmodulin binding protein Purkinje cell protein 4 (Sigma: HPA005792; 1:200) were used. Incubations with primary antibodies were allowed to proceed for at least 24 hr under mild shaking at 4°C in free-floating sections. Incubations with primary antibodies were followed by detection with secondary antibodies coupled to different fluorophores (Alexa 488, 546 and 633; Invitrogen). Secondary antibodies were diluted (1:500) in PBS-X and the reaction allowed to proceed for two hours in the dark at RT. For multiple antibody labeling, antibodies raised in different host species were used. For visualizing cell nuclei, sections were counterstained with DAPI (Molecular Probes: R37606). After the staining procedure, sections were mounted on gelatin coated glass slides with Vectashield mounting medium (Vectorlabs: H-1000).

Image acquisition

An Olympus BX51 microscope (Olympus, Shinjuku Tokyo, Japan) equipped with a motorized stage (LUDL Electronics, Hawthorne NY) and a z-encoder (Heidenhain, Shaumburg IL, USA), was used for bright field microscopy. Images were captured using a MBF CX9000 (Optronics, Goleta CA) camera using Neurolucida or StereoInvestigator (MBF Bioscience, Williston VT, USA). A Leica DM5500B epifluorescence microscope with a Leica DFC345 FX camera (Leica Microsystems, Mannheim, Germany) was used to image the immunofluorescent sections. Alexa fluorophores were excited using the appropriate filters (Alexa 350 – A4, Alexa 488 – L5, Alexa 546 – N3, Alexa 633 – Y5). Fluorescent images were acquired in monochrome, and color maps were applied to the images post acquisition. Post hoc linear brightness and contrast adjustment were applied uniformly to the image under analysis.

Analysis of layer width

To determine the width of different layers of the medial entorhinal cortex, we prepared parasagittal sections and stained them for calbindin-immunoreactivity, Purkinje cell protein-immunoreactivity and DAPI. Measurements were taken from dorsal, medial and ventral parts of each section analyzed using Leica Application Suite AF (Leica Microsystems, Mannheim, Germany).

Analysis of spatial periodicity

To determine the spatial periodicity of calbindin+ patches, we determined spatial autocorrelations. The spatial autocorrelogram was based on Pearson’s product moment correlation coefficient (Sargolini et al., 2006).

where, is the autocorrelation between pixels or bins with spatial offset τand τ is the monochromatic image without smoothing, n is the number of overlapping pixels. Autocorrelations were not estimated for lags of τand τ where n<20. Grid scores were calculated, as previously described (Ray et al., 2014), and can vary from −2 to 2.

Analysis of spatial overlap

To determine the degree of overlap between doublecortin and calbindin or reelin, we determined spatial crosscorrelations. Spatial crosscorrelations were determined based on Pearson’s product moment correlation coefficient.

where, is the cross-correlation between the monochromatic images f1 and f2 without smoothing. n is the number of pixels in the image. The Pearson’s cross-correlation coefficient can vary from -1 (anti-correlated) through 0 (un-correlated) to 1 (correlated).

For analysis of dorso-ventral variation in overlap between doublecortin with calbindin, two regions of the same size were selected from a section double-stained for calbindin and doublecortin. One region was selected from the dorsal half of the section and another from the ventral half and the regions were represented as pairs. Where, due to section damage, it was not possible to obtain regions from both dorsal and ventral parts, the data was presented as unpaired.

For analysis of variation in overlap between doublecortin and calbindin/reelin, comparisons were performed between the same regions from a section triple stained for calbindin, reelin and doublecortin.

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Structural Development and Dorsoventral Maturation of the Medial Entorhinal Cortex" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Howard Eichenbaum as the Reviewing Editor and Eve Marder as the Senior Editor.

The following individuals involved in review of your submission have agreed to reveal their identity: Thomas Van Groen, Andre Fenton, and Rosamund Langston (peer reviewers).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This paper provides important new insights into the developmental circuitry of the medial entorhinal and parasubiculum circuitry, of high importance to considerable current research on the functional organization of these brain areas.

Essential revisions:

The reviewers had several general concerns about explanation of the findings and many recommendations for improvement of the data presentation. These are detailed below.

