by a further 3 to 6 days [two-way analysis of
variance (ANOVA), neuronal subpopulation ×
Postnatal Day, including comparisons at every
segment along the MEC: F7,32 > 22.3, P < 0.0001].
The progression of maturation from dorsal to
ventral MEC was also observed in PV-expressing
interneurons in superficial and deep layers (direct
comparison between block I and more ventral
segments: Group × Segment: F7,32 > 14.2, P <
0.0001; fig. S5).
To investigate whether the instructive signal
that drives maturation through the transverse
circuit is also responsible for maturation of the
MEC, we silenced layer 2 cells in the MEC using
inhibitory DREADDs in the same manner as in
the transverse study. Silencing excitatory neu-
rons in MEC-L2 did not change DCX levels in
stellate cells at any dorsoventral level of the MEC
[Group (silenced versus control): F1,32 = 2.54, P =
0.81; Group × Segment: F7,32 = 0.42, P = 0.87; Fig.
3C, left]. In contrast, the maturation-related
Donato et al., Science 355, eaai8178 (2017) 17 March 2017 4 of 10
Fig. 3. Dorsoventral topographical maturation of MEC is driven by
excitatory activity in L2. (A) The fraction of DCX− neurons was determined at eight positions of equal dimension (each 375 mm in length) along
the dorsoventral axis of MEC (positions I to VIII) in a single sagittal section
(NeuN+ cells, red). Dorsal and ventral borders of MEC were identified as sharp
transitions in L2 cell density [position 0 corresponding to the MEC-postrhinal
cortex transition (MEC-POR); position 3000 corresponding to MEC-LEC tran-sition]. The length of segment VIII was adjusted for early postnatal sections
(P8 to P14) to account for change in brain size (therefore, the MEC-to-LEC
transition always fell into this block). The total number of NeuN+ cells in each
block did not change significantly between P5 and P90 (except for block VIII).
(B) Cumulative fractions of DCX− neurons in superficial and deep layers in
every segment along the dorsoventral MEC axis. Stellate and pyramidal cells
were distinguished on the basis of reelin and calbindin expression (>45,000
neurons from three animals per time point). (C) Fraction of DCX− excitatory
neurons and PV+ inhibitory neurons after silencing excitatory neurons selectively in MEC-L2 (green) or widely across retrohippocampal cortices (orange)
between P14 and P20. Controls were pooled as in Fig. 2 (>210,000 neurons
from at least three animals per treatment group). (D) Similar estimates of
DCX and PVexpression after silencing excitatory neurons in MEC-L2 (green)
or retrohippocampal cortices (orange) between P11 and P14 (>60,000 neurons
from at least three animals per treatment group).