When periphery matters: Enhanced reactivity of 8-oxa-1,4-dithiaspiro[4.5]decane-7,9-dione and 9-oxa-1,5-dithiaspiro[5.5]undecane-8,10-dione in the Castagnoli-Cushman reaction with imines

Article abstract of DOI:10.1016/j.tetlet.2020.152658

8-Oxa-1,4-dithiaspiro[4.5]decane-7,9-dione and 9-oxa-1,5-dithiaspiro[5.5]undecane-8,10-dione, cyclic anhydrides formed in situ from the respective dicarboxylic acids in the presence of acetic anhydride, possess an enhanced reactivity and display a broad substrate scope in the Castagnoli-Cushman reaction with imines.

Full text of DOI:10.1016/j.tetlet.2020.152658

Neuron
Article
Microcircuits of Functionally Identified Neurons
in the Rat Medial Entorhinal Cortex
Andrea Burgalossi, * Lucas Herfst, Moritz von Heimendahl, Henning F o¨ rste, Kurt Haskic, Martin Schmidt,2
1
,
1,3
1
2,4
2
1
,
and Michael Brecht *
1
Bernstein Center for Computational Neuroscience, Humboldt University of Berlin, Philippstr. 13 Haus 6, 10115 Berlin, Germany
2Institut f u¨ r Konstruktion, Mikro- und Medizintechnik, Fachgebiet Mikro- und Feinger a¨ te, Technische Universit a¨ t Berlin, Hardenbergstr. 36,
Sekretariat EW1-2,10623 Berlin, Germany
3Present address: Department of Urology, Maastricht University Medical Centre, POB 5800, P. Debeyelaan 25, 6202 AZ Maastricht,
The Netherlands
4Present address: Optris GmbH, Ferdinand-Buisson-Str. 14, 13127 Berlin, Germany
*Correspondence: andrea.burgalossi@bccn-berlin.de (A.B.), michael.brecht@bccn-berlin.de (M.B.)
DOI 10.1016/j.neuron.2011.04.003
SUMMARY
2008) have been described. Second, the large-scale anatomy
of the hippocampal and parahippocampal regions is well
Extracellular recordings have elucidated spatial described (van Strien et al., 2009; Suzuki and Amaral, 2004).
neural representations without identifying underlying Superficial entorhinal layers project to the hippocampal forma-
tion, whereas deep layers receive hippocampal feedback (van
Strien et al., 2009). Neuronal morphologies of entorhinal cortex
microcircuits. We labeled neurons juxtacellularly in
medial entorhinal cortex of freely moving rats with a
friction-based, pipette-stabilizationsystem. Inalinear
maze novel to the animals, spatial firing of superficial
layer neurons was reminiscent of grid cell activity.
Layer 2 stellate cells showed stronger theta modula-
have been carefully characterized (Lingenh o¨ hl and Finch, 1991;
Klink and Alonso, 1997; Witter and Amaral, 2004; Quilichini
et al., 2010). The architecture of medial entorhinal cortex is char-
acterized by clusters of neurons in cytochrome oxidase-rich
patches in layer 2 (Klingler 1948; Hevner and Wong-Riley,
tion than layer 3 neurons, and both fired during the
ascending phase of field potential theta. Deep-layer
1992; Solodkin and Van Hoesen, 1996). Third, the cognitive
map theory is a powerful conceptual framework relating spatial
neurons showed little or no activity. Layer 2 stellate cognition to the hippocampus (O’Keefe and Nadel, 1978) and
cells resided in hundreds of small patches. At the parahippocampal regions (O’Keefe and Burgess, 2005).
dorsomedial entorhinal border, we identified larger
putative parasubicular) patches, which contained
Medial entorhinal cortex is a major input-output structure of
the hippocampus (Burwell 2000; Suzuki and Amaral, 2004).
The coexistence of grid, head-direction, and border cells sug-
gested that the entorhinal network might be able to integrate
these signals to compute an updated metric representation of
position in space (Sargolini et al., 2006; Witter and Moser,
(
polarized head-direction selective neurons firing
during the descending theta phase. Three axon
systems interconnected patches: centrifugal axons
from superficial cells to single large patches, centri-
petal axons from large-patch cells to single small
patches, and circumcurrent axons interconnecting
large patches. Our microcircuit analysis during
2006; Moser and Moser, 2008; Derdikman and Moser, 2010).
Despite the key role of medial entorhinal cortex in rodent
spatial cognition, we still lack a mechanistic understanding of
how individual neurons contribute to spatial representations.
behavior reveals modularity of entorhinal processing. Entorhinal microcircuits remained poorly defined because extra-
cellular recordings fail to identify the recorded neuronal elements
INTRODUCTION
(Chorev et al., 2009). We therefore developed a method for
recording and labeling neurons in awake behaving animals
Understanding how cognitive functions map onto neural circuits and studied the anatomical properties of functionally identified
is a fundamental goal of neuroscience. For most cognitive oper- neurons in relation to the patchy architecture of medial entorhinal
ations this goal is not within reach, but in rodent spatial cognition cortex. Our data show the existence of well-defined functional
there have been three impressive advances. First, physiological microcircuits, characterized by selective axonal interconnec-
studies on hippocampal and parahippocampal neurons have tions between cortical patches.
revealed rich and abstract representations of space. In partic-
ular, earlier studies identified place cells in the hippocampus RESULTS
(
O’Keefe and Dostrovsky, 1971) and head-direction cells in
the anterior thalamus (Taube and Muller, 1998) and the presubic- Recording and Identification of Neurons in Freely
ulum (Taube et al., 1990a, 1990b; for a review, see Taube, 2007). Moving Animals
Moreover, in the medial entorhinal cortex, grid cells with tessel- We aimed at identifying microcircuits associated with spatial
lating spatial discharges (Hafting et al., 2005), head-direction representations in medial entorhinal cortex. Head-anchored
cells (Sargolini et al., 2006), and border cells (Solstad et al., whole-cell recordings (Lee et al., 2006, 2009; Epsztein et al.,
Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc. 773

Neuron
Entorhinal Microcircuits
Pipette Holder
A
B
Signaling Wire
Glass Rod
Pipette
Pipette Guide
Titanium Nitride
Magnesium
Acrylic Base
Acrylic Seal
Agarose
F
~
1 N
Plastic Ring
f
Bone
2
9.0 mm
Neuron
Brain
Figure 1. A Friction-Based Pipette-Positioning Device
A) The top panel shows a disassembled pipette-positioning device. The pipette guide is implanted on the head. Bottom left is a view of an assembled pipette-
(
positioning device; bottom right is a schematic of the friction interface between pipette holder (magnesium) and pipette guide coating (titanium nitride). The static
friction force is about 1 N.
