J Neurophysiol 110: 795– 806, 2013.
First published May 15, 2013; doi:10.1152/jn.00071.2013.
Common excitatory synaptic inputs to electrically connected cortical fastspiking cell networks
Takeshi Otsuka and Yasuo Kawaguchi
Division of Cerebral Circuitry, National Institute for Physiological Sciences, Okazaki, Aichi, Japan; Department of
Physiological Sciences, Graduate University for Advanced Studies (SOKENDAI), Okazaki, Aichi, Japan; and JST, CREST,
Tokyo, Japan
Submitted 29 January 2013; accepted in final form 15 May 2013
fast-spiking cell; gap junction; cortex; pyramidal cell
THE NEOCORTEX IS COMPOSED of various types of excitatory and
inhibitory cells, which form intricate networks (Kawaguchi
and Kubota 1997; Markram et al. 2004). Although inhibitory
interneurons account for only ⬃20% of total neocortical cells,
these cells play important roles in activity regulation of neocortical circuits. Excitatory pyramidal cell activity is regulated
at various domains by GABAergic inputs from specific interneuron types. Dysfunction of inhibitory systems induces
abnormal electrical activity patterns and causes various pathologies such as epilepsy, anxiety, depression, and schizophrenia
(Lewis et al. 2005; Powell et al. 2003; Sanacora et al. 2000).
Parvalbumin-positive fast-spiking (FS) cells constitute
⬃40 –50% of cortical inhibitory interneurons (Uematsu et al.
2008). They innervate the soma and proximal dendrites of
pyramidal cells, indicating a powerful impact on pyramidal cell
spiking activities (Somogyi et al. 1998). Reciprocal excitatory
and inhibitory connections between pyramidal and FS cells
have been proposed as cellular mechanisms underlying the
temporal patterning of population activity and the generation of
oscillations related to high-order brain functions (Buzsaki and
Chrobak 1995; Traub et al. 1997). The electrical coupling
between FS cells is a distinctive feature of the cell type. FS
cells selectively interconnect with each other through gap
Address for reprint requests and other correspondence: T. Otsuka, Div. of
Cerebral Circuitry, National Inst. for Physiological Sciences, 5-1 MyodaijiHigashiyama, Okazaki, Aichi 444-8787, Japan (e-mail: otsuka@nips.ac.jp).
www.jn.org
junctions and form dendritic net structures extending to different functional columns (Amitai et al. 2002; Fukuda et al. 2006;
Galarreta and Hestrin 1999; Gibson et al. 1999). Electrical
coupling between cells is crucial for the regulation of FS cell
activities including synchronous spike discharges in response
to synaptic inputs (Galarreta and Hestrin 2001b). However,
how excitatory pyramidal cells innervate a group of electrically
connected FS cells has remained largely unexplored.
Cortical cells including FS interneurons are also known to
adopt different membrane potential states according to behavior modifications (Gentet et al. 2010; Steriade et al. 2001).
Cortical cells are continuously depolarized in the awake attentive state and oscillate between depolarized and hyperpolarized
potentials in the inattentive state (Gentet et al. 2010). Furthermore, during deep sleep membrane potentials of cortical cells
synchronously fluctuate between discrete depolarizing events,
called “Up state,” and inter-Up state hyperpolarization, known
as “Down state” (Contreras and Steriade 1995). Although
functional properties of gap junctions between FS cells have
been well investigated (Galarreta and Hestrin 2001a), how
electrical communication between FS cells depends on network state remains unknown.
Here we investigated excitatory synaptic input patterns from
pyramidal cells to electrically connected FS cell networks in
frontal layer V (L5). We found that pyramidal cells frequently
coinnervate electrically interconnected FS cells. In the depolarized state, spike activities in FS cells suppressed nearby
electrically connected FS cells. In the hyperpolarized state,
however, either sub- or suprathreshold inputs induced excitatory potentials in postsynaptic FS cells. Although FS cells are
globally connected with electrical synapses, our experimental
and simulation results suggest that FS cell networks are locally
regulated, especially in the depolarized state, by shared inputs
from pyramidal cells to electrically coupled cells.
MATERIALS AND METHODS
Electrophysiology. All experiments were carried out under a protocol approved by the Experimental Animal Care Committees, Okazaki National Research Institutes. To identify inhibitory interneurons
in slice preparations easily, VGAT-Venus transgenic rats expressing
fluorescent protein Venus in ␥-aminobutyric acid (GABA)ergic neurons were used (Uematsu et al. 2008). VGAT-Venus transgenic rats
were generated by Drs. Y. Yanagawa, M. Hirabayashi, and Y. Kawaguchi at the National Institute for Physiological Sciences, using
pCS2-Venus provided by Dr. A. Miyawaki. VGAT-Venus rats are
distributed by The National BioResource Project for the Rat in Japan
(http://www.anim.med.kyoto-u.ac.jp:80/nbr/default.aspx). Brain
slices from wild-type Wistar rats were also obtained. Slices were
0022-3077/13 Copyright © 2013 the American Physiological Society
795
Downloaded from http://jn.physiology.org/ by 10.220.33.5 on September 30, 2016
Otsuka T, Kawaguchi Y. Common excitatory synaptic inputs
to electrically connected cortical fast-spiking cell networks. J
Neurophysiol 110: 795– 806, 2013. First published May 15, 2013;
doi:10.1152/jn.00071.2013.—Cortical fast-spiking (FS) interneurons are electrically interconnected through gap junctions and form
dendritic net structures extending over different functional columns.
Here we investigated how pyramidal cells regulate FS cell network
activity. Using paired recordings and glutamate puff stimulations, we
found that FS cell pairs connected by electrical synapses shared
common inputs from surrounding pyramidal cells more frequently
than those unconnected or connected only by chemical synapses.
Experimental and simulation results suggest that activity spread
evoked by common inputs to electrically connected FS cells depends
on network state. When cells were in the depolarized state, common
inputs to electrically connected cells enhanced spike induction and
induced inhibitory effects in surrounding FS cells. By contrast, in the
hyperpolarized state, either sub- or suprathreshold inputs produced
depolarizing potentials in nearby cells. Our results suggest that globally connected FS cell networks are locally regulated by pyramidal
cells in an electrical connection- and network state-dependent manner.
