Download Cell-Type Specific Properties of Pyramidal

Cerebral Cortex April 2010;20:826--836
doi:10.1093/cercor/bhp152
Advance Access publication July 30, 2009
Cell-Type Specific Properties of Pyramidal
Neurons in Neocortex Underlying a Layout
that Is Modifiable Depending on the
Cortical Area
To understand sensory representation in cortex, it is crucial to identify
its constituent cellular components based on cell-type--specific
criteria. With the identification of cell types, an important question
can be addressed: to what degree does the cellular properties of
neurons depend on cortical location? We tested this question using
pyramidal neurons in layer 5 (L5) because of their role in providing
major cortical output to subcortical targets. Recently developed
transgenic mice with cell-type--specific enhanced green fluorescent
protein labeling of neuronal subtypes allow reliable identification of 2
cortical cell types in L5 throughout the entire neocortex. A
comprehensive investigation of anatomical and functional properties
of these 2 cell types in visual and somatosensory cortex demonstrates
that, with important exceptions, most properties appear to be celltype--specific rather than dependent on cortical area. This result
suggests that although cortical output neurons share a basic layout
throughout the sensory cortex, fine differences in properties are tuned
to the cortical area in which neurons reside.
Keywords: BAC transgenic, connectivity, dendrite morphology, neocortex,
pyramidal cell
Introduction
The organization of the cortex into layers of different cell-types
is a stereotypic attribute with functional consequences. For
example, sensory responses in somatosensory cortex and visual
cortex (VC) are layer and cell-type specific (Gilbert and Wiesel
1979; Armstrong-James et al. 1992; Brecht and Sakmann 2002;
Martinez et al. 2005; de Kock et al. 2007) and a correlation
between morphology and response property can be drawn. In
somatosensory (vibrissal) cortex (BC), in rodents, the thicktufted and the slender-tufted pyramidal neurons in layer 5 (L5)
respond differently to tactile (whisker deflection) stimuli
(Manns et al. 2004; de Kock et al. 2007). The functional
difference between slender- and thick-tufted pyramidal neurons
raises several questions. First, what are the defining characteristics of these neurons in terms of cell-type--specific properties
in dendrite morphology, intrinsic excitability, axon projection
target, gene expression, and properties of synaptic transmission?
Second, are these properties unique for a cortical area or
retained in different cortical areas? Classification of L5 pyramidal
neurons has relied on a combination of soma--dendrite
morphology, intrinsic electrophysiological properties, gene
expression pattern, as well as projection targets (Hevner et al.
2003; Molnar and Cheung 2006; Morishima and Kawaguchi
2006; Nelson et al. 2006; Sugino et al. 2006; Le Be et al. 2007;
Watakabe et al. 2007). A scheme that has emerged is the
correlation between soma--dendrite morphology and projection
Ó The Author 2009. Published by Oxford University Press. All rights reserved.
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Alexander Groh1, Hanno S. Meyer2, Eric F. Schmidt3,
Nathaniel Heintz3, Bert Sakmann1,4 and Patrik Krieger5
1
Institute for Neuroscience of the Technical University
Munich, Biedersteiner Strasse 29, 80802 Munich, Germany,
2
Max-Planck-Institut für medizinische Forschung, Jahnstrasse
29, D-69120 Heidelberg, Germany, 3Laboratory of Molecular
Biology, The Rockefeller University, New York, NY 10065, USA,
4
Max Planck Institute for Neurobiology, Am Klopferspitz 18,
82152 Martinsried, Germany and 5Department of
Neuroscience, Karolinska Institutet, SE-17177 Stockholm,
Sweden
targets (Hübener and Bolz 1988; Hübener et al. 1990; Larkman
and Mason 1990; Mason and Larkman 1990; Hattox and Nelson
2007). Uncertainties, however, of cell identities remain when
correlating parameters from individual studies. The identification of genetically labeled neurons throughout the neocortex
enabled in the present work the combined study of many
parameters and their dependency on the sensory modality.
We have characterized 2 populations of L5 pyramidal
neurons genetically labeled with enhanced green fluorescent
protein (EGFP) and compared each cell type in somatosensory
cortex with the corresponding cell type in VC. We used 2
mouse lines for the 2 L5 cell types expressing EGFP under the
control of either a promoter for a transcription factor (ets
variant gene [etv] 1) or the promoter for a glycosyltransferase
(glycosyltransferase 25 domain containing [glt] 2). Each is
specifically expressed in a subpopulation of the slender- and
thick-tufted pyramidal neurons, respectively.
We show that in both somatosensory cortex and VC, cell-type-specific properties define 2 populations of tufted L5 pyramidal
neurons. Each subpopulation retains its basic properties between
cortices. In both cortices, the 2 different populations can be
clearly distinguished based on anatomical and functional parameters. However, when comparing a given cell-type between the 2
cortices, some parameters are characteristically tuned to the
cortical area. These differences suggest that tactile and visual
stimuli are processed by similar local neuronal circuits that,
however, show variations depending on cortical region.
Materials and Methods
Experiments were performed on Glt25d2 bacterial artificial chromosome (BAC)-EGFP transgenic mice and Etv1 BAC-EGFP transgenic mice
obtained from the GENSAT project. Information on the genes and
expression data at the GENSAT Web site (http://www.gensat.org). See
also Gong et al. (2007) and Doyle et al. (2008) for a brief description of
the Etv1and Glt25d2 BAC-EGFP lines and other BAC lines. Experiments were performed on mice with both parents BAC-EGFP mice and
mice with one parent BAC-EGFP and one wild-type C57Bl/6. Experiments were conducted in accordance with the German animal welfare
guidelines.
Stereotaxic Injection
Stereotaxic injections, using isofluorane anesthesia, were done at
postnatal day 60--120. Cholera toxin subunit B (Molecular Probes)
conjugated to Alexa 555 (pseudo color red) or Alexa 647 (pseudo color
blue) for retrograde labeling was mixed with a solution containing
adeno-associated viral particles (AAVs) encoding for dTomato (AAVdTomato) for anterograde labeling. In total, 50- to 100-nL solution,
containing tracers, was slowly injected into target sites (pontine
nucleus, caudate putamen, and posterior medial thalamic nucleus
[POm]) with the following coordinates (mm) relative to bregma,
midline, and dura: caudate putamen: 0.8, 1.8, –3.6; pontine nucleus: –3.6,
0.55, –5.3; and POm: –2.0, 1.15, –2.9. To avoid inadvertent diffusion of
tracer from the intended target area, the tracer injection volume was
kept to a minimum. The resulting fraction of retrogradely labeled cells
is, thus, unlikely to reflect all cortical cells projecting into the target.