(B) The left view shows an implanted pipette-positioning device with the manipulator-driven glass rod that advanced the construct; right view shows how after
staining the recorded neuron, the glass rod is pulled up, and the recording configuration is further stabilized by an acrylic seal and addition of water to the friction
interface (not shown) to increase the static friction.
2
010) can in principle achieve this goal, but low success rates with functional neuronal activity (Wong-Riley, 1989; Wong-Riley
make it difficult to recover neurons in sufficient numbers. We et al., 1998). In medial entorhinal cortex, histochemical staining
addressed this issue by a new method for recording and labeling for cytochrome oxidase activity revealed two types of patches:
neurons juxtacellularly (Pinault, 1994, 1996). A head-mountable, ‘‘small’’ patches, which were restricted to layer 2; and ‘‘large’’
friction-based device held the pipette very rigidly, protecting the patches (Figures 2A and 2B) at the dorsal and medial borders
recording against mechanical disturbances (Figures 1A and 1B). of medial entorhinal cortex.
We stabilized recordings by head anchoring the pipette with
As pointed out in detail in the Discussion, the large patches at
acrylic and applying water to the friction interface (Figure 1B). the medial border of medial entorhinal cortex correspond to what
We worked with untrained animals that were initially anesthetized has been classically referred to as the superficial layers of the
during staining and stabilization and then received an antidote parasubiculum (Caballero-Bleda and Witter, 1993). The large
against the anesthetic (Lee et al., 2006). Animals typically woke patches at the dorsal border of medial entorhinal cortex,
uprelativelyabruptlyabout2–3minafteradministrationoftheanti- however, seem to not have been fully identified in previous
dote and explored the maze (average distance traveled = 513 ± studies (Witter and Amaral, 2004; Boccara et al., 2010). Since
4
62 cm; see Figures S1A and S1C available online). Because of the medial and dorsal large patches are continuous and
the lack of training and perhaps also due to the wake-up proce- cytoarchitectonically similar, we consider them to be one—puta-
dures, animals sometimes showed only limited spatial explora- tively parasubicular—structure and refer to them as large
tion. We therefore chose to evaluate spatial firing properties not patches.
inanopenfieldbut insteadina linear‘‘O’’ maze, where wetypically Often but not always the large patches could be divided in two
obtained good spatial coverage of the maze (average turns = 3.9 ± vertically split subpatches (Figures 2A and 2B). Quantification of
2
.7). A fraction of freely moving juxtacellular recordings (ꢀ30%) cytochrome oxidase activity levels revealed a clear periodicity of
was terminated deliberately to improve the rate and quality of patches (Figures S2A and S2B), which were visible along the
the cell recovery (average recording length = 330 ± 316 s; see entire mediolateral extent of medial entorhinal cortex (Figures
Supplemental Experimental Procedures and Figure S1B). These S2C–S2E).
procedures allowed us to record spiking activity from 46 identified
To further characterize the organization of medial entorhinal
neurons in medial entorhinal cortex (see representative spike cortex, we stained alternating parasagittal, horizontal, or tangen-
waveforms in Figure S1D), in 39 of which axons were visualized tial sections for cytochrome oxidase activity, Nissl, and myelin.
and traced for distances of up to 6 mm from the soma. In most Differences in cell size, density, soma morphology, and cyto-
recordings (65%, see Supplemental Experimental Procedures), chrome oxidase activity confirmed the existence of the two types
animals sampled each location more than twice. In order to be of patches (Figures 2A–2C). Areas of higher cell density in layer 2,
able to judge the spatial consistency of neural activity, we as visualized by Nissl staining, coincided with the patches
restricted the assessment of spatial modulation and head-direc- identified by cytochrome oxidase activity staining (Figure 2C),
tion selectivity tothis subset ofrecordings fromidentified neurons. and the patchy organization was typically more obvious in cyto-
chrome oxidase than in Nissl stains.
Large and Small Patches in Medial Entorhinal Cortex
Large patches showed strong cytochrome oxidase reactivity,
Staining for cytochrome oxidase activity has revealed clear probably reflecting a constitutively high metabolic activity. They
anatomical patterns across several brain areas, which correlated differed strikingly from the surrounding cortical sheet and
774 Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc.

Neuron
Entorhinal Microcircuits
Figure 2. Small and Large Patches in Medial Ento-
rhinal Cortex
A
D
V
(
A) Cytochrome oxidase activity staining of a parasagittal
section showing small patches in layer 2 and the large
patch at the dorsal-most border of medial entorhinal
cortex (indicated by the arrow). D, dorsal; V, ventral.
(
B) Drawing of layers, small layer 2 patches (light brown),
and the pair of large patches (dark brown) from the section
shown in (A).
B
C
5
00 m
(
C) Top view shows parasagittal section adjacent to (A)
L1
L2
L3
stained for Nissl substance. Bottom views illustrate high
magnification of the boxes indicated above with the cor-
responding cytochrome oxidase activity stains from the
section in (A).
L4
L5
L6
(
D) Drawing of neuronal somata (black) revealed by a Nissl
stain in the parasagittal section of the dorsal border of
medial entorhinal cortex shown in (C). Outlines of cyto-
chrome oxidase patches recognized in the adjacent
section (A) are superimposed for clarity.
(
E) Drawing of myelinated axons (black) revealed by
a myelin staining with superimposed cytochrome oxidase
activity staining pattern from an adjacent section.
(
F) Top view shows quantification of patch area along the
dorsoventral axis. Bottom view illustrates quantification of
maximal and minimal Feret diameters (maximal diameter
corresponding to the longest dimension across all orien-
tations and minimal diameter corresponding to that of the
smallest inscribed circle) of patches along the dorsoven-
tral axis. Data are from five different brains (bins of 25%;
1
00 m
1
00% = maximal axis length; see Figure S3). Error bars
indicate standard error.
G) Reconstructions of dendritic morphologies from five
(
neurons recovered from large patches (left) and seven
layer 2 stellate neurons (right) superimposed on the outline
of the patches. Care was taken to correctly position the
reconstructions relative to the border nearest to the soma
and to position the reconstructions such that the relative
position of the cell in its patch is reflected in the super-
position. Note the strong polarization of large patch cells
away from patch borders. Dendrites from layer 2 stellate
cells were also largely (though not perfectly) restricted to
their home patch within layer 2. See also Figure S4.