796
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
vation threshold and a faster activation time constant as features of
Kv3-type K⫹ currents, and IKd, which has a lower activation threshold
and a slower inactivation time constant. On the other hand, dendrites
of the model have only IK and Il. The membrane potentials of the
soma and dendrite of the FS model cell are described by
Cm
Cmd
dV
dt
dVd
dt
⫽ ⫺INa ⫺ IK ⫺ IKd ⫺ I1 ⫺ Idend1 ⫺ Idend2
⫽ ⫺IK ⫺ I1 ⫺ Isoma
where Cm and Cmd are the membrane capacitances of the soma and
dendrite and are assigned values of 1 and 0.3 ␮F/cm2, respectively; V
and Vd are the membrane potentials of the soma and dendrite; and
Isoma, Idend1, and Idend2 are the total currents of the soma and two
dendrites. Coupling conductance between the dendrite and the soma
was set to 0.4 mS. The ionic currents in the model are given by the
following Hodgkin-Huxley type equations:
INa ⫽ gNam3h共␯ ⫺ ␯Na兲
I K ⫽ g Kn 4共 ␯ ⫺ ␯ K兲
IKd ⫽ gKda2b共␯ ⫺ ␯K兲
I 1 ⫽ g 1共 ␯ ⫺ ␯ 1兲
where a, b, h, m, and n are activation and inactivation variables; vNa,
vK, and vl are the reversal potentials of the sodium, potassium, and
leak currents, respectively (in mV); and gNa, gK, gKd, and gl are the
maximal conductances (in mS/cm2). The gating kinetics of the ionic
conductances are governed by equations of the following form:
dw
dt
⫽
w ⬁共 ␯ 兲 ⫺ w
␶w
where w stands for one of a, b, h, m, and n, and the steady-state
activation and inactivation functions are given by
w⬁ ⫽
1
1 ⫹ exp关共␯ ⫺ ␪w兲 ⁄ kw兴
where ␪w and kw are the half-activation/half-inactivation voltage and
slope, respectively. The activation time constants (␶w in ms) for IK and
IKd are given by the following bell-shaped function:
␶w ⫽ ␶0x ⫹ ␶1x ⁄ 兵exp关⫺ 共v ⫺ ␪␶x1兲 ⁄ ␴1x 兴 ⫹ exp关⫺ 共␯ ⫺ ␪␶x2兲 ⁄ ␴2x 兴其
When membrane potentials are less than ⫺65mV, ␶w for IK and IKd
are assigned constant values of 0.6 and 1.0 ms, respectively. The
inactivation time constant for INa is given by the following sigmoidal
function:
␶h ⫽ 0.5 ⫹ 14 ⁄ 兵1 ⫹ exp关共v ⫹ 60兲 ⁄ 12兴其
The other activation and inactivation time constants were assumed
as constant (in ms). Parameter values used in simulations are given in
Table 1.
To examine activity transmissions between FS model cells, electrical connections were made on one of the dendritic compartments.
For chemical synaptic inputs, we assumed that transmitters were
released to the cleft for 1 ms when the membrane potential of the
presynaptic cell overshot the threshold potential (0 mV). During this
period, postsynaptic conductance change is given by the following
equation:
dr
dt
⫽ ␣共 1 ⫺ r兲 ⫺ ␤r
where ␣ and ␤ are the binding and unbinding rate of transmitters and
are assigned values of 0.3 and 0.1 ms⫺1, respectively. Transmitters are
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prepared from animals aged postnatal 19 –24 days, as described
previously (Otsuka and Kawaguchi 2008). Brains were cut in an
ice-cold solution containing (in mM) 90 N-methyl-D-glucamine, 40
choline Cl, 2 KCl, 1.25 NaH2PO4, 1.5 MgCl2, 0.5 CaCl2, 26 NaHCO3,
and 10 glucose (310 ⫾ 5 mosM, pH 7.4 adjusted with HCl). Slices
including frontal cortex (300-␮m thickness) were incubated in an
oxygenated artificial cerebrospinal fluid (ACSF) composed of (in
mM) 126 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 26
NaHCO3, and 10 glucose (310 ⫾ 5 mosM, pH 7.4; bubbled with 95%
O2-5% CO2) containing 0.2 mM ascorbic acid and 4 mM lactic acid
at room temperature. Whole cell recordings were obtained from
frontal L5 cells with an EPC-9 double amplifier (HEKA Elektronik,
Lambrecht/Pfalz, Germany). The bath solution temperature in the
recording chamber was adjusted to 30°C. The recording pipettes were
filled with a solution containing (in mM) 130 potassium methylsulfate, 0.5 EGTA, 2 MgCl2, 2 Na2ATP, 0.2 GTP, 20 HEPES, and 0.1
leupeptin with 0.75% biocytin (pH 7.2, 290 ⫾ 5 mosM). Glutamate
was focally applied to a L5 pyramidal cell to evoke a spike in the cell
(Otsuka and Kawaguchi 2008, 2009, 2011). Sodium glutamate (1
mM) was dissolved in ACSF and filled the same pipettes as those for
whole cell recordings. The glutamate-filled pipettes were positioned
within 5 ␮m from the cell soma. Glutamate was ejected from the
pipettes by air puffer (50-ms duration and pressure ⬍ 10 psi). We
stimulated cells generally located within 300 ␮m of recording cells,
because the primary cluster of pyramidal cell axons around their soma
is roughly 300 ␮m in diameter (Brown and Hestrin 2009; Gilbert and
Wiesel 1983). In experiments examining excitatory inputs from pyramidal cell subtypes, pyramidal cells projecting to the contralateral
frontal cortex [commissural (COM) cells] and the ipsilateral pontine
nuclei [corticopontine (CPn) cells] were identified with retrograde
fluorescent tracers injected in vivo into the target brain areas of
animals anesthetized with ketamine (40 mg/kg im) and xylazine (4
mg/kg im), as described previously (Otsuka and Kawaguchi 2011).
The injection coordinates of contralateral frontal cortex and ipsilateral
pontine nuclei were COM: 4, 1.5–2.5, 0.5– 0.8 and CPn: 5.6, 0.5–1, 9,
respectively (anterior to bregma, lateral to bregma, depth in mm).
After tracer injections, animals were fed for 2–3 days for recovery and
tracer transportation. Alexa Fluor 555-conjugated cholera toxin subunit B (Invitrogen) and rhodamine-labeled latex microspheres (Lumafluor) were used to distinguish labeled cells in the same preparation.