The relevant comparison is the relative fraction of retrogradely labeled
etv- versus glt-pyramids from injection into different targets and the
depth distribution of EGFP and retrogradely labeled cells.
Slice Preparation
Coronal slices containing primary somatosensory (barrel) cortex or VC
were prepared by placing the anterior part of the brain on a 10-deg slope
and then cutting perpendicular to the brain surface, discarding the tissue
caudal to the cut. The brain was subsequently glued with the cut surface
facing down, and 300-lm thick slices were cut. In a few experiments,
parasagittal slices (300 lm) containing VC were also used. Slices were
maintained at room temperature before recording. All experiments were
performed close to physiological temperatures (32--34 °C).
Electrophysiology
Patch pipettes for whole-cell recordings were filled with (in mM) 105
K-gluconate, 30 KCl, 10 phosphocreatine-Na2, 10 N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid (HEPES), 4 ATP-Mg, 0.3 GTP, with pH
adjusted to 7.3 with KOH (osmolarity 300 mOsm). Biocytin (2 mg/ml;
FLUKA) was included in the pipette to allow the morphology of the
neurons to be reconstructed in the fixed tissue. For cell-attached
stimulation, the pipette contained the following solution (in mM): 105
Na-gluconate, 30 NaCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, and
0.3 GTP (adjusted to pH 7.3 with NaOH). The extracellular solution
contained (in mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 25
glucose, 2 CaCl2, and 1 MgCl2, continuously bubbled with 95% O2 and
5% CO2. Membrane potential was not corrected for junction potential.
Dual whole-cell recordings to investigate synaptic connectivity 1) in
barrel cortex (BC) were made on 19 Etv1 BAC-EGFP transgenic mice
with an average age of 22 days (range: 18--25) days and on 14 Glt25d2
BAC-EGFP transgenic mice, average age 25 days (range: 16--39 days) 2)
in VC (including both primary and secondary VC) were performed on 7
Etv1 BAC-EGFP transgenic mice, average age 23 days (range: 19--25
days) and 2 Glt25d2 BAC-EGFP transgenic mice (age 21 and 22 days).
One pipette was used to obtain whole-cell recordings from one cell and
the second pipette was used to search for synaptically connected
neurons. The search pipette was used for cell-attached stimulation or
whole-cell recording. Only cell pairs located within 20 lm of one
another in the z-plane were tested. Cell pairs with no synaptic
connection in either direction were counted as 2 unconnected pairs,
pairs with a unidirectional connection were counted as one connected
pair and one unconnected pair, and pairs with a bidirectional
connection were counted as 2 connected pairs. The horizontal distance
(x-axis) between neurons was measured from the center of the soma,
with the vertical y-axis aligned to the apical dendrite. Recordings were
made with an Axoclamp2B amplifier (Axon Instruments, Union City,
CA), filtered at 3 kHz and sampled at 10 kHz using custom written
software (Igor; WaveMetrics, Lake Oswego, OR).
Histological Procedures
Slices were fixed in 100 mM PBS containing 4% paraformaldehyde and kept
at 4 °C for 1--2 days. Slices were stained for cytochrome oxidase, to visualize
barrels, and biocytin to visualize cell morphology. Biocytin-labeled neurons
were visualized using the avidin:biotinylated horseradish peroxidase
complex (Vectastain ABC kit, Vector Laboratories, CA). The location of
the neurons within the BC could, thus, be confirmed, and neurons were
reconstructed in 3D with the Neurolucida software (MicroBrightField,
Colchester, VT) using an Olympus (BX 51) or Zeiss microscope with an oil
immersion 3100/1.4 numerical aperture (NA) objective.
Quantification of Dendrite Morphology, Intrinsic Membrane
Properties, and Cell Counting
Apical tuft dendrite was defined in BC as the dendritic profiles in the
upper 10% of the pia--white matter distance and upper 15% of the pia--
white matter distance in VC. This corresponds approximately to layer 1.
Apical dendrite tuft width was the maximum horizontal width. Primary
obliques were dendrites bifurcating from the main apical dendrite and
located between the soma and the major apical dendrite bifurcation.
‘‘Nodes’’ are bifurcation or trifurcation points. Soma and dendrite
parameters were calculated with Neurolucida Explorer (MicroBrightField). Fluorescently labeled cells were counted manually using the
AMIRA (Visage Imaging, Germany version 4.0; Mercury Computer
Systems) software. Threshold current was the minimum current to
elicit at least one action potential at resting membrane potential.
Frequency adaptation was calculated by measuring the average time
interval between successive spikes elicited using three 1-s pulses with
current in the range 200--400 pA, in steps of 20 pA. The average
interspike time intervals (ISIs), excluding the 2 first ISIs, was
normalized to the average of the third ISI and plotted as a function of
the ISI number (ISI #). Subsequently, a linear regression line was fitted.
The slope of the line is taken as the ‘‘adaptation index.’’ An adaptation
index of ‘‘10,’’ thus, means that on average the time interval increases
with 10 percentage points between successive spikes.
Two-Photon Imaging
EGFP-labeled cells were identified in the living slice using two-photon
microscopy. Two-photon excitation was made by a Ti:Sa-Laser (MIRA
900F; Coherent, Santa Clara, CA) set at 820--840 nm and pumped by
a solid-state laser (Verdi 5W; Coherent). Scanning was performed using
a resonant scanning unit (TCS-SP2RS; Leica Microsystems, Mannheim,
Germany) mounted on an upright microscope (DMLFS; Leica Microsystems) equipped with a 340 objective (HCX APO, 340, NA 0.8; Leica).
A dichroic mirror (560DCXR) split the fluorescence to one detector
(bandpass filter HQ525/50M) for green fluorescence from EGFPexpressing L5 pyramidal neurons and to one detector (bandpass filter,
HQ610/75M) recording red fluorescence from Alexa Fluor 594. Online
superposition of fluorescence and infrared contrast-enhanced image
allowed targeted recordings from fluorescently labeled neurons.