Reviewer #1:

The manuscript by Ray and Brecht is quite interesting and relatively well written. The Introduction, Methods and Discussion are fine, even if the Discussion is a bit short. The Methods are good, but could be more detailed. The Results, however, need to be substantially revised or improved. One minor issue is that "superficial layers" need to be defined, even if most people know those are layers I to III, another issue are the tangential sections, that should be more clearly explained in the text. If a cortical area changes in size, by definition neuronal density decreases, this needs to be more clearly stated and discussed. The biggest issue is the presentation of the figures in the Results. Figure 1 shows changes in the size of the superficial layers, but also includes the deep layers. Figure 2: the autocorrelation images need to be the same size as the insets of the immunohistochemical staining images to be acceptable and interpretable. The figure orientation is needed in Figure 2A; what is where? For instance, what is the band of bright staining on the right side of the image? The acetylcholinesterase activity staining is of low quality (at least in the images provided). Similarly, in Figure 3, images showing the sections at equal magnification would be helpful (orientation?). Figure 5 is of too low quality to be interpretable, doublecortin is supposed to stain neurons, which is not very visible in this figure. Taken together, this will make the data more understandable for readers; in the current version the images are not exceedingly helpful.

Reviewer #2:

General assessment:

This is a clear and valuable study, describing the anatomical and developmental expression of calbindin and other molecular markers of pyramidal cells in the MEC and parasubiculum across developmental time from late gestation to young adulthood. These cells are important elements of the microcircuitry that generates spatially-tuned discharge. Whether the histochemically identified Calbindin+ cells correspond to functionally defined grid cells and/or border cells is controversial, which makes the present study an important contribution to the debate. The debate is crucial to notions of how the hippocampal-entorhinal system generates and computes information about space from the different functional components that have been identified such as distances, locations, directions, borders and speed. The development of the functional cell classes has been studied in recent years to constrain the theories, but which functionally-identified cells correspond to which histochemical and morphological cell classes is still unclear and clarification of this issue will provide important knowledge of the circuit wiring diagram that will drive theories of neural computation that are grounded in structure-function relationships. In summary, this manuscript is an important contribution.

Summary of concerns:

The manuscript is clearly written and the studies appear to be done carefully, and the procedures are straightforward. The data look clear and the interpretations of the measurements are not controversial. There are however a number of improvements I will suggest that will make the report more accessible to the general readership of eLife.

1) The analyses and interpretation of the histochemical images depend strongly on knowledge of the anatomical topography of the region, which non-specialists will not be. Whether the sections are tangential or parasaggital, and knowledge of the precise cutting angles is important even for the specialist. Consequently, it would be valuable to provide a 3-D model of the brain or just the cortical region with indications of the tangential and parasaggital planes and to use these on the Figures as a short-hand to help orient the reader. I know this is asking too much, but I will mention it to make my point: a 3-D CLARITY image of the immunolabeled cells would go a long way towards showing this very cool grid-like organization to the non-specialist and specialist alike. A 3-D model could accomplish the same.

2) I was disappointed not to see analysis of the stellate ocean cells, the coexistence of which in the region, but outside the pyramidal patches, is the source of the controversy. The authors should explain in the manuscript why parallel analyses were not performed.

3) I like the Discussion, which is appropriately driven by the findings. Again, for the general readership of eLife, I suggest the Discussion be expanded to more explicitly include the differential hippocampal and neocortical connectivity of the stellate and pyramidal cells, a discussion of what is known about their function properties, and why it is important to understanding how information about space is computed.

Reviewer #3:

Although structural maturation has been shown for the hippocampus this was lacking in the literature for entorhinal cortex despite the huge recent interest in the spatially selective cell types found there, so this paper is timely and of high theoretical importance.

The Introduction focuses on Layer II of MEC and is very brief – a little more attention to the cell types of Layer III and PaS and their electrophysiological characteristics, and what makes them different to the Layer II neurons would be nice since quite a lot of the results focus on these and not on layer II.

Also how does one identify stellate neurons and their developmental profile, and why is this not done? Also why are deeper layers of MEC not analysed? These are not suggestions for more experiments, just requests to explain briefly why they were not included here.

A bit more information about wolframin would be good – the authors initially state it is co-localised with calbindin but from their results this is not always the case?