Data are represented as means ⫾ SD, and statistical differences
between samples were tested by ANOVA with Tukey post hoc tests,
unless otherwise specified. Significance was accepted at P ⬍ 0.05.
Morphological analysis. Slices containing cells intracellularly labeled with biocytin were fixed with a solution containing 4% paraformaldehyde, 1.25% glutaraldehyde, and 0.2% picric acid in phosphate buffer (PB) and resectioned at a thickness of 50 ␮m. Neurons
labeled with biocytin were visualized by the avidin-biotin-horseradish
peroxidase reaction. After staining, sections were postfixed in 1%
OsO4 in PB containing 7% glucose and coverslipped with Epon after
dehydration.
A Neurolucida system (MicroBrightField, Williston, VT) was used
for a reconstruction of the stained cell. Stained cells were observed
with a ⫻60 objective lens followed by ⫻1.25 magnification. After
reconstruction, morphological data were exported as text files that
contained three-dimensional coordinates of dendrites. The minimum
distances between dendrites of recorded cell pairs were calculated for
each point of dendrites. The mean interpoint distance of reconstructed
dendrites was 1.14 ⫾ 0.32 ␮m.
Computational simulation. Our FS cell model is based on
Golomb’s model with several modifications (Golomb et al. 2007). The
model was constructed as three compartments (soma and 2 dendrites).
The soma of the model has a sodium current (INa), potassium currents,
and a leak current (Il). Because Kv3- and Kv1-type K⫹ currents (IKd)
are crucial for FS cell firing properties (Goldberg et al. 2008), the
soma compartment of the model contains two types of K⫹ currents: a
delayed-rectifier K⫹ current (IK), which has a relatively higher acti-
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
Table 1. Basic set of parameter values for FS cell model
Value
gNa
gK
gKd
gl
gK-dend
gl-dend
VNa
VK
Vl
␪a
␪b
␪m
␪h
␪n
ka
kb
km
kh
kn
␶m
␶b
␶0n
␶1n
␶0b
␶1b
␪b␶1
␪b␶2
␪n␶1
␪n␶2
␴1n
␴2n
␴1b
␴2b
112.5
225
1.9
0.25
30
0.01
30
⫺80
⫺65
⫺45
⫺70
⫺24
⫺58.3
⫺12.4
⫺20
6
⫺12.2
6.7
⫺6.8
1
100
0.3
10.4
1.0
60
⫺60
3
⫺14.6
1.3
⫺8.6
33
⫺5
55
then removed from the cleft. Postsynaptic conductance change for this
period is given by the following equation:
dr
dt
⫽ ⫺ ␤ 2r
where ␤2 is the removal rate of transmitters and is set to 0.3 ms⫺1.
Reversal potential for GABA was set to ⫺58 mV, which was
determined experimentally in cortical FS cells with gramicidin-perforated patch-clamp recordings (Martina et al. 2001). Synaptic delay
was set to 1 ms. Chemical synapses were assumed to form on the
soma compartment.
The FS cell network model was constructed from 50 model cells as
described above. Random distribution of cells within a square of 500
␮m each side was assumed. The coordinates of cells were determined
by random numbers from 1 to 500. In experimental observations, FS
cell pairs connected electrically and chemically were frequently obtained when distances between recorded cells were within 150 ␮m
(Fig. 1). Therefore, model cells were assumed to have a chance of
forming electrical and chemical connections with cells located within
150 ␮m. Postsynaptic cells were randomly selected from candidates
with a connection probability of 0.44 ⫾ 0.06 for electrical connections. Chemical synaptic connection probabilities were 0.214 ⫾ 0.144
(1-way) and 0.132 ⫾ 0.122 (reciprocal) among electrically connected
cells and 0.133 ⫾ 0.07 (1-way) in electrically uncoupled cells. No
reciprocal connections were formed between electrically unconnected
cells. If no candidates for postsynaptic cells were found within a
150-␮m distance, we assumed that these cells formed connections
with two adjacent cells. For simplicity, we used constant values for
the electrical synaptic conductance and the maximum GABAergic
synapse conductance in all connections, which were set at 0.02 and
0.3 mS, respectively. External current inputs were applied to the soma
compartment.
All simulations reported here were performed with Visual Studio
(Microsoft). Programs were written in C⫹⫹ language. Differential
equations were solved by a fourth-order Runge-Kutta algorithm (time
step, 0.01 ms).
RESULTS
To investigate how pyramidal cells regulate FS cell networks, we obtained dual whole cell recordings from L5 FS
cells, which are mostly the basket cell type in the rat cortex
(Kawaguchi 1995; Kawaguchi and Kubota 1993; Markram et
al. 2004). We did not observe the chandelier cell type in our
recordings, visualized by intracellular staining with biocytin.
Electrical couplings among FS cells were examined by injection of negative current pulse into one of the two cells (Fig.
1A). Hyperpolarization induced by negative current pulse injection produced a simultaneous small hyperpolarization in the
other cell. These voltage changes were reciprocal, indicating
the presence of electrical connections between the cells. The
coupling coefficient of electrical connections among FS cells,
calculated from the ratio of voltage changes in response to the
negative current pulse injection to one of the two cells, was
5.58 ⫾ 2.16% (n ⫽ 42 cell pairs; Fig. 1B). Electrical connection probabilities among FS cells depended on the distance
between the soma. We frequently observed electrically connected cell pairs when the distance between recorded cells was
within 150 ␮m (Fig. 1C). No electrical couplings were observed in FS/non-FS cell pairs (n ⫽ 52 cell pairs examined).
Chemical synaptic connections between FS cells were frequently observed in electrically connected cell pairs compared
with electrically uncoupled FS/FS pairs (Fig. 1D; P ⬍ 0.01,
␹2-test). In addition, reciprocally connected cell pairs were
found only among electrically connected pairs. These observations are consistent with previous studies (Amitai et al. 2002;
Galarreta and Hestrin 1999, 2001b; Gibson et al. 1999). Thus
L5 FS cells in the frontal cortex frequently interconnect
through gap junctions, similar to those in other cortical areas.
Excitatory synaptic inputs to FS cells. Cortical pyramidal
and FS cells interact highly with each other through reciprocal
synaptic connections within the layer (Otsuka and Kawaguchi
2009; Yoshimura and Callaway 2005). Therefore, we examined synaptic connections from pyramidal to FS cells within
L5, using glutamate puff stimulations of pyramidal cells (Otsuka and Kawaguchi 2008, 2009, 2011).