Statistical Analysis
Two-tailed unpaired t-tests, and one-way analysis of variance (ANOVA)
using Bonferroni posttests, were done with GraphPad Prism 4 (GraphPad Software, CA). Significant differences in the morphological data and
the intrinsic properties were determined with one-way ANOVA and
Bonferroni posttests unless otherwise stated. Data are reported as mean
± standard deviation (SD), unless stated otherwise.
Numerical Analysis of Cell Densities in a Barrel Column
The distribution of L5 etv-expressing pyramidal neurons (etv-pyramids)
and L5 glt-expressing pyramidal neurons (glt-pyramids) was determined by manual cell counting of all EGFP-labeled cells in 2 barrel
columns in one brain slice from each of 3 etv-mice and 2 glt-mice (age
25--28 days). No correction was done for tissue shrinkage. GAD67 and
NeuN immunohistochemistry was used to estimate column and layer
borders. A detailed description of the immunocytochemistry and
analytical tools developed for the numerical analysis of cells in
a barrel column will be presented elsewhere (Meyer HS, Wimmer VC,
Schwarz D, Sakmann B, and Helmstaedter M, unpublished data).
Results
Soma Depth Location and Dendrite Morphology of 2
Genotypically Defined L5 Pyramidal Neurons in Primary
Somatosensory (Barrel) Cortex
Genetic labeling of cortical cells using the BAC method (Gong
et al. 2002, 2003, 2007; Doyle et al. 2008) has generated several
lines of transgenic mice with fluorescent labeling of potentially
specific classes of neurons. To investigate subpopulations of L5
pyramidal neurons in different cortical areas, we have used 2
BAC transgenic mouse lines where expression of Glt25d2 (glt)
or Etv1 (etv) was reported with EGFP expression. Expression of
Cerebral Cortex April 2010, V 20 N 4 827
these 2 genes is shown to identify L5 pyramidal neurons in the
primary somatosensory (barrel) cortex (Fig. 1A,B), VC (Fig. 3),
and other neocortical areas (data not shown). For each mouse
line, an average density map was calculated showing the 2D
soma distribution in 2 neighboring barrel columns (Fig. 1C). L5
etv-pyramids were located predominately in L5A (Fig. 1C;
median depth from pia 610 lm) and glt-pyramids in L5B
(Fig. 1C; median depth from pia 690 lm). In the horizontal
column axis, the highest density was located in the center of
the barrel columns (Fig. 1C). In somatosensory cortex, etv- and
glt-pyramids are, thus, expressed in 2 distinct sublayers. The
peak cell densities are approximately 80 lm apart.
A profile in 1D representing the ratio of etv- and glt-pyramids
to all excitatory neurons in a barrel column shows that at the
peak cell density, the etv- and glt-pyramids represent approximately 55% of the excitatory neurons (Fig. 1D). The fraction of
etv-pyramids and glt-pyramids to the total number of neurons
was estimated to 29.0 ± 3.1% in L5A (etv) and 12.3 ± 3.5% in
L5B (glt). The average neuron density in each layer was higher
(Supplementary Table 1) than previously reported for mouse
somatosensory cortex (White and Peters 1993).
In the acute brain slice preparation, EGFP-labeled etv- and gltpyramids were filled and subsequently stained for biocytin, and
their dendrite morphology was reconstructed and quantified.
Figure 2 (and Supplementary Fig. 1) illustrates examples of
reconstructed etv- (Fig. 2A) and glt-pyramids (Fig. 2B). In both
populations, the apical dendrites extended to layer 1. Compared
with the glt-pyramids, the etv-pyramids have a narrower apical
dendrite tuft width (etv-pyramids: 157 ± 54 lm, n = 29; gltpyramids 356 ± 84 lm, n = 19) and had fewer primary apical
oblique dendrites (etv-pyramids: 3.7 ± 2.2 primary branches; gltpyramids: 8.9 ± 2.6 primary branches). The apical dendrite
Figure 1. EGFP-labeled etv-pyramids and glt-pyramids in L5 of the somatosensory BC. Confocal images of EGFP-labeled pyramidal cells in brain slices (coronal section) from
transgenic mice carrying the BAC constructs for the (A) etv1 or (B) glt2. Red dotted line added for comparison of the relative depth of the 2 populations. Data in (C) and (D) was
obtained from 50-lm thick coronal slices from 3 etv-mice and 2 glt-mice. (C) The soma positions of EGFP-labeled cells in the 2 most medial barrel columns per slice were marked
manually in 3D and their average density distribution (bin size: 50 3 50 lm2) in the plane of slicing was plotted. Layer borders (outlined with black dots and lines) were derived
independently of soma distribution using GAD67 and NeuN immunohistochemistry. The 2D cell-density maps show that etv-pyramids were located in L5A and glt-pyramids in
L5B. Contours (black lines superimposed on the cell-density distribution) show isodensity lines at 80% (for etv) or 70% (for glt) of the respective peak density. Along the horizontal
column axis, both cell populations had their peak density in the center of the barrel column. (D) Excitatory neuron density was estimated by subtracting GAD67-positve neuron
density from NeuN-positive neuron density. The 1D etv- and glt-pyramid density profiles were then divided by the respective excitatory neuron density profiles, yielding the ratio of
etv- and glt-pyramids to all excitatory neurons at different depth (etv, black line; glt, blue line). The distances were normalized to the pia--white matter distance and registered to
a mean column height to plot etv- and glt-pyramid ratio profiles from different animals on the same axis.
828 Genetically Labeled L5 Pyramidal Neurons
d
Groh et al.
diameter at the base was for glt-pyramids 4.3 ± 1.3 lm, which
was wider (P < 0.0001, unpaired t-test) than for etv-pyramids
(2.7 ± 1.2 lm). Furthermore, for glt-pyramids, the average total
apical dendrite length (4990 ± 1052 lm) was longer than the
average total basal dendrite length (2585 ± 1038 lm; Fig. 2C). In
contrast, for etv-pyramids, the average total apical dendrite
length (2363 ± 823 lm) was shorter (P = 0.0044, unpaired t-test)
than the average total basal dendrite length (3006 ± 994 lm; Fig.
2C). The apical tuft dendrite is the most obvious discriminating
morphological parameter for the 2 cell-types with etv-pyramids
having a shorter total length, fewer nodes, and a smaller width
compared with the glt-pyramids. For the basal dendrite, etvpyramids have more nodes compared with glt-pyramids (Table
1). glt- and etv-pyramids are, thus, morphologically different cell
types corresponding to thick-tufted and slender-tufted pyramidal neurons, respectively (Markram et al. 1997; Schubert et al.
