Recent studies have demonstrated that radial glial cells are the major neuronal progenitors in the developing neocortex 13–17
. In addition to their well-characterized role in guiding the radial migration of postmitotic neurons 18
, radial glial cells divide asymmetrically to self-renew and to give rise to neurons. Consecutive asymmetric cell divisions of individual radial glial cells produce a number of clonally related neurons that migrate radially into the cortical plate. This results in a columnar arrangement of neocortical neurons – the ontogenetic radial clone 13, 14, 19, 20
. It has previously been suggested that “ontogenetic columns become the basic processing unit in the adult cortex” 19
; however, this assertion is based largely on their anatomical similarity, i.e. the vertical arrangement of neurons. In fact, virtually nothing is known about the functional development of ontogenetic radial clones in the neocortex.
To study the functional development of ontogenetic radial clones in the developing neocortex, we injected retroviruses expressing enhanced green fluorescence protein (EGFP) through the uterus into the ventricle of mouse embryos around 12 to13 days after conception (embryonic day 12 to 13, E12–E13) () – the period during which the peak phase of neocortical neurogenesis begins. After injection, the uterus was placed back to allow the embryos to come to term. Previous studies have shown that about half of the retroviral integration events occur in the self-renewing daughter cells 21
, resulting in the labelling of neocortical progenitor cells and their radially aligned clonal progeny 13, 20
. Similarly, we found that intraventricular injection of a low titre EGFP-expressing retrovirus at E12–E13 reliably labelled the radial clones of cells (), while additionally labelling some scattered cells (data not shown) 22, 23
, throughout the developing neocortex. In this study we focused our analysis on the isolated radial clones that consisted of sister cells ( and Supplementary Fig. S1
Morphological development of ontogenetic radial clones of excitatory neurons in the neocortex
In brain sections at various embryonic stages we frequently observed individual radial clones that comprised a radial glial mother cell (, red arrowhead) and a cluster of additional cells (~4–6 cells per clone at E18, , white arrowheads), all of which were in contact with the radial glial fibre of the mother radial glial cell. As development progressed into postnatal stages, the frequency of radial clones observed in individual brain sections decreased. Nonetheless, we often found isolated radial clones with two or more cells in individual sections that did not have any scattered cells in close proximity ( and Supplementary Fig. S1
), suggesting that the cells in these radial clones are lineage-related sisters. Nearly all of the cells in the labelled radial clones exhibited the morphological features of an excitatory neuron – a radially oriented pyramid-shaped cell body and a major apical process that progressively branched () and harboured dendritic spines (data not shown) as they developed.
We proceeded to perform whole-cell patch clamp recordings to assess the physiological development of these ontogenetic radial clones in the developing neocortex during postnatal stages (Supplementary Fig. S2
). As time progressed, the input resistance of neurons in the radial clone decreased (data not shown) and their resting membrane potential (RMP) became progressively more hyperpolarized (Supplementary Fig. S2 a and b
), indicating a maturation of membrane conductance. Furthermore, as development progressed, the threshold for firing action potential decreased drastically, whereas the maximum firing rate increased significantly (Supplementary Fig. S2 c and d
Synapse formation is a key step in the functional development of neurons in the brain. To assess synapses formed onto the excitatory neurons in ontogenetic radial clones, we examined the spontaneous miniature excitatory postsynaptic currents (mini-EPSCs) (Supplementary Fig. S2 e–k
). While the mean amplitude of mini-EPSCs detected at different developmental stages remained similar (Supplementary Fig. S2 e and f
), the frequency increased drastically as development progressed (Supplementary Fig. S2 e and g
). Moreover, the rise and decay of mini-EPSCs speeded up significantly with development (). These results suggest progressive formation and maturation of synapses onto the excitatory neurons in ontogenetic radial clones during postnatal development.