Whole cell or cell-attached recordings were obtained from
L5 pyramidal cells to examine whether glutamate puff application reliably induces a spike in the L5 pyramidal cell (Fig. 2A). While
spontaneous spiking activity was not observed at either whole
cell or cell-attached mode in the slice preparation, a focal
glutamate puff application (1 mM, 50 ms, 5–10 psi) reliably
evoked a single spike in L5 pyramidal cells, similar to those in
L2/3 pyramidal cells (Otsuka and Kawaguchi 2008). The
latency of spike measured from the onset of puff stimulation to
the spike peak was 57.92 ⫾ 14.21 ms (n ⫽ 5 and 12 in whole
cell and cell-attached modes, respectively). The standard deviation of delays for individual stimulations, representing the
variability of the spike timing, was 7.49 ⫾ 3.47 ms. Glutamateinduced spikes disappeared when the pipette was moved
slightly away from the soma. Spikes were completely abolished when puff pipettes were positioned 7.8 ⫾ 1.7 ␮m from
the soma (n ⫽ 3 and 9 in whole cell and cell-attached modes,
respectively). Moreover, we obtained recordings from two
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Parameter
797
798
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
A
Cell 1
B
Cell 2
15
40 mV
CC (%)
Fig. 1. Electrical connections between fast-spiking (FS) cells. A: dual recordings from FS cells.
Top: firing patterns of FS cells in response to the
depolarizing current pulse injection. Middle and
bottom: simultaneous hyperpolarization of 2 FS
cells when negative current was applied to 1 of
them. B: coupling coefficient (CC) of electrical
connections among FS cells (n ⫽ 42 cell pairs).
C: histograms of the probability of electrical connection as a function of the distance between
cells. D: % of cell pairs connected 1-way or
reciprocally by chemical synapses in electrically
connected (⫹) and unconnected (⫺) pairs.
10
-65 mV
-65 mV
0.5 s
5
4 mV
10 mV
0
0.2 s
D
Probability (%)
60
40
20
(2/25)
(15/35)
(15/37)
(10/29)
(0/16)
0
50
150
100
200
250
A
B
glu
glu
cell 1 cell 2
-
(9/42)
one way
reciprocal
(7/84)
0
10
20
30
40
%
stimulated cell induced EPSCs in the postsynaptic cells (n ⫽
7/7 cases). Thus focal glutamate puff stimulation of L5 pyramidal cells can be used for detecting monosynaptic connections in L5 in the same way as those from L2/3 to L5 cells
(Otsuka and Kawaguchi 2008, 2009, 2011).
We calculated connection probabilities from L5 pyramidal
to L5 interneurons in individual cells as the number of stimulated cells evoking EPSCs with constant latency (Fig. 3A)
divided by the total number of stimulated cells. The mean
number of stimulated L5 pyramidal cells for each L5 interneuron was 38.24 ⫾ 11.3. Connection probability from pyramidal
to FS cells was 0.284 ⫾ 0.073 (n ⫽ 124). We also examined
connection probability from L5 pyramidal to non-FS interneurons for comparison. We obtained recordings from both low-
C
Layer V
glu
50 mV
(5/42)
(pair No.)
Distance (µm)
closely located L5 pyramidal cells in whole cell or cellattached mode while applying glutamate to either of the two
cells. We found that glutamate puff application evoked a single
spike in one cell that we targeted but not in the other cell (n ⫽
5 cell pairs; Fig. 2B). These results suggest that glutamate puff
stimulation of a L5 pyramidal cell would induce excitatory
postsynaptic currents (EPSCs) with relatively constant latency
when a stimulated L5 pyramidal cell innervates recording cells.
As expected, glutamate puff stimulation of a L5 pyramidal cell
in some cases induced EPSCs with relatively constant latency
in the recording L5 cell (Fig. 2C, left). To confirm whether a
stimulated L5 cell induced EPSCs via monosynaptic connections to the recording cell, we obtained whole cell recordings
from the stimulated cell (Fig. 2C, right). Spike induction in the
+
C
to Cell ell 2 to Cell
2
1
Layer V
glu
cell 1 cell 2
50
0
20
10
0
spike disappearance
distance (µm)
100
delay SD (ms)
delay (ms)
-65 mV
0.1 s
cell 1
40 mV
15
cell 1
50 mV
10
15 pA
0.1 s
5
cell 2
20 ms
cell 2
0
10pA
20 ms
Fig. 2. Excitatory postsynaptic current (EPSC) induction in L5 cells by glutamate puff stimulation of L5 pyramidal cells. A: responses to glutamate puff applied
to a L5 pyramidal cell. Arrow indicates timing of puff stimulation onset. Ten traces of trials in a cell are superimposed. Bottom left and middle: means and
standard deviations of spike induction latencies from puff onset to the spike peak calculated for trial-to-trial variability in individual cells (box plots; n ⫽ 17 cells,
5 cells in whole cell and 12 cells in cell-attached mode). Bottom right: minimum distances from the soma to pipette where spike production completely failed
(box plots; n ⫽ 12 cells, 3 cells in whole cell and 9 cells in cell-attached mode). B: dual recordings from L5 pyramidal cells close to each other while applying
glutamate to either of the 2 cells. Arrow indicates the timing of glutamate puff application. Five traces were superimposed. Single spike was evoked by puff
application only in 1 cell that we selected for stimulation (n ⫽ 5 cell pairs, 3 pairs in whole cell and 2 pairs in cell-attached mode). C: confirmation of EPSC
induction by glutamate puffs to a L5 pyramidal cell by following dual whole cell recordings from the stimulated L5 pyramidal cell. Left: examples of EPSC
induction by glutamate puff to a L5 pyramidal cell. Three traces of trials in a cell are shown. Right: dual whole cell recordings from the glutamate-stimulated
L5 cell in addition to the recording cell on left. Monosynaptic connections were confirmed in examined cases (n ⫽ 7/7).