E
D
F
60
50
L1
L2
L3
L1
L2
L3
40
30
20
10
0
L4
L5
L4
L5
L6
L6
200
m
patches, but myelination did not extend into
their broad dorsal part (Figure 2E). The small
layer 2 patches were also often surrounded by
myelinated fibers (data not shown). The archi-
tecture of small patches changed along the
dorsoventral axis: cell size and myelination
decreased (data not shown), while patch size
increased (Figure 2F; Figure S3). Cells in large
patches appeared to be smaller than adjacent
neurons in small layer 2 patches (Figure 2D)
4
00
00
00
G
3
2
Max. Feret Diameter
5
0 m
100
Min. Feret Diameter
and had
strongly polarized away from the patch border
Figure 2G). Within layer 2 the dendrites of layer
stellate cells were also largely but not exclu-
sively restricted to their home patch, while
a unique dendritic morphology,
0
(
2
distorted the cortical lamination (Figure 2D). Their broad dorsal they extended more broadly in layer 1 (Figure 2G; Figure S4).
part extended into layer 1, and their ventral part tapered out Interestingly, patch diameters seemed to be within the range of
toward layer 4. Many myelinated axons originated from these the deep-to-superficial ‘‘input clusters’’ widths reported by
Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc. 775

Neuron
Entorhinal Microcircuits
F
A
Centrifugal Axon
Centrifugal Axon
RS ctx
RS ctx
L1
L2
L1
L2
L3
L4
L5
L6
L3
L4
L5
P
D
V
200 m
A
L6
D
I
B
G
H
1
mV
0.5 mV
0.2 s
0
.2 s
C
E
J
5
2
.9 Hz
.4 s
Max. 10 Hz
6.2 Hz
2.4 s
Max. 11 Hz
Figure 3. Identified Neurons from the Superficial Layers of Medial Entorhinal Cortex
A) Reconstruction of the dendritic (red) and axonal (blue) morphology of a layer 2 spiny stellate neuron with a superimposed outline of cytochrome oxidase activity
(
stain that identified small layer 2 (light brown) and a pair of large patches (dark brown) in this parasagittal section. Centrifugal axonal projection is indicated. The
dashed line demarcates the border of medial entorhinal cortex with retrosplenial cortex (RS ctx). D, dorsal; V, ventral; A, anterior; P, posterior.
(
(
(
(
(
B) Firing locations of spikes (red) and trajectory of the rat (gray) during running in clockwise direction in a 120 3 60 cm ‘‘O’’-shaped maze.
C) Color-coded rate map for the spikes in (B) (bins of 2.5 3 2.5 cm, maximum firing rate indicated above).
D) High-pass filtered spike trace recorded during freely moving behavior.
E) Firing rate (black) and occupancy times (gray) as a function of head direction. Peak firing rate and minimum occupancy time are indicated.
F–J) Recording from a layer 3 pyramidal neuron. Conventions are the same in (A)/(F), (B)/(G), (C)/(H), (D)/(I), and (E)/(J), respectively. Scale bar is the same for (A)
and (F). Axon thickness has been increased for display purposes. Note that both neurons displayed multiple spatially separated firing peaks.
The discharge pattern of the neurons in individual laps is shown in Figure S5.
Beed et al. (2010) for stellate cells (ꢀ200 mm at midlevel of medial descending axon and several long axon collaterals could be
entorhinal cortex), suggesting a possible correlation between identified (Figure 3A). One of these collaterals targeted a large
patches and interlaminar inputs in medial entorhinal cortex.
patch; as this collateral ran from the inner part of medial entorhi-
nal cortex to its border, we refer to it as the ‘‘centrifugal’’ axon.
This cell showed multiple spatially separated firing peaks
Spatial Firing Behavior of Superficial Neurons
Next, we studied the response properties of identified neurons in (Figures 3B and 3C), a firing behavior similar to grid cells in linear
medial entorhinal cortex while the rat explored a novel environ- environments (Hafting et al., 2008; Brun et al., 2008; Mizuseki
ment. Moreover, to relate neuronal morphology to the patchy et al., 2009). This neuron discharged in bursts whose occurrence
cortical organization, we double stained brain sections for biocy- was often modulated at the theta frequency (Figure 3D). In four
tin and cytochrome oxidase activity.
out of four layer 2 neurons where spatial modulation of activity
A recording experiment from a layer 2 spiny stellate cell is could be assessed (see Supplemental Experimental Proce-
shown in Figures 3A–3E. Staining for cytochrome oxidase dures), we observed multiple spatial firing peaks. Head-direction
activity revealed that the neuron was located in a small layer 2 tuning was measured as the normalized average vector of the
patch (Figure 3A). In layer 2, axon and dendrites were largely circular distribution of firing rates (see Experimental Procedures).
but not completely restricted to the patch, whereas dendrites This neuron showed little or no head-direction selectivity (head-
extended beyond the territory above the patch in layer 1. A direction index = 0.24; Figure 3E).
776 Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc.

Neuron
Entorhinal Microcircuits
Figures 3F–3J show a recording of a spiny layer 3 pyramidal apical dendrite extending toward the pia without an elaborate
cell (Figure 3F). The dendrites arborized in layer 1 and 3 with tuft. The axon arborized extensively in layers 3–6, and a single
few dendritic segments extending in layer 2. Also in this cell, descending axon collateral was identified (Figure 4A). The
a centrifugal axon collateral targeted a large patch. This cell neuron showed very little activity during exploration but became
showed multiple spatially separated firing peaks reminiscent of slightly more active during resting (Figures 4B–4E). Given the low
grid cells (Figures 3G and 3H). Consistent with previous work level of activity and the bias of spiking toward resting, it remained
(
Hafting et al., 2005; Sargolini et al., 2006), a fraction of layer 3 unclear if the discharge pattern reflected a true spatial modula-
cells (four out of nine) showed a similar spatial activity pattern. tion (Figures 4B and 4C) or head-direction selectivity.
The neuron’s spike discharges were regular and not modulated Figures 4F–4J show a recording of a spiny layer 6 multipolar
at the theta frequency (Figure 3I). This neuron showed little selec- cell with long thin dendrites and an axon that extended within
tivity for head direction (head-direction index = 0.05; Figure 3J). deep layers (Figure 4F). Much like the layer 5 cell described
Several observations indicate that the multiple firing peaks above, this layer 6 neuron showed very little activity and became
observed in the linear maze reflected a consistent spatial modu- more active during rest (Figures 4G–4J). Because the firing rate
lation. First, when we plotted individual laps of the recorded was very low, spatial modulation and head-direction selectivity
neurons, we found spatially repeating firing peaks in 8 out of could not be assessed in a meaningful way.