2001, 2006; Larsen and Callaway 2006; de Kock et al. 2007).
L5 in VC was further confirmed by crossing etv-mice with gltmice (Fig. 4). In the glt/etv-hybrid mice, the 2 cell types can be
separated because glt-pyramids have EGFP fluorescence coupled
to ribosomal proteins in the nucleolus (visible as a bright spot in
the soma; Fig. 4B), whereas etv-pyramids have a homogenously
labeled soma and brighter EGFP-labeled apical dendrites. In the
glt/etv-hybrid mice, the layer separation in BC (Fig. 4A,D) and
intermingling in VC was evident (Fig. 4C,E). This raises the
possibility that synaptic inputs to the basal dendrites of these 2
cell types are coming from different sources in BC, whereas in
VC, glt- and etv-pyramids share inputs (e.g., from the pulvinar).
Similar to BC for glt-pyramids (Fig. 3E), the total apical
dendrite length (3867 ± 1288 lm, n = 15) was significantly
longer than the total basal dendrite length (2640 ± 792), and for
the etv-pyramids (Fig. 3E), the total apical dendrite length
(2264 ± 647 lm, n = 21) was shorter than the total basal
dendrite length (2730 ± 773. Compared with BC, the etvpyramids in VC had on average the same number (4) of oblique
dendrites (P = 0.39, unpaired t-test). glt-Pyramids in VC and BC
both had on average 9 oblique dendrites. etv-Pyramids appear as
slender-tufted (apical dendrite tuft width: 182 ± 50 lm; apical
dendrite diameter: 2.7 ± 0.7 lm) and glt-pyramids as thick-tufted
pyramidal neurons (apical dendrite tuft width: 290 ± 92 lm; apical
dendrite diameter: 2.9 ± 1.1 lm). Similar to BC, the dendritic tuft is
Soma Depth Location and Dendrite Morphology of etvand glt-Pyramids in VC
Specific EGFP labeling of 2 pyramid populations in L5 was also
observed in VC (Fig. 3). In contrast to the somatosensory cortex,
the somata of etv- and glt-pyramids are more intermingled in L5
(Compare Fig. 1 and Fig. 3). Intermingling of the soma position in
Figure 2. Morphology of etv- and glt-pyramids in BC; etv- and glt-pyramids were filled with biocytin in the acute slice and reconstructed. (A) Slender-tufted etv-pyramids in layer
5A have a narrow apical dendrite tuft and few oblique dendrites. A part of the axon is shown to illustrate that projections were ipsilateral and contralateral. (B) Thick-tufted gltpyramids have a broad apical tuft and relatively more oblique dendrites. A part of the axon is shown to illustrate that the glt-pyramids had an ipsilateral projection only. (C) On
average, the total apical dendrite length was longer than total basal dendrite length for glt- but not for etv-pyramids. See also Table 1 and supplementary figure 2.
Table 1
Summary of intrinsic properties and morphology of etv- and glt-pyramids in BC and VC
Membrane
potential (mV)
BC
etv
68 ± 5
glt
64 ± 4
VC
etv
68 ± 5
glt
66 ± 4
Comparison between cell types and cortical
etv, BC vs. VC
glt, BC vs. VC
glt vs. etv in BC
glt vs. etv in VC
Input
resistance
(MX)
Threshold
current (pA)
Adaptation
index
Total
apical tuft
length (lm)
No. of
nodes in
apical dendrite
(excluding
obliques)
Tuft width
(lm)
Total basal
length (lm)
No. of nodes
in basal
dendrite
No. of
primary
basal
dendrites
No. of
oblique
dendrites
86 ± 25
53 ± 17
109 ± 25
116 ± 44
regions
158
158
131
103
24 ± 25
1±1
17 ± 15
3±3
944
2323
1021
1784
12
19
11
14
157
356
182
290
3006
2585
2730
2640
29
19
22
21
7
8
8
7
3.7
8.9
4.1
9.3
±
±
±
±
9
11
73
16
± 366
± 757
± 421
± 725
±
±
±
±
7
6
5
5
±
±
±
±
54
84
50
92
±
±
±
±
994
1038
773
792
±
±
±
±
11
8
6
7
±
±
±
±
2
2
2
2
±
±
±
±
2.2
2.6
1.6
3.9
Note: Numbers are derived from pooled data and are given as mean ± SD. Graphical display indicating whether parameters are significantly different (n) or not different (h) for the compared groups.
Cerebral Cortex April 2010, V 20 N 4 829
the most obvious discriminator between the 2 cell types: etvpyramids have a shorter total length and narrower width
compared with glt-pyramid tufts. In contrast to BC, the basal
dendrites of glt- and etv-pyramids are indistinguishable for all
parameters tested here (Table 1). Taken together, the morphological properties show that glt- and etv-pyramids are clearly 2
different cell types in both cortical areas.
Thalamocortical Innervation Fields
To determine the thalamic inputs to etv- and glt-pyramids in
BC, we expressed dTomato via AAV injections into POm and
ventral posterior medial thalamic nucleus (VPM) in order to
map thalamocortical innervation fields. POm axons densely
overlapped with basal dendrites and soma of etv-pyramids
(Fig. 5A). Potentially also the apical tufts of both etv- and gltpyramids were densely innervated in L1 (Fig. 5B). VPM axons
were predominately located in layer 4 and lower L5B. They
more sparsely overlapped with basal dendrites and somata of
glt-pyramids (Fig. 5C). The preferential innervation of different
populations of L5 pyramidal neurons by VPM and POm axons
confirms that the different thalamic nuclei can be part of
separate thalamocortical circuits (Ahissar and Kleinfeld, 2003;
Bureau et al. 2006).
Projection Fields of Somatosensory L5 etv- and gltPyramids
The knowledge of cortical long-range projections to subcortical targets is relevant for elucidating the possible function
of corticofugal projections in, for example, driving behaviors
that depend on sensory input.