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C
Electrical connection
4 mV
(pair No.) 0
Layer V
Cell 1
10 mV
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
A
glutamate
Layer V
C
COM CPn
frontal
cortex
Layer V
Pons
50 pA
D
**
25
60
20
30
20
15
40
20
0
0
20
40
60
Pcom (%)
10
5
10
0
0
FS
LTS
RS
-40 -20
0
20
40
Pcom - Pcpn (%)
Fig. 3. Excitatory synaptic inputs from pyramidal to FS cells. A: detection of
EPSCs in FS cells by glutamate puff stimulations. Arrow indicates the timing
of stimulation onset. Ten traces are superimposed. B: connection probabilities
from pyramidal cells to FS, low-threshold spike (LTS), and regular-spiking
(RS) interneurons (n ⫽ 124, 27, and 24, respectively). Data are means ⫾ SD.
**P ⬍ 0.01. C: simultaneous retrograde tracer injections to contralateral
frontal cortex and ipsilateral pontine nucleus. Image shows cells labeled by
tracers in L5. Red, Alexa Fluor 555-conjugated cholera toxin subunit B
injected into contralateral frontal cortex; blue, fast blue injected into ipsilateral
pontine nuclei. Section thickness, 20 ␮m. D: histograms of the differences
between connection probabilities from L5 contralateral frontal cortex (COM)
and ipsilateral pontine nuclei (CPn) cells to individual FS cells (n ⫽ 40). In
physiological experiments, CPn cells were labeled by Alexa Fluor 555conjugated cholera toxin subunit B and COM cells by rhodamine-labeled latex
microspheres. Inset: relationship between connection probabilities from COM
and CPn cells in individual FS cells.
threshold spike (LTS), identified by the rebound burst firing in
response to negative current pulse injection, and regular-spiking (RS) interneurons. Connection probability from pyramidal
cells to LTS and RS cells was 0.167 ⫾ 0.08 and 0.146 ⫾ 0.04,
respectively (n ⫽ 27, 24). Consistent with our previous study
(Otsuka and Kawaguchi 2009), connection probability from L5
pyramidal to FS cells was higher than that to the non-FS cells
examined (P ⬍ 0.01; Fig. 3B). No significant difference was
found between the connection probabilities to LTS and RS interneurons. Therefore, we grouped LTS and RS interneurons
together as non-FS cells.
L5 pyramidal cells project to several brain regions, with
their distinct subpopulations involved in specific targets
(Reiner et al. 2003; Wise and Jones 1977). Pyramidal cells
form subnetworks in intra- and interlaminar connections, depending on projection subtypes (Brown and Hestrin 2009;
Morishima and Kawaguchi 2006; Morishima et al. 2011;
Otsuka and Kawaguchi 2008, 2011). Therefore we examined
whether FS cells receive synaptic inputs from specific pyramidal projection subtypes. Pyramidal cells projecting to the
contralateral cortex (COM cells) and ipsilateral pontine nuclei
(CPn cells) were simultaneously identified by fluorescent retrograde tracer injections (Fig. 3C). L5 COM and CPn cells did
not overlap, indicating that these cell types are separate populations (Hallman et al. 1988).
L5 COM and CPn cells were alternately stimulated by
glutamate puff stimulations to examine synaptic connections to
the recorded FS cell. We stimulated the same number of COM
and CPn cells in individual FS cells (25.4 ⫾ 2.8 cells for each
subtype) and then calculated the connection probability from
L5 COM and CPn cells to those FS cells. Connection probabilities from COM and CPn to FS cells were 0.293 ⫾ 0.116 and
0.307 ⫾ 0.11, respectively (n ⫽ 40). To examine connection
specificity, we obtained the difference between connection
probabilities from COM and CPn cells in individual FS cells.
The difference between probabilities from COM and CPn cells
in individual FS cells was nearly 0 (0.01 ⫾ 0.1, P ⫽ 0.41,
1-sample t-test) and showed normal distribution (Fig. 3D;
1-tailed P ⫽ 0.23, D’Agostino-Pearson omnibus test). These
results suggest that FS cells receive excitatory synaptic inputs
from L5 pyramidal cells in a subtype-independent manner.
Excitatory common inputs to FS cell networks. We next
investigated how pyramidal cells innervate electrically connected FS cells. Dual recordings were obtained from L5 FS
cells while glutamate puff stimulations were applied to surrounding pyramidal cells (Fig. 4A). If a pyramidal cell coinnervates recorded cells, synchronous inputs should be detected
(Otsuka and Kawaguchi 2008, 2009, 2011). Indeed, stimulations of some L5 pyramidal cells induced synchronous EPSCs
at constant latencies in both FS cells (Fig. 4A). We calculated
the common input probability of individual cell pairs by
dividing the number of cells evoking synchronous inputs in
recorded cells by the total number of stimulated cells. Common
input probability from pyramidal to FS/FS cell pairs depended
on the presence of electrical connections between FS cells (Fig.
4B) and was higher in electrically connected FS/FS cell pairs
(0.156 ⫾ 0.046, n ⫽ 16) than in unconnected FS/FS cell pairs
(0.073 ⫾ 0.020, n ⫽ 26, P ⬍ 0.01).
Common inputs to electrically connected FS/FS cell pairs
might just reflect the passive conduction of EPSCs induced in
one cell to the other cell through gap junctions. To examine
this possibility, we investigated excitatory inputs to FS cell
pairs that were electrically connected first but disconnected by
bath application of the gap junction blocker carbenoxolone
(100 ␮M; coupling coefficient ⫽ 7.65 ⫾ 3.85% and 0.5 ⫾
0.42% before and after carbenoxolone application, respectively, n ⫽ 5). Common input probability from pyramidal cells
to electrically connected cell pairs was 0.137 ⫾ 0.021 in the
presence of carbenoxolone, similar to that in electrically connected FS/FS cell pairs (Fig. 4B; P ⬎ 0.05). Thus common
inputs observed in our recordings are likely to be EPSCs
synchronously induced in individual cells.
We also examined synchronous inputs to FS/non-FS cell
pairs for comparison. Common input probability of FS/non-FS
cell pairs was 0.054 ⫾ 0.016, similar to that in electrically
unconnected FS/FS cell pairs (n ⫽ 16, P ⬎ 0.05). The total
number of stimulated cells in electrically connected FS/FS cell
pairs, unconnected FS/FS cell pairs, and FS/non-FS cell pairs
was 39.6 ⫾ 7.7, 40.1 ⫾ 6.7, and 39.6 ⫾ 6.1, respectively. In
addition, common input probabilities of electrically uncoupled
FS/FS cell pairs did not depend on the distance between cell
pairs. In FS/FS cell pairs located at a ⱖ50-␮m distance,
common input probability was 0.073 ⫾ 0.016 and 0.072 ⫾
0.022, respectively (n ⫽ 10 and 16, P ⬎ 0.05). Moreover, the
common input probability in FS/FS cell pairs connected only
by chemical synapses was 0.077 ⫾ 0.014 (n ⫽ 4), similar to
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Pcpn (%)
40
Cell No.