The figures are mainly well explained, and the layout of the individual images, with the data presented graphically, is intuitive and aesthetically pleasing. The images clearly depict what is described and the mean data that is shown in the graphs.

Figure titles and/or images should include whether they depict MEC or PaS (e.g. not clear from Figure 5 where it is referred to in the text as being MEC layer II AND PaS as far as I read.)

Discuss relevance of wolframin vs. calbindin and why they appear separately in PaS but are co-localised in pyramidal cells of MEC layer 2.

In the Discussion it is a very interesting theory that the dorso-ventral structural development profile may reflect the maturation of the range of the spatial navigation system however this would be quite difficult to study since coincident with this is the fact that rats explore further after their eyes open at the end of the second postnatal week and it would be difficult to manipulate this behaviour to occur earlier, although similar things have been achieved with experiments to open rat pups' eyelids earlier.

The following theory suggested in the fourth paragraph of the Discussion that the patchy doublecortin co-localised with calbindin may reflect different functional maturation of border and grid cells requires a little more explanation, I did not see any obvious link.

Overall the paper provides much-needed high-quality solid anatomical evidence for dorso-ventral structural maturation occurring in the MEC and PaS, similar to that seen in the hippocampus. This data should feed into various models of neuronal spatial representations and presents the possibility that subsets of grid cells may mature before others so maps could potentially be formed of smaller spaces before larger ones, and a full complement of grid cells may not be necessary to see grid-like firing, a hypothesis which is testable and intriguing.

Essential revisions:

The reviewers had several general concerns about explanation of the findings and many recommendations for improvement of the data presentation. These are detailed below. Reviewer #1:

The manuscript by Ray and Brecht is quite interesting and relatively well written. The Introduction, Methods and Discussion are fine, even if the Discussion is a bit short. The Methods are good, but could be more detailed.

We thank the reviewer for appreciating the manuscript. We agree with the reviewer’s assessment about the brevity of certain sections of the manuscript and have performed a significant revision to all sections of the manuscript to result in a more detailed and comprehensive revised manuscript.

The Results, however, need to be substantially revised or improved. One minor issue is that "superficial layers" need to be defined, even if most people know those are layers I to III, another issue are the tangential sections, that should be more clearly explained in the text.

We agree with the reviewer’s assessment and have implemented their suggestion.

Changes:

Results, first paragraph – We have addressed the definition of superficial layer.

Video 1 – We have added a novel schematic video that illustrates the tangential sectioning process and provides the reader with a clear perspective of how the tangential sections are obtained from the rodent brain, and how the structures seen in tangential sections relate to the brain in situ.

If a cortical area changes in size, by definition neuronal density decreases, this needs to be more clearly stated and discussed. The biggest issue is the presentation of the figures in the Results.

We agree with this criticism and thank the reviewer for the suggestion to include variation in depth in deep layers of the MEC with increasing age.

Changes:

Discussion, fourth paragraph – We discuss the effect of increasing brain size on neuronal density.

Figure 1D and Results, first paragraph – We have updated Figure 1D and the Results to include changes in size in the deep layers of MEC.

We thank the reviewer for pointing out this oversight.

Changes:

Figure 2A-F – We have ensured that the inset of the immunohistochemical stain and the autocorrelation images are of the same size. The scale bar in the autocorrelation image is half the length of the one in the immunohistochemical image, since the autocorrelation denotes twice the length and breadth of the immunohistochemical image.

Figure legends (all relevant figures) – We have denoted the orientation in one panel of every figure, and now we state in the figure legends that it applies to all the other panels unless explicitly noted.

Figure legend (Figure 2) – We mention in the figure legend that the bright green band pointed out by the reviewer denotes the parasubiculum.

The acetylcholinesterase activity staining is of low quality (at least in the images provided). Similarly, in

We agree with the reviewer’s criticism and have changed the layout of the figure to address the issue.

Changes:

Figure 2G-1 – We have changed the layout of the figure to enable better visualization and co-localization of acetylcholinesterase activity and calbindin immunoreactivity.

Figure legends (all relevant figures) – In Figure 3 and elsewhere, as noted above, we have updated the legend to state that the orientation illustrated in one panel applies to the entire figure unless explicitly stated.