1
3 layer 2/3 recordings, which qualified for spatial modulation
The 11 neurons recovered from deep layers (eight layer 5,
analysis (see Supplemental Experimental Procedures and three layer 6) showed remarkably low levels of activity. Five out
Figures S5A–S5C). Second, repeating firing fields occupied of 11 cells were completely silent, and the presence of these
only a small part of the arena (cumulative area <30%; see Fig- cells in the awake animal could only be verified by juxtacellular
ure S5E), suggesting that firing was spatially restricted. Third, current injection (see Supplemental Experimental Procedures).
the strong firing rate modulation observed in averaged rate Even though in eight cells long axons were identified, we did
maps (Figure S5F) together with the rare occurrence of single- not observe any centrifugal axon. Thus, the large patches
lap firing fields (Figure S5E) reinforces the observation that firing receive inputs from superficial but not from deep layers.
was spatially consistent (Figures S5A–S5C).
To quantify spatial consistency, we ranked cells according to Head-Direction Selectivity and Microcircuits of Neurons
the extent to which firing repeated in restricted spatial locations. in the Large Dorsal Patches
To this end, we computed the fraction of repeating firing fields (as The selective axonal projection to large patches prompted us to
opposed to single-lap firing fields) divided by the area they characterize these structures. Prior to juxtacellular labeling, we
covered (see Supplemental Experimental Procedures). In two identified large patches at the dorsal-most border of medial en-
layer 2 neurons, this ratio was larger than 5, in accordance with torhinal cortex through extracellular recordings in awake head-
a striking, spatially restricted firing pattern. In the case shown in fixed animals. The large patches could be located by a sudden
Figure S5A, one firing field was present in four out five laps, increase in field theta activity (see also Fyhn et al., 2008).
another one in three out five laps, and only little other activity was
Figures 5 and 6 show recordings from large patch neurons.
observed. In six further recordings this ratio (fraction of repeating The morphology of the identified neurons was different from cells
firing fields divided by the area they covered) was between 2 and in the rest of medial entorhinal cortex. Dendritic arbors were
4
, with most though not all firing fields appearing in multiple laps often small and restricted to the home patch. Axons arborized
Figures S5B and S5C). In the remaining five cells, this ratio locally and sent out a descending axon and two additional
was <2, and they did not show spatially consistent firing activity long-range collaterals (Figures 5A, 5B, 6A, 6B, 6G, and 6H). As
Figure S5D). These findings are consistent with the conclusions best visualized in the projection of the reconstructed neuron
(
(
of other authors (see Moser and Moser, 2008 for review) that onto a tangential plane (Figures 5B, 6B, and 6H), one collateral
a large fraction of cells in medial entorhinal cortex are spatially connected to many other large patches; we refer to this collateral
modulated. Giventhelimitedduration ofour recordings, however, as the ‘‘circumcurrent’’ axon as it ran mediolaterally along the
our assessment of spatial consistency remains preliminary.
border of entorhinal cortex. A second collateral targeted specif-
We labeled 21 spiny, putatively excitatory, neurons in superfi- ically (i.e., without branches) one or two small layer 2 patches
cial layers (nine layer 2, 11 layer 3; one cell was located at the and arborized within these structures. As this collateral ran
layer 2/3 border and was not included in the functional analysis). from the border of medial entorhinal cortex to its inner part, we
A large fraction of layer 2 cells could be classified as stellate refer to it as ‘‘centripetal’’ axon.
(
eight out of nine) and in layer 3 as pyramidal neurons (all eight
Some neurons were spatially broadly tuned (Figures 5C–5E,
cells that had complete dendritic morphology), confirming 6I, and 6J), while others showed a multipeaked firing behavior
previous work (Lingenh o¨ hl and Finch, 1991; Klink and Alonso, (Figures 6C and 6D). Neurons in the large patches had two
1
997). We observed a centrifugal axon in 15 out of 18 superficial striking physiological characteristics. First, spike discharges
layer cells with a well-filled axon. This projection was highly were typically strongly modulated in the theta frequency range
selective with axons traveling for long distances to branch in (Figures 6E and 6K). Second, compared to superficial layer cells,
single large patches.
these neurons displayed higher degrees of head-direction tuning
head-direction index = 0.39, Figure 5F; head-direction index =
(
Deep-Layer Neurons Are Largely Silent 0.38, Figure 6F; head-direction index = 0.77, Figure 6L).
Figures 4A–4E show a recording experiment from a layer 5 pyra- In a linear environment an artifactual impression of head-direc-
midal cell (Figure 4A) with dendrites in the deep layers and an tion selectivity could arise if an animal traversed the maze only in
Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc. 777

Neuron
Entorhinal Microcircuits
A
F
L1
L2
L3
L4
L1
L2
RS ctx
RS ctx
L3
L4
L5
L5
L6
L6
2
00
m
B
C
D
G
I
0
.2 s
.5 mV
1 mV
0.2 s
0
E
H
J
Max. 1.9 Hz
Max. 1.4 Hz
0
0
0
0
.8
.6
.4
.2
0
0
.2
.1
0
0
Rest Explor.
Rest Explor.
Figure 4. Identified Neurons from the Deep Layers of Medial Entorhinal Cortex
A) Reconstruction of the dendritic (red) and axonal (blue) morphology of a layer 5 pyramidal neuron with a superimposed outline of cytochrome oxidase activity
(
stain that identified small layer 2 (light brown) and large patches (dark brown) in this parasagittal section. The dashed line demarcates the border of medial
entorhinal cortex with retrosplenial cortex (RS ctx). D, dorsal; V, ventral; A, anterior; P, posterior.
(
(
(
(
(
B) Firing locations of spikes (red) and trajectory of the rat (gray) during running in clockwise direction in a 120 3 60 cm ‘‘O’’-shaped maze.
C) Color-coded rate map for the spikes in (B) (bins of 2.5 3 2.5 cm, peak firing rate indicated above).
D) High-pass filtered spike trace recorded during freely moving behavior.
E) Averaged activity during exploration and rest (defined as periods without locomotion).
F–J) Recording from a layer 6 multipolar neuron. Conventions are the same in (A)/(F), (B)/(G), (C)/(H), (D)/(I), and (E)/(J), respectively. Scale bar is the same for (A)
and (F). Axon thickness has been increased for display purposes. These neurons displayed higher activity during rest than while exploring the environment.
one direction and the cell’s firing was restricted to one arm of the Response Properties and Discharge Timing Differ
maze. Several lines of evidence indicate, however, that such an across Layers and Patches
explanation does not account for the head-direction selectivity We summarize the striking physiological differences between
observed in large patch cells: (1) firing of both large patch cells the entorhinal layers and patches in Figure 7; Figure S7. Cells
(
Figures 5 and 6) and other entorhinal neurons (Figure 3) was in layers 2 and 3 and the large patches were active during
typically not restricted to single arms of the maze; (2) firing varied exploration, whereas deep layers showed little spiking activity
at single locations as a function of head direction; and (3) in three (Figure 7A). Under our experimental conditions, head-direction
dorsal patch cells, where we sampled sufficient data from both selectivity was weak in layers 2 and 3, and low activity levels pre-
clockwise and counterclockwise running directions, firing loca- vented the assessment of head-direction selectivity in deep
tions jumped as a function of running direction (Figure S6; see layers. The strongest head-directional modulation of firing was
Figures 6I and 6J for an example), suggesting that these neurons observed from the population of large patch neurons (Figure 7B).
carry directional rather than positional information.