The subcortical targets of the 2 populations of pyramidal
neurons in BC were determined by injections of retrograde
tracers (in 5 animals) into striatum, POm, and pons (Fig. 6). Gltpyramids projected to ipsilateral pons (Fig. 6A) and POm (data
not shown) but not to striatum (Fig. 6A). The depth
distribution of glt-pyramids overlapped with retrogradely
labeled corticopontine cells but was well separated from
corticostriatal cells (Fig. 6E). Glt-pyramids (9 of 9 cells) at the
level of the white matter had only an ipsilaterally projecting
main axon, thus, projecting to thalamus and pons and also likely
to the spinal cord and trigeminal nucleus (Wise and Jones
1977). Etv-pyramids projected to ipsilateral striatum, and to
some extent to contralateral striatum (1%, 2 of 156 cells), but
not to pons (Fig. 6C) or POm (data not shown). In 5 etvpyramids, the axon could be traced to the white matter, where
4 of 5 cells had a bifurcating main axon (Figs. 2 and 8),
indicating both an ipsilateral and contralateral projection. The
majority of etv-pyramids in BC, thus, appear to project both
ipsilaterally and contralaterally. The retrograde labeling indicated, however, that a minority of the etv-pyramids project to
contralateral striatum and, thus, the contralateral projection,
presumably, targets mainly contralateral cortex. The depth
distribution of etv-pyramids overlapped with retrogradely
labeled corticostriatal cells but was largely separated from
corticopontine cells (Fig. 6F); etv is, thus, inferred to be
expressed in a subpopulation of L5A slender-tufted pyramidal
Figure 3. Layer distribution and dendrite morphology of EGFP-labeled etv-pyramids
and glt-pyramids in L5 of the VC. Confocal images of EGFP-labeled pyramidal neurons
in brain slices (coronal sections) of transgenic mice carrying the BAC constructs for
(A) etv or (B) glt. (C) In the acute brain slice, EGFP-labeled cells in L5 were filled with
biocytin and reconstructed. Slender-tufted etv-pyramids had a relatively narrow apical
830 Genetically Labeled L5 Pyramidal Neurons
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Groh et al.
dendrite tuft and few oblique dendrites. (D) Thick-tufted glt-pyramids had a broad
apical dendrite tuft and relatively more oblique dendrites. (E) On average, the total
apical dendrite length was larger than total basal dendrite length for glt- but not for
etv-pyramids. See also Table 1.
Figure 4. Layer distribution of etv- and glt-pyramids in glt--etv chimera mice. (A) Confocal images of EGFP-labeled pyramidal neurons in brain slices (coronal sections) showing
the layer separation in BC. (B) Enlarged section of the cells marked in (A); etv-pyramids are homogeneously EGFP labeled (triangles), whereas glt-pyramids have a labeled
nucleolus and more faintly labeled cell cytoplasm compared with the cell membrane (circles with dot). This difference was used to identify the different cells types. (C) In VC cells
were less well separated. (D) Symbols mark the position of etv- (triangle) and glt-pyramids (circle with dot) derived from the confocal image in (A). (E) Symbols mark the position
of etv- (triangle) and glt-pyramids (circle with dot) derived from the confocal image in (D). Scale bar in (A) applies to (A), (C), (D), and (E).
Figure 5. Thalamocortical innervation of etv- and glt-pyramids in BC. (A) POm axons (red) innervate L5A with an extensive overlap with somata and basal dendrites of etvpyramids (green). (B) POm axons (red) also densely project to layer 1. Blue cells are retrogradely labeled POm projection neurons. Note that POm axons are restricted to the upper
layer 5 (L5A), whereas cortical neurons projecting to POm are located in deep L5 and layer 6. (C) VPM axons (red) innervate L4 and glt-pyramids (green) in L5B, predominately the
deeper cells. Scale bars, 100 lm.
neurons projecting to the ipsilateral striatum and contralateral
cortex, with a minority projecting also to contralateral striatum
and with no projections to pons. The target specificity of etvand glt-pyramids is furthermore indicated by EGFP labeling in
striatum in etv-mice and in pons in glt-mice (Supplementary
Fig. 3).
Similarly, in VC, the glt-pyramids project to pons (Fig. 6B),
but not striatum (Fig. 6B), and etv-pyramids project to striatum
(Fig. 6D) but not to pons (Fig. 6D). Compared, however, with
BC, there were relatively fewer retrogradely labeled etvpyramids (Fig. 6C,D), and the depth distribution of etvpyramids did not overlap to the same degree with retrogradely
labeled corticostriatal cells (Fig. 6H). This difference could
indicate that, in VC, the etv-pyramids are a relatively small
subpopulation of the corticostriatal cells. We conclude that in
VC and BC slender-tufted L5 etv-pyramids are corticostriatal
and that the majority of thick-tufted L5 glt-pyramids are
corticopontine and thalamic projection neurons.
In BC, the depth distribution of retrogradely labeled
corticostriatal and corticopontine cells was well separated
(Fig. 6E,F), whereas in VC, there was a larger overlap
(Fig. 6G,H). This difference indicates that, at least in some
parts of the VC, the corticostriatal pyramidal neurons are to
a higher degree than in BC located at the same depth as
Cerebral Cortex April 2010, V 20 N 4 831
Figure 6. Retrograde labeling of cortical projection neurons. Retrograde tracer cholera toxin subunit B (CT-B) Alexa 555 (red) was injected in striatum, and CT-B Alexa 647 (blue)
was injected into pons in glt-mice (glt) and etv-mice (etv). Confocal images showing the distribution of labeled cells in BC and VC. All images are ipsilateral to the injection site.
Cell count numbers refer to the total and not only to the depicted image. The total number of cells counted depended on the size of the cell counting frame. Scale bars, 100 lm.
(A) In BC, glt-pyramids were double labeled by pons (blue) injection (16%, 202 of 1271 cells). Corticostriatal cells (red) were predominately found in the upper L5 and did not label
glt-pyramids (green). (B) In VC, the glt-pyramids (green) were intermingled with corticostriatal cells (red) but were only double labeled by the pons injection (13%; 44 of 337
cells). (C) etv-Pyramids in BC were double labeled by the striatum (red) injection (11%, 38 of 336 cells) but not by the pons injection (blue). (D) In VC, retrogradely labeled
corticopontine and corticostriatal cells were to a higher degree intermingled. etv-Pyramids were double labeled by the striatum (red) injection (5%, 9 of 188 cells) but not by the
pons injection (blue). (E--H) Distribution in VC and BC of etv-pyramids (etv), glt-pyramids (glt), retrogradely labeled corticostriatal (striatum), and corticopontine (pons) cells plotted
as a function of soma depth from the pia. Filled circle shows the median, and error bars show 25--75 percentiles.
corticopontine cells. This finding supports the observation that
somata of etv- and glt-pyramids are less well separated in VC
(Fig. 4). This is, thus, potentially an anatomical substrate for a less
specific thalamocortical innervation of infragranular neurons.
