In vivo two-photon imaging of the embryonic cortex reveals spontaneous ketamine-sensitive calcium activity

Prior to sensory experience spontaneous activity appears to play a fundamental role in the correct formation of prominent functional features of different cortical regions. The use of anaesthesia during pregnancy such as ketamine is largely considered to negatively affect neuronal development by interfering with synaptic transmission. Interestingly, the characteristics of spontaneous activity as well as the acute functional effects of maternal anaesthesia remain largely untested in the embryonic cortex in vivo. In the present work, we performed in vivo imaging of spontaneous calcium activity and cell motility in the marginal zone of the cortex of E14-15 embryos connected to the mother. We made use of a preparation where the blood circulation from the mother through the umbilical cord is preserved and fluctuations in intracellular calcium in the embryonic frontal cortex are acquired using two-photon imaging. We found that spontaneous transients were either sporadic or correlated in clusters of neuronal ensembles at this age. These events were not sensitive to maternal isoflurane anaesthesia but were strongly inhibited by acute in situ or maternal application of low concentration of the anaesthetic ketamine (a non-competitive antagonist of NMDA receptors). Moreover, simultaneous imaging of cell motility revealed a correlated strong sensitivity to ketamine. These results show that anaesthetic compounds can differ significantly in their impact on spontaneous early cortical activity as well as motility of cells in the marginal zone. The effects found in this study may be relevant in the etiology of heightened vulnerability to cerebral dysfunction associated with the use of ketamine during pregnancy.Peer reviewe

Geniculo-Cortical Projection Diversity Revealed within the Mouse Visual Thalamus.

The mouse dorsal lateral geniculate nucleus (dLGN) is an intermediary between retina and primary visual cortex (V1). Recent investigations are beginning to reveal regional complexity in mouse dLGN. Using local injections of retrograde tracers into V1 of adult and neonatal mice, we examined the developing organisation of geniculate projection columns: the population of dLGN-V1 projection neurons that converge in cortex. Serial sectioning of the dLGN enabled the distribution of labelled projection neurons to be reconstructed and collated within a common standardised space. This enabled us to determine: the organisation of cells within the dLGN-V1 projection columns; their internal organisation (topology); and their order relative to V1 (topography). Here, we report parameters of projection columns that are highly variable in young animals and refined in the adult, exhibiting profiles consistent with shell and core zones of the dLGN. Additionally, such profiles are disrupted in adult animals with reduced correlated spontaneous activity during development. Assessing the variability between groups with partial least squares regression suggests that 4-6 cryptic lamina may exist along the length of the projection column. Our findings further spotlight the diversity of the mouse dLGN--an increasingly important model system for understanding the pre-cortical organisation and processing of visual information. Furthermore, our approach of using standardised spaces and pooling information across many animals will enhance future functional studies of the dLGN.Funding was provided by a Wellcome Trust grant jointly awarded to IDT and SJE (083205, www.wellcome.ac.uk), and by MRC PhD Studentships awarded to MNL and ACH (http://www.mrc.ac.uk/).This is the final version of the article. It was first available from PLOS via http://dx.doi.org/10.1371/journal.pone.014484

Geniculo-cortical Projection Diversity Revealed within the Mouse Visual Thalamus Supplemental Data

Supplemental data for the PLOS One paper "Genuculo-cortical Projection Diversity Revealed within the Mouse Visual Thalamus" by Leiwe et al. <div><br></div><div>Files are compressed. Except for the raw data</div

Registration in Standard Space Protocol

<p>A protocol for the 3D registration of sectioned material into standardised space from the paper "Geniculo-cortical Projection Diversity Revealed within the Mouse Visual Thalamus" by Leiwe et al 2015</p

Form and Features of dLGN-V1 Projection Columns.