B
Connection probability (%)
0.1 s
799
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
Layer V
B
glutamate
n.s.
25
(non-selective cases)
C
pi
p = pi X pj
Relative to
assumed non-selective cases
**
15
(16)
(5)
10 ≥50µm <50µm
5
(16)
(10)
Cell 1
Cell 2
(pair No.)
n.s.
**
**
2
≥50µm <50µm
1
(16)
0
pj
0
FS / FS
FS/
non-FS
FS / FS
FS/
non-FS
Fig. 4. Connection specificity from pyramidal to FS cells. A, top: dual recording from FS/FS or FS/non-FS cell pairs during glutamate puff stimulation of
pyramidal cells. Bottom: simultaneous EPSCs induced in 2 cells by pyramidal stimulation. Three traces of trials in a cell pair are shown. Arrow, onset of puff
stimulation. B: common input probability from pyramidal cells in FS/FS and FS/non-FS cell pairs. FS/FS cell pairs were grouped into electrically connected
(E-connected) pairs in the presence and absence of the gap junction blocker carbenoxolone (100 ␮M) and unconnected pairs separated by a distance of ⱖ50 ␮m.
In electrically connected FS/FS pairs as well as those with carbenoxolone application, common input probability was higher than in unconnected pairs. Note that
FS/FS cell pairs connected only by chemical synapses were included as unconnected pairs. C: common input probabilities were normalized to those in the
nonselective cases. Inset: calculation of common input probability from pyramidal to interneuron pairs (pi and pj). Data in B and C are means ⫾ SD. **P ⬍ 0.01.
n.s., P ⬎ 0.05.
that in unconnected FS/FS pairs. We therefore grouped these
cell pairs connected chemically alone with electrically uncoupled FS/FS cell pairs.
Connection probabilities from pyramidal cells to interneurons differed between FS and non-FS cells. To compare common input probability between FS/FS and FS/non-FS cell
pairs, we normalized common input probabilities for different
postsynaptic interneuron types relative to the probabilities
expected for nonselective innervation to two interneurons. To
calculate the expected probabilities in nonselective cases, we
used individual connection probabilities to FS and non-FS cells
(Fig. 3B). The hypothetical common input probability of nonselective cases was calculated as p1 ⫻ p2, where p1 and p2 are
the experimentally obtained probabilities of individual interneurons receiving synaptic inputs from pyramidal cells (Fig.
4C, inset). Like the observed common input probability, the
normalized common input probability was higher in electrically connected FS/FS cell pairs (1.935 ⫾ 0.577; 1.708 ⫾
0.263 in the presence of carbenoxolone) than in unconnected
FS/FS cell pairs (0.899 ⫾ 0.278, ⬎50 ␮m and 0.91 ⫾ 0.195,
⬍50 ␮m) and in FS/non-FS cell pairs (1.138 ⫾ 0.345, P ⬍
0.01) (Fig. 4C). No significant difference was observed between the normalized common input probability in FS/non-FS
and electrically unconnected FS/FS cell pairs. These results
suggest that individual pyramidal cells preferentially coinnervate electrically connected FS cells.
The dendrites between FS cells connected electrically
through dendritic gap junctions may be adjacent to each other
and thus have a higher chance of forming synaptic connections
with the same presynaptic axons than those between uncoupled
cells. We therefore compared dendritic geometries between
electrically connected and unconnected cell pairs. Recorded
cell pairs were reconstructed (Fig. 5A), and minimal distances
between mutual dendrites were measured. The distance between the somata of electrically connected reconstructed FS/FS
cell pairs was 58.8 ⫾ 22.2 ␮m (n ⫽ 11), similar to that of
electrically unconnected FS/FS cell pairs (46.7 ⫾ 13.7 ␮m,
n ⫽ 9) and FS/non-FS cell pairs (46.6 ⫾ 16.9 ␮m, n ⫽ 9, P ⬎
0.05). Figure 5B shows the cumulative histograms of minimal
distances between dendrites in electrically connected and unconnected FS/FS and FS/non-FS cell pairs. Fifty percent cumulative probability of minimum distance between dendrites
of individual cell pairs was 40.1 ⫾ 11.8 and 40.3 ⫾ 10 ␮m for
electrically connected and unconnected FS/FS pairs, respectively, and 42.7 ⫾ 13.3 ␮m for FS/non-FS pairs, with no
significant differences between these groups (Fig. 5C). These
results suggest that electrical coupling itself is more important
to the formation of coinnervations from pyramidal to FS cells
than dendritic proximity.
Spike transmission between electrically connected FS cells.
Cortical FS cells highly interconnect through gap junctions and
form globally connected dendritic net structures (Amitai et al.
2002; Fukuda et al. 2006; Galarreta and Hestrin 2001a). Because pyramidal cells frequently coinnervated electrically connected FS cells, we examined how FS cell activities evoked by
excitatory inputs affect surrounding electrically connected FS
cells. Cortical FS cells demonstrate synchronous membrane
potential fluctuations according to brain state (Gentet et al.
2010; Steriade et al. 2001). We therefore examined activity
transmission to electrically connected postsynaptic FS cells in
depolarized and hyperpolarized states.
We injected brief current pulses (5-ms duration) into one of
two cells connected electrically but not chemically. Membrane
potentials of FS cells were controlled by DC current injections.
In the depolarized state, subthreshold current pulse injection
(just below spike threshold) to the presynaptic cell induced
only a small depolarization in the postsynaptic cell (Fig. 6B).
However, when spikes were evoked by current pulse injection
to the presynaptic cell, voltage responses of the postsynaptic
cell were biphasic: a transient small depolarization (spikelet
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E-connected
Carbenoxolone
50 ms
**
20
E-connected
50 pA
Common input probability (%)
3
E-connected
Carbenoxolone
A
E-connected
800
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
A
E-connected
FS
FS
non-FS
FS
FS
FS
100 µm
5 µm
C
100
80
60
FS/FS
FS/FS (+E)
FS/non-FS
40
20
0
0
50
100
150
200
50% min distance (µm)
Cumulative probability (%)
*
60
40
20
+E
(9)
0
(pair No.)