The projection patterns of etv- and glt-pyramids are in
agreement with those inferred from EGFP antibody staining
(http://www.gensat.org). The layer localization of retrogradely
labeled corticostriatal and corticopontine cells is similar to
previously reported results (Wise and Jones 1977; Wiesendanger and Wiesendanger 1982; Akintunde and Buxton 1992;
Jinno and Kosaka 2004; Hattox and Nelson 2007).
Intrinsic Membrane Properties of etv- and glt-Pyramids in
BC and VC
Cell-type and area specificity of intrinsic membrane properties
was then investigated. The resting membrane potential was –68
± 1 mV for etv-pyramids in both BC and VC. glt Neurons had
a resting membrane potential of –64 ± 1 mV in BC and –66 ± 1mV
in VC. Thus, in contrast to VC, in BC, etv- and glt-pyramids have
significantly different membrane potentials.
The input resistance (MOhm) for etv-pyramids was cortex
specific with 86 ± 4 (n = 45) in BC and 110 ± 4 (n = 33) in VC
(unpaired two-tailed t-test, P < 0.0001). Furthermore, input
resistances of etv- and glt-pyramids were indistinguishable in VC
but were different in BC (VC: etv 110 ± 4 MOhm and glt 116 ±
13 MOhm; BC: etv 86 ± 4 MOhm and glt 53 ± 3 MOhm).
Threshold current was dependent on the cortical area for the
glt-pyramids but not for etv-pyramids and was furthermore not
significantly different between glt-pyramids and etv-pyramids
in both areas (in pA) etv-pyramids 158 ± 9 in BC and 131 ± 12
in VC and 2) glt-pyramids 158 ± 11 in BC and 103 ± 5 in VC).
Spike frequency adaptation was quantified by measuring the
average time interval between successive spikes to calculate an
‘‘adaptation index’’ (see Materials and Methods). Adaptation
index was for: 1) etv-pyramids 23 ± 27 in BC and 17 ± 17 in VC.
832 Genetically Labeled L5 Pyramidal Neurons
d
Groh et al.
The relatively high adaptation index indicates that the interspike
interval increased between successive spikes (Fig. 7A,B; Supplementary Fig. 4A,B) and 2) glt-pyramids 1 ± 2 in BC and 2 ± 2 in
VC, indicating a close to constant interspike interval (Fig. 7C,D;
Supplementary Fig. 4C,D); etv- and glt-pyramids can, thus, be
referred to as adapting and nonadapting, respectively. Among
the glt-pyramids, approximately 50% and 20% of the cells were
intrinsically bursting in BC and VC, respectively (Fig. 7E,F;
Supplementary Fig. 4E,F). glt-Pyramids responding with bursts
of action potential (Fig. 7E,F; Supplementary Fig. 4E,F) were not
included in the calculation of the adaptation index.
A comparison between etv- and glt-pyramids showed that
etv-pyramids in BC were more hyperpolarized and had a higher
input resistance than glt-pyramids. The adaptation index was
higher for etv-pyramids in both areas. Threshold current was
cortex specific only for glt-pyramids, but input resistance was
cortex specific for both cell types. Intrinsic electrical properties are subject to both, region, and cell-type specificity: etvand glt-pyramids have cell-type--specific properties in both
sensory cortices, concomitantly showing a region specificity.
Monosynaptic Connections between etv-pyramids and gltpyramids
Paired whole-cell recordings were made to investigate cell-type
and area dependency of synaptic properties. A monosynaptic
connection between etv-pyramids in BC (Fig. 8, Supplementary
Fig. 5A) was found in 13 of 76 recorded pairs (17%; 9
unidirectional pairs and 2 bidirectional pairs). The average
monosynaptic excitatory postsynaptic potential (EPSP) amplitude was 0.39 ± 0.26 mV (range: 0.09--0.82 mV, n = 13 pairs),
with a coefficient of variation (CV) of 0.46 ± 0.11. For EPSP
amplitudes larger than 0.23 mV, the failure rate was low (range:
0--3%, n = 6), but increased for smaller EPSP amplitudes (failure
range: 8--28%, n = 3). The paired-pulse ratio at an interstimulus
Figure 7. Intrinsic electrical properties of etv-pyramids and glt-pyramids in BC and VC. Examples of action potential trains evoked by current injection in 6 different cells (A--F).
etv-Pyramids in BC (A) and VC (B) showed spike frequency adaptation. glt-Pyramids in BC (C) and VC (D) were either regular spiking with no frequency adaptation or intrinsically
bursting (E, F). Inset in (E, F) shows with a higher magnification that burst of spikes were elicited in intrinsically bursting cells. Scale bar in (A) applies to all figures, except time
scale for insets in (E) and (F). Square pulse shows current injection. Current was 300 pA in (A), (C), and (E) and 240 pA in (B), (D), and (F). See summary table 1 for numbers.
Figure 8. Synaptic transmission between slender-tufted etv-pyramids in BC and VC. (A) Two-photon image in the acute slice of EGFP-labeled etv-pyramids (upper panel) in BC.
The same field of view in differential interference contrast (lower panel). Star marks the same cells in both panels. (B) Reconstruction of 2 etv-pyramids. (C) The etv-pyramids
reconstructed in (B) were bidirectionally connected. Calibration: EPSP trace 0.2 mV, 100 ms; action potential (AP) trace 40 mV, 100 ms. (D) Amplitude ratios for etv-pyramid pairs
in BC between successive EPSPs and the first EPSP at different stimulation frequencies (EPSP2/EPSP1 5 2/1). Average of all tested connections (mean, SD for data points: 10 Hz:
2/1: 0.91 ± 0.19, 3/1: 0.66 ± 0.33, 4/1: 0.60 ± 0.16, 5/1: 0.53 ± 0.29; 20 Hz: 2/1: 0.79 ± 0.52, 3/1: 0.68 ± 0.44, 4/1: 0.54 ± 0.60, 5/1: 0.42 ± 0.39; 40 Hz: 2/1: 0.82 ±
0.38, 3/1: 0.74 ± 0.72, 4/1: 0.50 ± 0.59, 5/1: 0.35 ± 0.39). (E) Reconstruction of 2 connected etv-pyramids. (F) Eliciting action potentials in cell-1--evoked EPSPs in cell-2 that
decreased in amplitude with successive APs. Calibration: EPSP trace 1 mV, 200 ms; AP trace 100 mV, 200 ms. (G) Amplitude ratios for etv-pyramid pairs in VC between
successive EPSPs and the first EPSP at different stimulation frequencies (mean, SD for data points: 10 Hz: 2/1: 0.80 ± 0.36, 3/1: 0.75 ± 0.348, 4/1: 0.53 ± 0.27, 5/1: 0.55 ±
0.38; 20 Hz: 2/1: 0.62 ± 0.34, 3/1: 0.46 ± 0.28, 4/1: 0.36 ± 0.31, 5/1: 0.28 ± 0.21; 40 Hz: 2/1: 0.55 ± 0.32, 3/1: 0.35 ± 0.23, 4/1: 0.35 ± 0.35, 5/1: 0.30 ± 0.26).