Neurons in different entorhinal compartments showed pro-
The 14 neurons recovered from large patches formed very nounced differences in their temporal discharge patterns. To
similar microcircuits. In 10 out of 12 cells with a well-filled examine the spike timing relative to the theta rhythm, we ob-
axon, we observed both circumcurrent and centripetal axons; tained the phase of the theta oscillation from the field potential
in the remaining two only a circumcurrent axon was observed. signal, which we extracted from our juxtacellular recordings
In three neurons from large patches, we could follow a descend- (Figures 7C–7E). On average, firing of the layer 3 cell population
ing axon subcortically and observed branching in the presubic- was biased toward the ascending phase of the theta cycle (Fig-
ulum (data not shown).
ures 7F and 7G; Rayleigh average vector length = 0.092 and
778 Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc.

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Entorhinal Microcircuits
Centripetal Axon
Figure 5. Identified Neuron from a Large Patch
A) Reconstruction of the dendritic (red) and axonal (green)
A
B
(
Circumcurrent Axon
morphology of a cell recorded from a large patch with
a superimposed outline of cytochrome oxidase activity
stain that identified small layer 2 (light brown) and large
patches (dark brown) in this parasagittal section. The
centripetal axonal projection is indicated. The dashed line
demarcates the border of medial entorhinal cortex with
retrosplenial cortex (RS ctx). D, dorsal; V, ventral; A,
anterior; P, posterior.
L1
L2
Centripetal
Axon
RSctx
L3
P
(B) Projection of the reconstructed neuron onto a tangen-
tial plane. For display purposes, the neuron is shown
superimposed on a reconstruction of small layer 2 (black)
and large patches (brown) from a myelin stain of a
tangential section (prepared from another animal as
described in the Supplemental Experimental Procedures).
Care has been taken in the superposition to correctly align
the reconstruction with respect to large and small patches.
Axon thickness has been increased for display purposes.
The circumcurrent and centrifugal axonal projections are
indicated. Same conventions as in (A). D, dorsal; V, ventral;
M, medial; L, lateral.
L4
D
V
L5
L6
500 µm
A
D
L
M
200 µm
V
C
D
(
(
C) Firing locations of spikes (red) and trajectory of the rat
gray) during running in clockwise direction in a 120 3
6
0 cm ‘‘O’’-shaped maze.
D) Representative high-pass filtered spike trace recorded
during freely moving behavior.
E) Color-coded rate map for the spikes in (C) (bins of 2.5 3
.5 cm, maximum firing rate indicated above). This cell
displayed broadly distributed spatial activity.
F) Firing rate (black) and occupancy times (gray) as a
(
(
2
0
.5 mV
0
.2 s
(
function of head direction. Peak firing and minimum
occupancy time are indicated.
1
.8 Hz
5.4 s
E
Max. 2.0 Hz
F
1
layer 1 (Figure S7A). Interestingly, large patch
cells showed the strongest theta-phase locking
(
Figures 7F and 7G; Rayleigh average vector
length = 0.35; p < 0.003) and in contrast to
superficial neurons, showed maximal firing on
the descending phase of the theta cycle, near
greater than expected by chance; p < 0.002), with weak phase the trough (difference in average vector angle: layer 2 versus
ꢁ
locking to the field potential theta (Figure S7A). Individual layer large patch = 170 , p = 0.006; layer 3 versus large patch =
ꢁ
2
on average tended to fire preferentially on the ascending phase
cells showed stronger theta-phase locking (Figure S7A) and 167 , p = 0.004) (Figures 7F and 7G; Figure S7A).
Autocorrelation analysis indicated that theta modulation of
of the theta cycle (Figure 7G), in agreement with recent observa- activity was strong in layer 2 and weak in layer 3 cells (Figures
tions (Hafting et al., 2008). However, theta-phase preferences in S7A and S7B), consistent with differences of oscillatory
layer 2 were heterogeneous (Figure 7F; Figure S7A; Rayleigh discharge behavior described in vitro (Alonso and Klink, 1993;
vector not significant; p = 0.615). It appears that this heteroge- van der Linden and Lopes da Silva, 1998). In line with the strong
neity of theta-phase preferences might be at least in part theta modulation of the field potentials, the largest fraction of
accounted for by the laminar location of the tip of the recording theta-modulated cells was found in large patches (Figure S7B).
pipette, which we could identify in all of our recordings. The
polarity of the field potential theta signal is known to invert on Large-Scale Axonal Organization of Patches
the pial side of layer 2 (Alonso and Garc ı´ a-Austt, 1987; Chrobak In order to explore the axonal connectivity scheme across medial
and Buzs a´ ki, 1998; Hafting et al., 2008; Mizuseki et al., 2009) entorhinal cortex, we visualized the large-scale architecture of
(
Figure S7A). In all recordings from superficial layer neurons in axons traveling in layer 1 in ‘‘mass’’ myelin stains of tangential
which we observed firing preferences on the ascending phase sections (Figure 8). Large patches (dark brown) were identified
of the theta cycle, recording locations were well within layers 2 by cell somata clustering and by the clear myelination pattern
and 3. However, in two out of eight recordings from layer 2 cells, that surrounded these structures (Figure S8). Figure S8, which
in which we observed preferences on the descending theta shows serial sections through the dorsomedial part of medial
phase, it appeared that the recording pipette tip was located in entorhinal cortex, also illustrates that large patches seemed to
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Entorhinal Microcircuits
Centripetal Axon
A
G
L1
L2
L1
L2
RS ctx
P
RS ctx
L3
P
D
L3
L4
V
L4
L5
D
A
V
A
L5
L6
L6
200 µm
200 µm
H
Circumcurrent Axon
Circumcurrent Axon
B
Centripetal
Axon
D
V
L
M
5
00 µm
500 µm
C
E
I
K
CCW
CW
1 mV
.2 s
0
.5 mV
0
0
.2 s
D
F
1
J
L
Max. 5.0 Hz
3.9 Hz
8.2 Hz
17.1 s
Max 17 Hz
5.9 s
Figure 6. Head-Direction Selectivity and Microcircuits of Neurons in the Large Patches
A) Reconstruction of the dendritic (red) and axonal (green) morphology of a cell recorded from a large patch with a superimposed outline of cytochrome oxidase
(
activity stain that identified small layer 2 (light brown) and large patches (dark brown) in this parasagittal section. The centripetal axonal projection is indicated. The
dashed line demarcates the border of medial entorhinal cortex with retrosplenial cortex (RS ctx). D, dorsal; V, ventral; A, anterior; P, posterior.