Min distance (µm)
corresponding to the presynaptic cell spike) followed by longer
hyperpolarization corresponding to the spike afterhyperpolarization in the presynaptic cell (Fig. 6A, left). By contrast, in the
hyperpolarized state presynaptic cell activities evoked by supra- and subthreshold current pulse injections were transmitted
to the postsynaptic cell as excitatory potentials (Fig. 6A, right).
To quantify the voltage spread through gap junctions, we
measured the area of postsynaptic voltage responses (Fig. 6B).
In the hyperpolarized state, postsynaptic excitatory effects
increased linearly depending on current pulse amplitude. In the
depolarized state, spike discharge produced inhibitory effects
in the postsynaptic cell. These results suggest that presynaptic
activities induce either excitatory or inhibitory effects in the
postsynaptic cell depending on network state.
To examine further how FS cell activities are transmitted to
the electrically connected postsynaptic cell, we constructed an
FS cell model composed of three compartments, a soma and
two dendrites, with several Hodgkin-Huxley type conductances. A simple compartment model is sufficient to examine
electrical transmission between cells (Gibson et al. 2005). Our
model reproduced the firing patterns of FS cells observed
experimentally (Fig. 6C). We then connected model cells
electrically at a dendritic compartment. Like recordings in slice
experiments, hyperpolarization in one of the two model cells
produced simultaneous hyperpolarization in the other cell (data
not shown). The coupling coefficient between the model cells,
the ratio of voltage changes in response to negative current
pulse injection, was adjusted to 6%. We applied brief depolarizing current pulses (1-ms duration) to one of the model cells
to induce a spike. In the depolarized state, spike discharge in
the presynaptic cell induced mainly hyperpolarization in the
postsynaptic cell (Fig. 6D, left). In the hyperpolarized state,
(11)
FS/
FS
FS/
FS
(9)
FS/
non
-FS
presynaptic spikes evoked by current injections produced depolarizing potentials in the postsynaptic cell (Fig. 6D, right).
Thus our model of electrically coupled FS cells reproduced the
experimental observations. In both states, evoked potentials
were strongly attenuated in the disynaptic cell.
We examined the summation of two presynaptic cell activities in a common postsynaptic cell with a model of three FS
cells connected in series (Fig. 6E). When synchronous spikes
were induced in the two presynaptic cells by depolarizing
current pulse injections, voltage responses in the postsynaptic
cell were linearly summated in both the depolarized and
hyperpolarized states (Fig. 6E).
FS cells frequently form both electrical and chemical synaptic connections between them. To examine how GABAergic
chemical synapses cooperate with electrical connections in the
FS cell network, we included chemical synaptic connections in
our model. We assumed reversal potentials of GABAergic
synaptic inputs of ⫺58 mV (Martina et al. 2001) and a synaptic
delay of 1 ms. In the depolarized state, the inhibitory effect in
a postsynaptic cell elicited by presynaptic spike activity was
enhanced, with little or no effect on the spikelet (Fig. 6F, top).
In the hyperpolarized state, excitatory potentials in the postsynaptic cell were enhanced when the presynaptic cell was
discharged (Fig. 6F, bottom). Thus chemical synapses can
cooperate with electrical connections in both states.
Spread of activities in the FS cell network model. To examine the impacts of synchronous excitatory synaptic inputs on
electrically connected FS cell networks, we extended our
model to a larger network. We assumed that 50 FS cells were
randomly located within a square, 500 ␮m each side, which
approximately corresponds to the density of rat somatosensory
L5(6) parvalbumin-positive cells in a 100-␮m thickness (Ami-
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Fig. 5. Dendritic geometries of interneuron
pairs. A: Neurolucida reconstructions of electrically connected (center, E-connected) and
unconnected (left) FS/FS cell pairs and a FS/
non-FS cell pair (right). Arrows indicate putative gap junctions. Spatial overlap of 2 dendrites was found by observation with a ⫻100
objective lens, where gap junctions may be
formed. Inset: photograph obtained with ⫻100
objective lens in the region marked with asterisk. B: averaged cumulative probabilities of
minimum distance of dendrites of electrically
connected and unconnected FS/FS cell pairs
and FS/non-FS cell pairs (n ⫽ 11, 9, and 9,
respectively). C: histograms of half minimum
distance in individual cell pairs. No significant
difference was found. Data are means ⫾ SD.
*
B
801
802
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
A
Depolarized state
Hyperpolarized state
B
30
Area (mV*ms)
20
30 mV
-51 mV
-72 mV
10
0
-10
Hyperpolarized
state
-20
50 ms
-30
1 mV
-51 mV
-72 mV
-40
C
Model
D
Experiment
25% 50% sup sub
sup sup ra
ra
ra
Cell 1
0.3 s
0.3 s -65 mV
-64 mV
Cell 1
-65 mV
-64 mV
15 mV
-53 mV
-72 mV
Cell 2
30 mV
30 mV
Cell 2
-53 mV
-72 mV
Cell 3
-64 mV
-53 mV
0.5 mV
-72 mV
Depolarized state
E
Cell 1
Cell 3
Cell 1
Iext
F
Cell 3
40 mV
Hyperpolarized state
Iext
Cell 2
-53 mV
Cell 1
Cell 2
Cell 2
40 mV
-53 mV
-53mV
-53 mV
Depolarized state
Depolarized state
30 ms
1 mV
-72 mV
-72 mV
Hyperpolarized state
tai et al. 2002). In our experimental results, electrically connected FS cell pairs were frequently observed when the distance between somata was within 150 ␮m (Fig. 1C). We
therefore assumed that FS model cells have a chance to form
electrical connections with surrounding cells located within
150 ␮m (Fig. 7A). Postsynaptic cells were randomly selected
from those neighboring cells with connection probabilities
reflecting experimental data (Fig. 1, C and D). Chemical
synaptic connections between model cells were also included
in the network model in the same manner as electrical connections (Fig. 7B).