Synapse formation between sister excitatory neurons in ontogenetic radial clones
Having found that the neurons in ontogenetic radial clones actively form synapses, we set out to determine whether sister neurons in individual radial clones form synapses with each other. Simultaneous whole-cell recordings were performed on two EGFP-expressing sister neurons in individual radial clones, whose cell bodies were often more than 100 µm apart (). After the recordings were established, single as well as a train of action potentials were triggered in one of the neurons by current injection, while the other neuron was kept in voltage-clamp mode around −70 mV to record postsynaptic responses (). If the sister neurons are synaptically connected, action potentials triggered in the presynaptic neuron should evoke synaptic currents in the postsynaptic neuron. Indeed, we found that action potentials triggered in one neuron (neuron 1) reliably evoked postsynaptic currents in the other neuron (neuron 2, ), indicating that these two sister neurons in a radial clone are indeed connected. Moreover, we found that the connection between them was unidirectional, as action potentials triggered in neuron 2 failed to evoke detectable postsynaptic currents in neuron 1 (). Similar results were obtained when the postsynaptic neuron was kept in current-clamp mode (). The biophysical properties of these postsynaptic responses recorded between sister neurons in radial clones indicate that they are glutamate-receptor mediated EPSCs. This was corroborated by pharmacological experiments using picrotoxin, D-AP5, and NBQX () – the inhibitors of γ-aminobutyric acid (GABA)-A, N-methyl-D-aspartic acid (NMDA), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, respectively, as well as carbenoxolone and 18α-glycyrrhetinic acid – two commonly used blockers of gap junctions (Supplementary Fig. S3
Our results thus far suggest that sister excitatory neurons in individual radial clones in the developing neocortex develop unidirectional chemical synapses. We examined 101 pairs of sister neurons in individual radial clones at different developmental stages (). Whereas the rate of finding a connected pair of sister neurons in a radial clone was rather low around the first postnatal week (0 out of 34 at P1–5, and 2 out of 21 at P6–9), it increased dramatically around the second postnatal week (7 out of 20 at P10–13, and 9 out of 26 at P14–17), suggesting that the second postnatal week is a critical period for the development of synaptic connections between sister neurons in a radial clone. This time window coincides with the critical period of functional circuit development in the neocortex 24–26
A strong preference for synapse formation between sister excitatory neurons in ontogenetic radial clones
Previous studies suggested that the probability of detecting a synaptic connection between adjacent excitatory neurons in the mature neocortex is between 5% to 20% depending on the neocortical layer that they are located in and the neuronal subtype 4–7, 27
. Sister excitatory neurons in a radial clone in the developing neocortex are located quite far away from each other (a hundred to few hundred µm). Therefore, the likelihood of finding a synaptic connection between them would be far lower 5
. Nonetheless, we found that ~35% (16 out of 46) of sister excitatory neurons in a radial clone around P10 to P17 were connected, suggesting a propensity for radially aligned sister excitatory neurons to form synapses with each other.
To directly test this, we performed quadruple whole-cell recordings on two EGFP-expressing sister neurons in individual radial clones and two non-EGFP-expressing neurons adjacent to the EGFP-expressing sister neurons on the same side in the developing neocortex at P10–P17 ( and Supplementary Fig. S4
). We targeted non-EGFP-expressing neighbouring neurons with radially aligned pyramid-shaped cell bodies because they were most likely to be excitatory neurons and the progeny of different progenitor cells. Furthermore, the morphology and biophysical properties of these control neurons confirmed that they were excitatory neurons (). Hence, they served as adjacent non-sibling controls. Once all four recordings were established, action potentials were sequentially triggered in one of the four neurons and the postsynaptic responses were then measured in the other three neurons to probe synapses formed among them. As shown in , when action potentials were triggered in EGFP-expressing neuron 1, glutamate receptor-mediated EPSCs were recorded only in its sister neuron 3 (). And despite the nearly complete overlap of the dendritic arbours of adjacent neurons 3 and 4 (), no EPSC was detected in the non-sibling neuron 4 (). Furthermore, action potentials triggered in all three remaining neurons (neuron 2, 3 and 4) failed to evoke any detectable EPSCs in the other three neurons (). These results suggest that a unidirectional synaptic connection selectively develops between two sister excitatory neurons, but not between non-sisters, in the developing neocortex ().