We agree with the referee’s criticism, about the need for a higher magnification image to visualize doublecortin localization in a single neuron. However, since other reviewers found the layout of these images quite intuitive, we decided not to change Figure 5 (now Figure 6) and instead added a novel Figure 7 to address this issue.

Changes:

Figure 7 – We have now added a novel Figure 7, as a result of our new experiments, which shows at higher magnification the co-localization of doublecortin in the cell membrane of the cells, with calbindin.

This figure also illustrates reelin which mark stellate cells in layer 2 of MEC, in response to queries raised by the other reviewers and quantifies the differential co-localization of doublecortin with calbindin and reelin during early postnatal development.

Reviewer #2:

Summary of concerns: The manuscript is clearly written and the studies appear to be done carefully, and the procedures are straightforward. The data look clear and the interpretations of the measurements are not controversial. There are however a number of improvements I will suggest that will make the report more accessible to the general readership of eLife. 1) The analyses and interpretation of the histochemical images depend strongly on knowledge of the anatomical topography of the region, which non-specialists will not be. Whether the sections are tangential or parasaggital, and knowledge of the precise cutting angles is important even for the specialist. Consequently, it would be valuable to provide a 3-D model of the brain or just the cortical region with indications of the tangential and parasaggital planes and to use these on the Figures as a short-hand to help orient the reader. I know this is asking too much, but I will mention it to make my point: a 3-D CLARITY image of the immunolabeled cells would go a long way towards showing this very cool grid-like organization to the non-specialist and specialist alike. A 3-D model could accomplish the same. We thank the reviewer for the idea of a 3D model, and have included a novel schematic video (Video 1) to address these concerns.

Changes:

Video 1 – We have added this video, which illustrates the tangential sectioning process and provides the reader with a clear perspective of how the tangential sections are obtained from the rodent brain, and how the grid-like structures seen in tangential sections relate to the brain in situ.

2) I was disappointed not to see analysis of the stellate ocean cells, the coexistence of which in the region, but outside the pyramidal patches, is the source of the controversy. The authors should explain in the manuscript why parallel analyses were not performed. This comment is similar to the criticism of another reviewer and we think the omission of stellate cell development was a major flaw in the previous version of our manuscript. We have performed a series of experiments to address this concern, and now provide a detailed perspective of the development of stellate cells.

Changes:

Figure 2—figure supplement 1 – New figure, illustrating the spatial layout of reelin+ stellate cells during development.

Figure 7 – We have added this new figure, illustrating the co-localization of doublecortin with pyramidal but not stellate cells during early postnatal development.

We have also made appropriate changes in the Abstract, Results and Discussion sections of the manuscript to reflect these novel results.

In addition, we found an increasing expression of reelin in layer 3 neurons with development, which we have highlighted in new Figure 4 and new Figure 5—figure supplement 1.

3) I like the Discussion, which is appropriately driven by the findings. Again, for the general readership of eLife, I suggest the Discussion be expanded to more explicitly include the differential hippocampal and neocortical connectivity of the stellate and pyramidal cells, a discussion of what is known about their function properties, and why it is important to understanding how information about space is computed. We thank the reviewer for the appreciation and the idea to highlight the differential connectivity profiles of pyramidal and stellate cells and its impact in spatial information processing.

Changes:

Discussion, seventh paragraph – Discussion on different projection patterns of pyramidal and stellate cells.

Reviewer #3:

Although structural maturation has been shown for the hippocampus this was lacking in the literature for entorhinal cortex despite the huge recent interest in the spatially selective cell types found there, so this paper is timely and of high theoretical importance. The Introduction focuses on Layer II of MEC and is very brief – a little more attention to the cell types of Layer III and PaS and their electrophysiological characteristics, and what makes them different to the Layer II neurons would be nice since quite a lot of the results focus on these and not on layer II. We thank the reviewer for pointing out this oversight, and we have expanded the Introduction to include the characteristics of cells in layer 3 and parasubiculum in addition to layer 2.

Changes:

Introduction, fourth paragraph – We have included an introduction on layer 3 and the functional characteristics of the cells found there.

Introduction, fifth paragraph – We have included an introduction on parasubicuulum and the functional characteristics of the cells found there.