Cerebral Cortex April 2010, V 20 N 4 833
interval of 100 ms was 0.91 ± 0.19. The connection was
depressing in the 10- to 40-Hz stimulation range (Fig. 8C,D).
Synaptic connectivity between glt-pyramids in BC (Supplementary Fig. 6) was less extensively investigated, but the data
suggest that these cells are connected with a lower connection
probability (6%; 2 connected pairs of 35) compared with etvpyramids.
To compare synaptic properties within a pyramidal cell
population in different cortical areas, we also made whole-cell
recordings from pairs of etv-pyramids in the VC (Fig. 8E--G,
Supplementary Fig. 5B). A monosynaptic connection was found
in 6 of 33 (18%) recorded pairs. The average monosynaptic
amplitude was 0.82 ± 0.94 mV (range: 0.08--2.3 mV, n = 6 pairs),
with a CV of 0.43 ± 0.20. For EPSP amplitudes larger than 0.23
mV, the failure rate was low (range 0--3.8%, n = 4) but increased
for smaller EPSP amplitudes (failure: 9 and 18%, n = 2). Similar to
the BC, the etv--etv pyramid connection was depressing in the
10- to 40-Hz stimulation range (Fig. 8G). The paired-pulse ratio
at an interstimulus interval of 100 ms was 0.80 ± 0.36. The
similar synaptic properties and connectivity (chi-square test
0.89) for connected etv-pyramids in BC and VC suggest that
within a population of cells synaptic properties may be retained
across different cortical areas. Furthermore, corticostriatal etvpyramids appeared more interconnected than corticopontine/
thalamic glt-pyramids.
Comparison between Cortices
The majority of properties of etv- and glt-pyramids were similar
in VC compared with the properties of the corresponding cell
type in BC, although with some notable differences outlined
below. A given cell type shows variations in dendrite geometry
depending on the cortical region. Compared with BC, etv cells
in VC have fewer basal dendrite nodes, but the same total
length. The basal dendrite tree of glt-pyramids is similar in BC
and VC, but in VC, the tuft dendrite is shorter and has fewer
nodes (Table 1). The soma depth was also characteristic for the
cortical region with a clear separation of glt- and etv-pyramids
in BC and the tendency to share the layer in VC. Taken
together, the anatomical data suggest that etv- and glt-pyramids
are more similar to each other in VC and more distinct in BC.
Differences between the cortical regions could also be
observed for intrinsic electrical properties. Both cell types
had a lower input resistance and needed larger currents to
reach spiking threshold in BC. In summary, the differences for
a given cell type in different areas suggests that a basic cell
layout is fine tuned to the cortical area and by the sensory
system, respectively.
Discussion
Untangling the diversity of cortical cell types requires the
combination of genetic targeting, quantification of cell anatomy
and physiology, and eventually the molecular characterization
of the different cell types (Luo et al. 2008). We have
undertaken this approach to provide a detailed histological
and physiological characterization of L5 neocortical cell types
using BAC-transgenic mice. This allows evaluation of the extent
of conservation of cellular properties in different cortical areas
and the influence of the sensory modality on L5 neurons. In
both VC and somatosensory cortex, the Etv1 line had selective
EGFP expression in ‘‘slender-tufted’’ L5 pyramidal neurons, and
the Glt25d2 line had selective EGFP-expression in ‘‘thick834 Genetically Labeled L5 Pyramidal Neurons
d
Groh et al.
tufted’’ L5 pyramidal neurons. Etv-pyramids in both somatosensory cortex and VC share characteristic properties compared with glt-pyramids, being more hyperpolarized, having
a higher input resistance, an adapting spiking pattern, and
being more densely interconnected. In BC, the slender-tufted
corticostriatal etv-pyramids were predominately found in
upper L5 and the thick-tufted corticopontine/thalamic gltpyramids in lower L5. In contrast, in VC, the 2 populations
were not well separated, suggesting a more common thalamic
innervation of these cell types in VC but not in BC. A cell
classification in L5 based on soma--dendrite morphology,
intrinsic electrical excitability, properties of synaptic transmission and gene expression can, thus, differentiate between
cells with different projection targets. The 2 genetically defined
L5 pyramidal neuron types were found to have in general
conserved properties in BC and VC, with the notable
anatomical exceptions and, thus, a dependency on the sensory
modality (touch or vision), in apical dendrite shape and layer
organization.
Subpopulations of Pyramidal Neurons in L5
In BC, 16% of all neurons located in L5 were etv-pyramids and
9% were glt-pyramids. Within the layer, however, the local
density of labeled pyramidal neurons was significantly higher
(Fig. 1C,D). By means of retrograde labeling, the fraction of
corticostriatal or corticothalamic cells in L5 of mouse
somatosensory cortex has been estimated to 26% and 8%,
respectively, of the total number of neurons (Hattox and
Nelson 2007). Because fewer etv-pyramids are labeled than this
estimated total fraction of corticostriatal cells, it is likely that
Etv1 labels a subpopulation of corticostriatal (slender-tufted)
pyramidal neurons. Because etv-pyramids have callosally
projecting axons, they are likely to be part of the intratelencephalically projecting type of corticostriatal neurons
(Wilson 1987; Reiner et al. 2003). The thalamic POm projection
of glt-pyramids is, most likely, a collateral of the main axon
projecting predominately to pons and POm, but also to other
brainstem nuclei and the spinal cord (Veinante et al. 2000),
suggesting an anatomical substrate for efference copy of motor
instructions to POm.