(B) Projection of the reconstructed neuron onto a tangential plane. For display purposes, the reconstruction is shown superimposed on a myelin stain from
a tangential section (prepared from another animal) that shows small layer 2 (black) and large patches (brown). Care has been taken in the superposition to
correctly align the reconstruction with respect to large and small patches. Axon thickness has been increased for display purposes. The circumcurrent and
centripetal axonal projections are indicated. Same conventions as in (A). D, dorsal; V, ventral; M, medial; L, lateral.
(
(
(
(
C) Firing locations of spikes (red) and trajectory of the rat (gray) during running in clockwise direction in a 120 3 60 cm ‘‘O’’-shaped maze.
D) Color-coded rate maps for the spikes in (C) (bins of 2.5 3 2.5 cm, maximum firing rate indicated above).
E) Representative high-pass filtered spike trace recorded during freely moving behavior.
F) Firing rate (black) and occupancy times (gray) as a function of head direction. Head-direction selectivity was computed for the entire recording trial, including
both clockwise (C) and counterclockwise (not shown) running directions. Peak firing rate and minimum occupancy time are indicated.
G–L) Conventions are the same in (A)/(G), (B)/(H), (C)/(I), (D)/(J), (E)/(K),and (F)/(L), respectively. Note that in this cell only a circumcurrent axon was identified (H).
Firing locations of spikes (I) and color-coded rate maps (J) are shown separately for clockwise (CW) and counterclockwise (CCW) running directions.
(
780 Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc.

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Entorhinal Microcircuits
form a continuum with the parasubiculum (Shipley, 1974, 1975;
K o¨ hler, 1984; Caballero-Bleda and Witter, 1993; Witter and
Amaral, 2004). Myelin stainings revealed a striking regularity of
layer 1 axonal fibers, organized in axonal bundles running along
the dorsomedial to ventrolateral axis (Figure 8A). We traced
putative centrifugal axons originating above the territory of small
layer 2 patches (blue, Figures 8A and 8C), and we drew a large
number of axons that surrounded a single large patch (green,
Figures 8A and 8B). As in most identified cells from large patches
(Figures 5 and 6), putative circumcurrent axons to dorsolateral
neighboring patches were longer and more prominent than
axons extending toward the ventromedial ones (green, Figures
A
7
6
5
4
3
2
1
0
B
1
*
*
*
*
*
0
.8
.6
.4
.2
**
0
0
0
*
0
L2 L3 L5/6 LP
L2
L3
LP
8
A and 8B). A schematic overview of the position of medial ento-
Layer 2
C
rhinal patches in the rat brain is shown in Figure 8D. Overall, the
circumcurrent axons surrounding large dorsal patches were
much more numerous than the circumcurrent axons surrounding
medioventral patches. Mass myelin stains appear to be consis-
tent with our single-cell reconstruction data and suggest a global
organization of three long-range axon systems in medial entorhi-
nal cortex: (1) centrifugal and (2) centripetal axons, which recip-
rocally connect large and small patches; and (3) circumcurrent
axons, which connect large patches along the mediolateral
axis. Because mass myelin stains cannot ascertain the origin
and endpoint of axonal projections, additional tracing work will
be required to firmly establish the reciprocal nature of centrifugal
and centripetal connections.
1
mV
Layer 3
D
0
.5 mV
DISCUSSION
To our knowledge, we provided the first microcircuit description
of functionally identified cells in medial entorhinal cortex.
Friction-based pipette-stabilization and juxtacellular labeling
allowed the combined analysis of neural activity, circuitry, and
behavior. We demonstrated efficient (>50% success rates) cell
recovery with excellent morphology from freely moving animals.
The success of the friction-based device is almost certainly
related to the rigidity of the pipette positioning. As the pipette
is securely locked into position, the resulting recordings with-
stand mechanical disturbances. As only a handful of neurons
1
mV
Large Patch
E
200 ms
9
0°
G
(B) Head-direction index for cells in L2 (n = 5), L3 (n = 10), and LP (n = 10). Head-
direction tuning was quantified as the normalized average vector of the circular
distribution of firing rate. L3 and LP differed significantly. Circles indicate mean
values. One (*), two (**), and three (***) asterisks indicate significance at the
F
1
35°
45°
0
.05, 0.01, and 0.001 levels, respectively.
1
80°
0°
(C–E) Representative raw spike traces (black, bottom) recorded for layer 2 (C),
layer 3 (D), and large patch cells (E) during 1.5 s of running behavior. Super-
imposed on top (gray) is the band-pass filtered (4–10 Hz) field potential trace.
The amplitude of the filtered trace has been increased 2-fold for display
purposes. Note the differences in spike timing between layer 2/3 and large
patch neurons.
0.5
1
2
25°
315°
Layer 2
Layer 3
Large Patch
2
70°
0
180 360
540 720
(
F) Polar plots of layer 2 (red), layer 3 (green), and large patch cells (blue)
Theta Phase (deg)
ꢁ
ꢁ
ꢁ
representing both the preferred phase (peak = 0 , 360 ; trough = 180 ) and the
modulation strength (average vector, proportional to the distance from the
center) for each single neuron.
Figure 7. Response Properties across Layers and Patches
A) Average firing rates for all recorded cells in layer 2 (L2, n = 9; one neuron
(
(G) Relative phase preference for layer 2, layer 3, and large patch cells. The
phase of the theta cycle is shown above (dotted line). Two cycles are shown.
Note the difference in phase preferences of large patch cells compared to
superficial layer neurons. Colors as in (F).
was silent), layer 3 (L3, n = 11), layer 5-6 (L5-6, n = 11; five neurons were silent),
and large patches (LP, n = 14). Error bars indicate standard error. Average
firing rates differed significantly between L5-6 and every other group.
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Entorhinal Microcircuits
Figure 8. Large-Scale Architecture of Layer
A
B
1
Axons in Medial Entorhinal Cortex
A) Reconstruction of the architecture of medial
(
D
V
entorhinal cortex from serial tangential sections
stained for myelin. Large patches at the dorsal-
most border of medial entorhinal cortex were de-
tected by massive axon bundles on the dorsal
surface of each patch; subdivisions between pairs
of patches were visible in a subset of them but are
not indicated. Most of the borders of medial large
patches were difficult to discern in this prepara-
tion, and their outlines have been dashed. Small
layer 2 patches were detected in mid-depth of
layer 2 as clusters of white somata surrounded by
myelin. For consistency with other panels, we
illustrate large patches in dark brown and small
layer 2 patches in light brown. Layer 1 was densely
populated with axon bundles, and roughly twice
as many axon bundles as small patches were
detected. Ten bundles originating above small
patches (putative centrifugal axons; blue) and
axons associated with one large patch (putative
centripetal and circumcurrent axons; green) were
drawn. The reconstruction was performed across
twelve 40 mm sections stained for myelin. The
reconstruction encompasses most but not all of
medial entorhinal cortex; in particular, it appeared
that small and large patches were missed laterally,
probably because of the cortical curvature and
their poor alignment with the sectioning angle.