We analyzed a total of 179 pairs of radially aligned EGFP-expressing sister excitatory neurons and their neighbouring non-sibling neurons (). Among them, 36.9% (65 out of 179) of sister neurons in a radial clone were connected. By contrast, only 6.3% (9 out of 143) of radially situated non-sister excitatory neurons (one EGFP-expressing and one non-EGFP-expressing) were connected (). These results clearly suggest that sister excitatory neurons in a radial clone have a strong preference to form synapses with each other rather than with adjacent non-sister neurons, and the rate of connectivity between distant, radially situated non-sister excitatory neurons is rather low. Consistent with this low rate of connectivity, only 5.5% (4 out 73) of any two randomly selected radially situated non-EGFP-expressing neurons over at least 100 µm apart were connected ().
Previous studies have revealed the overall organization of the excitatory neuron microcircuit in the mature neocortex 28, 29
. Thalamic input enters primarily into layer 4 (the first station of sensory processing). Layer 4 excitatory neurons send ascending projections to pyramidal neurons in layer 2/3 (the second station of columnar processing), which provide a prevalent descending projection to layer 5/6 pyramidal neurons (the third station of columnar processing). Descending and ascending excitatory connections also exist between layer 4 and layer 5/6. In addition to the sister neurons radially situated in layer 2/3 and layer 5/6 (), our dataset of quadruple recordings contained radially aligned sister neurons located in layer 4 and layer 5/6 (, and Supplementary Fig. S5a
) as well as in layer 2/3 and layer 4 (, and Supplementary Fig. S5b
). These experiments allowed us to address the interlaminar direction preference of synaptic connectivity formed within ontogenetic radial clones of excitatory neurons. We found that 15 out of 21 connected sister excitatory neuron pairs located in layer 2/3 and layer 5/6, and 10 out of 14 pairs of those located in layer 4 and layer 5/6 formed synapses in the descending direction (i.e. from layer 2/3 to layer 5/6 and from layer 4 to layer 5/6), whereas 15 out of 19 pairs of connected sister excitatory neurons located in layer 2/3 and layer 4 formed synapses in the ascending direction (i.e. from layer 4 to layer 2/3, ). These results suggest that the synaptic connection formed among sister excitatory neurons in ontogenetic radial clones is rather specific. Moreover, these results demonstrate that the specificity of synaptic connectivity formed within ontogenetic radial clones of excitatory neurons in the developing neocortex is highly similar to that in the mature neocortex.
A highly specific microcircuit forms among sister excitatory neurons in ontogenetic radial clones
The concept of the column has cast a dominant influence on our understanding of the functional organization of the neocortex 1, 2
. From its inception, the concept of the functional column has been considered on both a macroscopic as well as a microscopic scale. However, most of our knowledge about functional columns and neocortical maps comes from measurements with limited spatial resolution. Recent in vivo
imaging studies elegantly demonstrated that even adjacent neurons can have distinct physiological properties 9–11
, indicating that neocortical maps are built with single-neuron precision. In this study, we found that sister excitatory neurons in individual radial clones in the developing mouse neocortex preferentially develop highly specific synaptic connections with each other, creating radial columnar microarchitectures of interconnected neuron ensembles with single-neuron resolution. The high degree of similarity in the direction of interlaminar connectivity between the synapses formed within individual ontogenetic radial clones and those observed in the mature neocortex suggests that these radial clones contribute to the formation of precise functional columnar architectures in the neocortex. Along this line, variations in the neurogenesis and emergence of ontogenetic radial clones during early neocortical development 30
may underlie differences that have been observed in the functional organization of the mature neocortex across species 10