In comparison to related work on the classification of
pyramidal neurons in L5, the dendrite morphology and spike
frequency adaptation of etv- and glt-pyramids are similar to
those found in retrogradely labeled corticostriatal and corticothalamic pyramidal neurons, respectively (Hattox and Nelson
2007). Furthermore, potassium channel Kv3.1-expressing L5
pyramidal neurons (Akemann et al. 2004) which, with respect
to their dendrite morphology and spike frequency adaptation,
are similar to the etv-pyramids are described here, with one
noticeably difference. The Kv3.1-expressing cells made local
intracortical connections only and did not have axons
projecting to subcortical targets. Taken together with our
results, this comparison suggests that slender-tufted pyramidal
neurons in L5 are a very heterogeneous group of neurons. It
may consist of at least 2 subpopulations, corticocortical and
corticofugal projecting neurons (Ivy and Killackey 1981;
Hattox and Nelson 2007; Le Be et al. 2007). According to the
anatomical classification by Tsiola et al. (2003), the L5 gltpyramids in VC appear to fall in the class denoted ‘‘subgroup
1A’’ and the L5 etv-pyramids in the class denoted ‘‘subgroup
1B.’’ Pyramidal cells in L5 that are organized in cell clusters
(Krieger et al. 2007) are similar to the glt-pyramids; these
clustered pyramidal neurons were located in lower L5 and
projected to POm and pons. The less significant bundling of
apical dendrites originating from glt-pyramids suggests, however, at most a partial overlap between the cell types. Notably,
the glt- and etv-pyramids project to pons/POm or striatum,
respectively, but not to both areas. There were, however, non-EGFP-labeled cells in L5 that were double labeled by tracer
injections into pons and striatum (data not shown; compare
Levesque et al. 1996). A possible difference in the cell-type-specific properties of etv- and glt-pyramids in other cortices,
notably the motor cortex (Miller et al. 2008), remains to be
investigated. Genetically defined cell types, thus, enable
a detailed investigation of epigenetic factors controlling the
formation of local cortical circuits in different cortical areas.
Using Genetic Markers to Understand the Contribution of
Different Cell Types to the Animal’s Behavioral Repertoire
Genetic markers that define specific cell-types can be used to
selectively manipulate cortical circuits. This will aid to understand the contribution of a particular pyramidal cell type in
driving behaviors. Conditional silencing, for example, of pyramidal neurons in L5 could unravel their contribution to learning
and execution of behaviors. The transcription factor Etv1 (also
referred to as ER81) has been shown to participate in neurogenesis in the mouse olfactory bulb (Stenman et al. 2003) and
circuit formation in the spinal cord (Arber et al. 2000). Taken
together with the present results, this indicates that etvdependent proteins can participate in the formation of neuronal
circuits in different areas of the central nervous system.
The restricted expression of etv in L5 is consistent with
previous studies (Xu et al. 2000; Sugitani et al. 2002; Beggs et al.
2003; Hasegawa et al. 2004; Watakabe et al. 2007) and with the
in situ hybridization data documented in the Allen Brain Atlas
(http://www.brain-map.org). However, expression differs in
essential aspects from previous reports of dendrite morphology, functional properties, and projection targets of etvexpressing cells. Yoneshima et al. (2006) reported that ER81
was expressed in non-tufted cells, was equally distributed in L5,
and was expressed in neurons projecting to the spinal cord or
superior colliculus, whereas Sugino et al. (2006) report that
ER81 mRNA was detected in non-adapting L5 and L6 cells. The
reasons for these discrepancies remain unclear. We note that
the restricted expression to L5 reported for the Etv1 EGFPreporter mice used in the present study, and that reported for
an independently generated Etv1 BAC Cre recombinase driver
line (Gong et al. 2007) agrees well.
Cell-Type Specific Properties in Somatosensory and VC
In both somatosensory cortex and VC, etv-pyramids were
distinct from glt-pyramids on the basis of the soma--dendrite
morphology, intrinsic membrane properties, and projection
target. Furthermore, the properties of synaptic transmission
between etv-pyramids in the 2 cortices were similar, with
a connectivity ratio of approximately 20%. The connectivity for
glt-pyramids in BC was estimated, based only on a limited
sample, to 6% which is similar to the 10% estimated for
clustered thick-tufted pyramidal neurons in L5 (Krieger et al.
2007). In rat VC, the connectivity ratio for thick-tufted L5
pyramidal neurons has also been estimated to approximately
10% (Song et al. 2005). A similar difference in connection
probability for L5 pyramidal neurons projecting to different
targets has been observed in rat neocortex (Morishima and
Kawaguchi 2006; Le Be et al. 2007).
Comparing etv and glt pyramidal neurons, we find that they
are clearly distinguishable based on morphology, intrinsic, and
synaptic properties and long-range circuitry. However, when
comparing individual cell types in BC and VC, we find that the
difference between cell types (etv vs. glt) is much more
pronounced than the differences of these cell types between
different cortical areas. The individual cell types are, thus,
influenced by the cortical region they belong to, but the overall
layout of etv- and glt-pyramids is retained. Our results, thus,
raise the intriguing possibility that genes can be identified that
characterize cell types which not only share morphology but
are also incorporated in comparable local circuits with
a stereotypic layout in different cortical areas. It can, thus, be
concluded that for a genetically defined neuron population,
cell-type--specific properties such as soma--dendrite morphology, intrinsic electrical excitability, projection target, and
properties of synaptic transmission are not necessarily unique
for a cortical area. They may be retained, with some noted
modification, between sensory cortical areas, and possibly
other neocortical areas.
Supplementary Material
Supplementary Table 1 and Figs 1--6 can be found at: http://
www.cercor.oxfordjournals.org/.
Notes
We thank Randy Bruno and Gilad Silberberg for comments on the
manuscript, Rolf Sprengel for AAV-dTomato, and Marlies Kaiser for
excellent technical assistance. This work was supported by Åke
Wibergs stiftelse to P.K. Author contributions: Conceived and designed
the experiments: A.G., H.S.M., B.S., and P.K. Performed the experiments:
A.G., H.S.M., and P.K. Analyzed the data: A.G., H.S.M., B.S., and P.K.
Contributed reagents/materials/analysis tools: H.S.M., E.S., and N.H.
Wrote the paper: A.G., B.S., and P.K. Conflict of Interest: None declared.
Address correspondence to Patrik Krieger, Nobel Institute for
Neurophysiology, Department of Neuroscience, Karolinska Institutet,
SE-171 77 Stockholm, Sweden. Email: [email protected].
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