<p>(A-D) Location of V1 injections (mm relative to Lambda) and positions of the labelled column on the pial surface (scale bar 200μm) for four groups: Adult Wild Type (WT), n = 16; Adult β2^-/-, n = 9; P6 WT, n = 14, P12 WT, n = 13. Coordinates are relative to lambda. (E-G) Reconstructed paths of dLGN-V1 projection columns collapsed onto coronal (E), horizontal (F), and sagittal (G). Columns extend from the pial surface (⭕) to the ventral boundary of the dLGN (●). (H) Distribution of injection volumes in each group, Kruskal Wallis test, with Dunn’s Multiple Comparison Post Hoc Tests, *p<0.05, ** p<0.01. (I) Column lengths. (J) Number of cells per column. (I & J) One-way ANOVA with Bonferroni post-hoc tests: * p<0.05; ** p<0.01; *** p<0.001. (K) Number of cells per 5^th percentile of unit column. Data presented as mean ±SEM. Grey shading in this and subsequent figures indicate the shell core boundary at approximately 30% of the projection column. Two-way ANOVA with Bonferroni post-hoc tests relative to pial end: *** p<0.001. (L) Spread of cells from the centre of mass of each column per 5^th percentile. Data presented as mean ±SEM. Values are corrected for different sized dLGNs by scaling to the WT dLGN. (M) Cumulative histogram illustrating the numbers of cells of a given spread (see L). Kruskal Wallis test, with Dunn’s Multiple Comparison Post-hoc tests compared to WT: ** p<0.01. (N) Fitted Rayleigh distributions to cumulative data in M, normalised to the area under each curve.</p

Three dimensional reconstruction of dLGN-to-V1 projection columns.

<p>(A) Fluorescent <i>RetroBeads</i> were injected into V1 and transported into thalamo-cortical projection neurons within the dLGN. (B) Bright-field images of sections registered to 3D standardized space (MRI). Cross-hairs are colour-coded for each orthogonal view. Yellow dashed lines outline the boundary of the dLGN. (C) Reconstructed locations of labelled somas within the boundaries of the standardized dLGN space (mesh). Left inset: summarised trajectory of the dLGN projection column that extends from the pial surface (⭕) to the ventral boundary of the dLGN (●). Right inset: spread of cells along normalised columns (based on boundary positions: ⭕->●). Scale bars are 250μm.</p

Dynamic Topological and Topographical Order in Geniculo-Cortical Projection Columns.

<p>Generating maps of dLGN columns for each 5^th percentile along its length enables estimations of organisation and order. (A) Example of a best-fit plane (normal vector: blue-line) for WT projection columns at 10^th percentile (0.1) position. Inset: collapsed view orthogonal to the plane–end on normal vector. Scale bar is 100μm. (B) Example of registration (expansion—E; rotation - θ) between neighbouring (0.1 and 0.05) WT planes. (C) Cumulative rotation and (D) expansion of each 5^th percentile map aligned to the pial plane (0). (E) Internal topological order within each group compared to the preceding 5^th percentile position. Note, a normalised P_t of 1 represents complete disorder while 0 is perfect order. (F) Degree of topological order between the pial and ventral maps of the dLGN. Monte Carlo permutation tests: * (p<0.05)—statistically significant order. (G) Schematic: correspondence of WT V1 injection sites to dLGN pial plane (0). (H) Degree of topographical order between V1 and the dLGN at each 5^th percentile. Monte Carlo permutation tests: * (p<0.05)—statistically significant order.</p

PLS Regression Suggests Complex Cryptic Organisation.

<p>(A) Normalised profiles (zero mean and unit variance) representing the numbers of cells; spread of cells and normalised P_t representing topological and topographical order for all groups (WT, P6, P12 and β2^-/-). Colour coding is arbitrary except that the β2^-/- group are dashed lines. (B) PLS regression; the degree of variance within A is increasingly explained by an increasing number of latents. Curves represent the explained variance for the combined WT, P6 and P12 groups (solid line) and all groups combined together (dashed). (C) Cross-validation error (Mean Squared Error—MSE) as a function of the number of latents retained in the model. WT, P6 and P12 groups (solid line) and all groups (dashed).</p