Note that axons could only be followed for limited
distances and that axon thickness has been
increased for display purposes. D, dorsal;
V, ventral; M, medial; L, lateral.
L
M
5
00 µm
50 µm
C
50µm
D
(
B) Myelin-stained inset showing putative circum-
current axons. Magnification of the marked
section in (A).
(
C) Myelin-stained inset showing bundles of
putative centrifugal axons in layer 1. Magnification
of the marked section in (A).
(
D) Schematic representation of the patchy orga-
nization of medial entorhinal cortex in a posterior-
lateral view of the rat brain (based on the same
reconstruction shown in A). To give
a more
complete overview, additional large and small
patches have been added dorsolaterally, which
were not reconstructed from the sections used for
(
A) because they could not be well visualized as
a result of the curvature of the brain. The rhinal
fissure is indicated.
have been recovered from freely moving mammals previously, ing and Brecht., 2008; Voigt et al., 2008; Houweling et al., 2010),
our approach opens up a new frontier in microcircuit research. making it possible to explore the effects of single-cell stimula-
Beyond cell identification, our methodology provides additional tions on freely moving animal behaviors. A major limitation of
advantages over conventional extracellular recording tech- the current method is the use of the antagonizable anesthesia.
niques. First, it allows unequivocal unit separation, given the Although under the same conditions as the present study hippo-
remarkably high signal-to-noise ratio of the recorded spike campal physiology seemed to be preserved to a large extent
signals (average signal-to-noise = 11.64 ± 6.9; see Supplemental (e.g., occurrence and properties of place cells were unaltered)
Experimental Procedures and Figure S1D). Second, it allows (Lee et al., 2006, 2009; Epsztein et al., 2010), we cannot exclude
recording and identifying silent cells, whose impact on cortical potential residual effects of the anesthesia after antagonization.
computations is poorly understood. Third, activity of the In the present study we made three key observations. First, we
recorded cells can be manipulated by current injections (Houwel- identified two types of patches in medial entorhinal cortex: small
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Entorhinal Microcircuits
patches in layer 2 and large patches at the dorsomedial border. direction selectivity in an ‘‘O’’-shaped linear arena, and we
Second, we identified the connections between patches. Super- cannot exclude that our directionality measures were influenced
ficial layer cells connect to single large patches (via centrifugal by the special geometry of this environment, in particular by the
axons), whereas cells from these structures connect back to constraints imposed on the rat’s heading direction. Neverthe-
small patches (via centripetal axons) and to other large patches less, under these experimental settings we could record head-
(
via circumcurrent axons). Third, we characterized the functional direction cells, whose directional tuning was stable across
properties of identified cells in the different compartments of clockwise and counterclockwise running directions and sharply
medial entorhinal cortex. Altogether, our data point to a congru- tuned to a narrow angle (Figures 6I–6L; Figure S6). Further work
ence of connectivity and responses with cortical patches, is needed to extend these findings to open field environments,
implying that these units—described not only in rats but also in where spatial and head-directional modulation have been most
monkeys and humans (Hevner and Wong-Riley, 1992; Solodkin extensively studied (Taube et al., 1990a; Taube and Muller,
and Van Hoesen, 1996)—shape entorhinal processing in a funda- 1998; Sargolini et al., 2006). Consistent with our findings,
mental way.
head-direction cells have been recorded from the dorsal-most
Grid cells display a periodic and regular hexagonal firing regions of medial entorhinal cortex in mice (Fyhn et al., 2008)
pattern while the rat explores an open field (Hafting et al., and rats (Sargolini et al., 2006).
2
005). Typically, such strict periodicity of firing pattern is not
Theta rhythm is the most prominent oscillatory pattern in
observed in linear environments (Hafting et al., 2008; Brun the hippocampal-entorhinal system and is thought to provide
et al., 2008; Mizuseki et al., 2009), which makes it impossible temporal windows for efficient neuronal communication (Dragoi
under our conditions to certify if a cell is a grid cell or not. The and Buzs a´ ki, 2006; Sirota et al., 2008; Mizuseki et al., 2009). In
largest fraction of grid cells were recorded from superficial layers this respect, the timing of neuronal discharges relative to the
of medial entorhinal cortex (Hafting et al., 2005; Sargolini et al., underlying theta rhythm can provide important insights into
2
006; Boccara et al., 2010). In agreement with these studies, the mechanisms that regulate temporal coordination within the
we also observed a large fraction of cells with multipeaked firing network (Mizuseki et al., 2009). Interestingly, the spike-theta
behavior in layer 2 and 3, a firing pattern that is reminiscent of phase relationship was strikingly different between superficial
grid cells tested in linear environments (Hafting et al., 2008; layer and large patch cells. While the first appeared to fire pref-
Brun et al., 2008; Mizuseki et al., 2009). The spatial firing across erentially on the ascending phase of the theta cycle (Figure 7F
laps was highly stable in some cells (Figure S5A) but less repro- and 7G), large patch cells showed the strongest theta-phase
ducible in other neurons (Figures S5B and S5C). The complete locking and opposite theta-phase preferences from superficial
novelty of the environment might contribute to the instability of layer cells, with maximal firing on the descending phase of the
spatial firing, as previously suggested (Hafting et al., 2005; Lang- theta cycle, near the trough (Figures 7F and 7G; Figure S7A).
ston et al., 2010; Wills et al., 2010). Finally, we cannot rule out Our data on the theta-phase preferences of layer 2 and 3 cells
that external, potentially uncontrolled nonspatial stimuli contrib- are consistent with recent findings (Hafting et al., 2008) but
uted to the observed spatial modulation because we did not appear not to be in line with the work of Mizuseki et al. (2009).
perform spatial manipulations (such as cue-card rotation exper- Several possibilities could account for the latter discrepancy,
iments) that address such possibilities.
including our relatively small sample and the variability of
We describe a system of large patches at the dorsal border of theta-phase preferences among the layer 2 (Figure 7F; Fig-
medial entorhinal cortex, which covers the entire mediolateral ure S7A) and layer 3 populations (Mizuseki et al., 2009).