Also how does one identify stellate neurons and their developmental profile, and why is this not done? Also why are deeper layers of MEC not analysed? These are not suggestions for more experiments, just requests to explain briefly why they were not included here. This comment is similar to a query raised by another reviewer, and we have performed a series of novel experiments to explore the development of stellates by using the extracellular matrix protein Reelin. We have performed a series of experiments to address this concern, and now provide a detailed perspective of the development of stellate cells. For deep layers, we added the development of their thickness during development, congruent with the recommendations of another reviewer.

Changes:

Figure 2—figure supplement 1 – New figure, illustrating the spatial layout of reelin+ stellate cells during development.

Figure 7 – New figure, illustrating the co-localization of doublecortin with pyramidal but not stellate cells during early postnatal development.

Figure 1D; Results, first paragraph– We have updated Figure 1D and the Results to include changes in size in the deep layers of MEC.

We have also made appropriate changes in the Abstract, Results and Discussion sections of the manuscript to reflect these novel results.

In addition, we found an increasing expression of reelin in layer 3 neurons with development, which we have highlighted in new Figure 4 and new Figure 5—figure supplement 1.

A bit more information about wolframin would be good – the authors initially state it is co-localised with calbindin but from their results this is not always the case? We have now expanded our discussion on wolframin on its co-localization with calbindin in layer 2 of MEC but not the developed parasubiculum.

Changes:

Discussion, fifth paragraph – We expanded the Discussion to include how studies target specific cell-types using their molecular profiles, and how experiments towards understanding the specific roles of these proteins might provide insights in understanding the functional differences in these cells.

The figures are mainly well explained, and the layout of the individual images, with the data presented graphically, is intuitive and aesthetically pleasing. The images clearly depict what is described and the mean data that is shown in the graphs.

We thank the reviewer for the appreciation of the figures.

Figure titles and/or images should include whether they depict MEC or PaS (e.g. not clear from We thank the reviewer for pointing out this oversight, and have now corrected the figure title and legend.

Discuss relevance of wolframin vs. calbindin and why they appear separately in PaS but are co-localised in pyramidal cells of MEC layer 2.As noted above, we provide a more comprehensive discussion on wolframin and calbindin, and their possible functions.

Changes:

Discussion, fifth paragraph – Discussion on the roles of wolframin and calbindin.

In the Discussion it is a very interesting theory that the dorso-ventral structural development profile may reflect the maturation of the range of the spatial navigation system however this would be quite difficult to study since coincident with this is the fact that rats explore further after their eyes open at the end of the second postnatal week and it would be difficult to manipulate this behaviour to occur earlier, although similar things have been achieved with experiments to open rat pups' eyelids earlier. We thank the reviewer for finding the theory interesting, and have now discussed in greater detail how it can be tested by the early eyelid opening experiments, as pointed out by the reviewer.

Changes:

Discussion, sixth paragraph – Discussion on possible experiments to test dorso-ventral maturation hypothesis.

The following theory suggested in the fourth paragraph of the Discussion that the patchy doublecortin co-localised with calbindin may reflect different functional maturation of border and grid cells requires a little more explanation, I did not see any obvious link. We apologize for not having adequately clarified this point, and have now addressed this issue in greater detail by performing a series of experiments to simultaneously analyze the co-localization of the immature neuronal marker doublecortin, with pyramidal cell marker calbindin, and stellate cell marker reelin in layer 2 of MEC. We find a differential neuronal maturation profile, which indicates that the structural development of pyramidal and stellate cells, closely mirrors the differential functional maturation profiles of grid and border cells respectively.

Changes:

Figure 7 – New figure, illustrating the co-localization of doublecortin with pyramidal and not stellate cells during early postnatal development.

We have also made appropriate changes in the Abstract, Results and Discussion sections of the manuscript to reflect these novel results.

Overall the paper provides much-needed high-quality solid anatomical evidence for dorso-ventral structural maturation occurring in the MEC and PaS, similar to that seen in the hippocampus. This data should feed into various models of neuronal spatial representations and presents the possibility that subsets of grid cells may mature before others so maps could potentially be formed of smaller spaces before larger ones, and a full complement of grid cells may not be necessary to see grid-like firing, a hypothesis which is testable and intriguing.

We thank the reviewer for the comprehensive summary of our manuscript, which mirrors our enthusiasm.