extent of medial entorhinal cortex and overlaps more medially
Our data confirm and extend previous observations on laminar
with the parasubiculum (Caballero-Bleda and Witter, 1993). differences in entorhinal cortex (Hafting et al., 2005; Sargolini
Because of their similarity and continuity with the parasubicu- et al., 2006). Our observations on a prevalence of spiny stellate
lum, we suggest that the large dorsal patches should best be morphologies and spatially modulated responses in layer 2
viewed as an extension of this structure. We note, however, confirm previous morphological (Klink and Alonso, 1997) and
that while previous authors described the parasubiculum as physiological (Hafting et al., 2005; Sargolini et al., 2006) studies.
a multilayered structure (Witter and Amaral, 2004; Boccara Deep-layer neurons were largely silent during exploration. This
et al., 2010), we found no evidence that the large dorsal patches result differs from the extracellular recording data of Sargolini
were associated in any way with deep cortical layers. To our et al. (2006), which indicated substantial deep-layer activity.
knowledge, the large dorsal patches have been largely unrecog- We note, however, that most of the deep-layer cells recorded
nized previously. Classically, the dorsal border of medial entorhi- in our study were silent and could, therefore, not have been de-
nal cortex had been defined by the sudden increase in layer 2 tected with extracellular recordings. Moreover, we used naive
width, which extends into layer 1 and forms a ‘‘club-like thick- animals exploring a novel environment, whereas the recordings
ening’’ (Amaral and Witter, 1989; Insausti et al., 1997). Here, of Sargolini et al. (2006) were done in rats with prior spatial
we provide several lines of evidence suggesting that this experience. Our results suggest that deep layers—and presum-
dorsal-most structure is organized in patches and contains ably also the hippocampal feedback that arrives in these layers
a distinct neuronal subpopulation from the rest of medial entorhi- (van Strien et al., 2009)—might not initially contribute to the
nal cortex. Cells in this structure have unique morphology and representation of a novel environment.
connectivity, are strongly theta modulated, show different
In medial entorhinal cortex, position, speed, and directional
theta-phase preferences, and are more head-direction selective information are integrated to generate an updated metric repre-
than superficial layer neurons. In our study we assessed head- sentation of space (McNaughton et al., 2006; Moser and Moser,
Neuron 70, 773–786, May 26, 2011 ª2011 Elsevier Inc. 783

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Entorhinal Microcircuits
2
008). In line with anatomical and structural features, like period-
(Fyhn et al., 2008; Derdikman et al., 2009). Details are provided in the Supple-
mental Experimental Procedures.
icities in cell densities, dendritic clusters (Ikeda et al., 1989), and
molecular markers (Solodkin and Van Hoesen, 1996; Suzuki and
Porteros, 2002), the patchy organization of medial entorhinal
cortex further supports the existence of segregated functional
modules, as previously suggested (Witter and Moser, 2006).
This is in line with evidence that grid spacing and orientation
are typically identical from grid cells recorded from the same
location, while they change along the dorsoventral axis in
a discontinuous fashion (Hafting et al., 2005; Fyhn et al., 2007;
H. Stensland et al., 2010, Abstr. Soc. Neurosci., abstract). Inter-
estingly, implementing an attractor-like modular organization
within the grid cell network makes it possible to represent space
in parallel at different spatial scales (Witter and Moser, 2006). In
this context it is worth mentioning that indications for grid cell
networks have been observed also in humans (Doeller et al.,
Histological Analysis
To reveal the morphology of juxtacellularly labeled cells, 100–150 mm thick
brain slices were processed with the avidin-biotin-peroxidase method (Lee
et al., 2006, 2009; Epsztein et al., 2010). Cytochrome oxidase and Nissl stain-
ings were performed as described previously (Wong-Riley 1979; Brecht and
Sakmann, 2002). For myelin stainings a variation of the gold-chloride protocol
(Schmued 1990) was used. Details are provided in the Supplemental Experi-
mental Procedures.
Data Analysis
To assess spatial modulation of spiking activity, space was discretized into
pixels of 2.5 3 2.5 cm bins, and color-coded firing maps were plotted.
Head-direction tuning was measured as the length of the average vector of
the circular distribution of firing rates. The head-direction index of a cell was
defined as the vector length divided by average firing rate across the circular
distribution. Theta modulation of spiking activity was quantified by measuring
the maximum of the autocorrelation function’s power spectrum between 4 and
2
010), where the patchy organization of entorhinal cortex is
most prominent (Hevner and Wong-Riley, 1992). The cytochrome
oxidase-rich patches of entorhinal cortex are known to be
destroyed in Alzheimer’s disease (Solodkin and Van Hoesen,
1
0 Hz. For spike-theta phase analysis, juxtacellular signals were band-pass
filtered at 4–10 Hz, and a Hilbert transform was used to determine the instan-
ꢁ
ꢁ
taneous theta phase of the filtered theta wave (peaks = 0 , 360 and troughs =
1
996), and we wonder if the destruction of patches is related to
ꢁ
1
80 ). Then, each spike was assigned the theta phase of the Hilbert transform
the loss of spatial orientation and awareness in Alzheimer’s
patients (Cherrier et al., 2001).
at the time of that spike. Details are provided in the Supplemental Experimental
Procedures.
The localized connections between large patch cells and small
patches suggest that each small patch might receive very selec-
tive and specific head-directional input for local computation.
The focal head-directional input to small layer 2 patches is
remarkable, since both our data and the results from previous
studies (Sargolini et al., 2006) indicate that layer 2 cells express
little or no head-direction selectivity. Therefore, since layer 2
cells do not simply inherit head-direction selectivity via centrip-
etal axons, we wonder if head-directional information is trans-
formed in layer 2 patches in a way that generates allocentric
coding. More specifically, head-directional information could
be used to generate allocentric coding by rotating (and thus
allocentrically stabilizing) the grid pattern with head turns against
egocentric coordinates. The circumcurrent axons form another
prominent long-range circuit. We speculate that the circumcur-
rent axons might impose a global constraint that unifies direc-
tional information across large patch neurons to a single head
direction.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and eight figures and can be found with this article online at doi:10.1016/
j.neuron.2011.04.003.
ACKNOWLEDGMENTS
We thank Alison Barth, Prateep Beed, Rajnish Rao, Dietmar Schmitz, John
Tukker, and Jason Wolfe for comments on the manuscript, and Brigitte
Geue, Carolin Mende, Mike Kunert, Undine Schneeweiß, and Arnold Stern
for outstanding technical assistance. This work was supported by Neurocure,
the Bernstein Center for Computational Neuroscience (BMBF) and Humboldt
University, the EU Biotact-grant, and the Neuro-behavior ERC grant.
Accepted: April 4, 2011
Published: May 25, 2011
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tion, pipette anchoring, and anesthesia/wake-up procedures were performed
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cortex (left hemisphere), a small craniotomy (ꢀ2–4 mm diameter) was made
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