As synchronous excitatory inputs, EPSC-like currents were
applied to a set of cells. The rise and decay kinetics of currents
were based on values of experimentally obtained EPSCs in L5
pyramidal/FS cell pairs (rise time, 0.8 ms; decay time constant,
4.3 ms) (Otsuka and Kawaguchi 2009). Suprathreshold inputs,
enough to induce a spike in a given cell in the depolarized
state, were applied to one cell, and subthreshold inputs, with
strength just below the spike threshold when solely applied to
30 ms
1 mV
50 ms
50 ms
-72 mV
30 ms
Cell 1
Iext
Cell 2
ra
Cell 3
Iext
-65 mV
sup
-72 mV
Hyperpolarized state
the model, were simultaneously applied to a set of electrically
connected cells (coupled inputs) or unconnected cells (decoupled inputs). When synchronous inputs were applied to
electrically connected cells in the depolarized state, a spike was
generated in all input-receiving cells, even those receiving a
subthreshold input (Fig. 7C, arrow). These activities then
spread to electrically connected surrounding cells and induced
inhibitory effects in these cells (Fig. 7C, coupled input case in
the depolarized state). These inhibitions were enhanced when
chemical synaptic connections were included in the model
circuit (Fig. 7C, E ⫹ Chem connections). On the other hand,
synchronous inputs to a set of electrically unconnected cells in
the depolarized state induced spikes only in suprathreshold
input-receiving cells, not in subthreshold input-receiving cells.
As a result, we observed both inhibitory and excitatory effects
in surrounding cells (Fig. 7C, decoupled input case). Thus in
the coupled input case the transmitted potentials in the depolarized state were mostly hyperpolarized (Fig. 7D). By contrast, in the uncoupled case transmitted potentials were depo-
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Fig. 6. Activity transmissions between electrically connected FS cells. A: dual recordings
from FS cells connected electrically but not
chemically. Top: current pulse was applied to
1 of 2 cells in depolarized and hyperpolarized
states. Bottom: postsynaptic voltage responses.
Membrane potentials were adjusted by DC
current injections. B: histograms of integrated
postsynaptic voltage responses in depolarized
and hyperpolarized states in response to supra- and subthreshold current pulse injections
to the presynaptic cell. 50% supra and 25%
supra, 50% and 25% amplitude of the suprathreshold current pulse; sub, subthreshold
current, current just below the spike threshold. C: voltage responses of model and FS
cells to depolarizing current pulse injections.
Current pulse duration, 1 s; intensity 2, 3, and
5 ␮A/cm2 in the model, 100, 150, 300 pA in
a FS cell. D: 3 model FS cells were connected
in series. Top: at different membrane potentials, current pulses (1-ms duration, Iext) were
applied to cell 1 to induce a spike. Middle and
bottom: voltage responses of cells 2 and 3.
E: effects of simultaneous spike induction in
cells 1 and 3 on the potential of cells electrically connected to both cells. Right: cell 2
voltage. Black line, synchronous firing of
cells 1 and 3; gray line, firing of only 1 cell.
F: a chemical synapse was included in the
model (top). Left: spike discharge at different
membrane states. Right: postsynaptic cell
voltage responses. Gray line, postsynaptic
voltage responses of cell 2 without a chemical synapse.
Depolarized
state
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
500
400
400
400
300
300
300
200
150 µm
100
200
200
100
100
0
0
0
0
100
200
300
400
0
500
100
200
C
EPSC-like
current
Coupled Inputs
1
3
(µm)
2*
400
500
one way
reciprocal
E + Chem
connections
1
*
40 mV
*
2
2*
*
*
40 ms
*
3
4
3
*
2 mV
300
1
*
*
3
200
E connections
40 ms
4
2
100
Decoupled Inputs
*
40 mV
*
*
0
4
4
2 mV
0
1
1
10 mV
10 mV
400
1
1
2
(µm)
2
40 ms
40 ms
2
2
200
3
3
1 mV
3
3
200
(µm)
EPSC-like current
injected cell
> 10
Depolarized
state
10
Area (mV*ms)
0
Area (mV*ms)
D
400
0
-10
-20
coupled
decoupled
-30
200
400
(µm)
5 ~ 10
-10 ~ 5
10
Depolarized
state
10
20
30
Number of cell
40
200
(µm)
400
0
200
(µm)
400
< -20 (mV*msec)
-20 ~ -10
Hyperpolarized
state
Hyperpolarized
state
60
20
0
-10
-20
-30
-40
10
75%
supra
E + Chem
-50
0
0
Area (mV*ms)
0
Area (mV*ms)
0
1 mV
10
20
30
Number of cell
40
0
10
20
supra
0
0
0
40
20
30
40
Number of cell
0
10
20
30
40
Number of cell
Fig. 7. Spatial voltage spread in an electrically connected FS network model. A: distribution of 50 FS model cells. Electrical and chemical connections were formed among cells
located within a 150-␮m distance. B: electrical and chemical synaptic connection patterns between model cells. Note that electrical and chemical synaptic connections were made
on dendritic and somatic compartments, respectively. Left: red lines show electrical connections. Right: black and red arrows show chemical synaptic connections from pre- to
postsynaptic cells (1-way) and reciprocal connections between cells. C: voltage responses of cells in the network model in the depolarized and hyperpolarized states. Sub- and
suprathreshold EPSC-like currents were simultaneously applied to 3 electrically connected (coupled input case) and unconnected (decoupled input case) cell pairs (6 cells),
respectively. Red filled circle, suprathreshold current applied; red filled circle with *, subthreshold current applied; arrow, cell generating a spike by subthreshold input. Left:
network of electrically connected FS cells (E-connections). Right: network with both electrical and chemical connections (E ⫹ Chem connections). Voltage responses in
individual cells were integrated and represented as colors. Numbers beside cells correspond to those of traces shown on right. DC currents were applied to the soma compartment
of all cells (1.7 and ⫺2.0 ␮A/␮m2) to induce depolarized (top) and hyperpolarized (bottom) states. Note the more prevalent hyperpolarization among FS cells in the coupled input
case in the depolarized state but no qualitative differences between the coupled and decoupled input patterns in the hyperpolarized state. D: cumulative histograms of voltage
responses evoked by synchronous inputs in depolarized and hyperpolarized states (44 cells; cells receiving synchronous inputs were excluded). In the hyperpolarized state, the
same amplitude of currents was applied to receiving cells in both cases. Supra, suprathreshold input enough to induce a spike; 75% supra, 75% amplitude of suprathreshold current.
larized in some connections and less hyperpolarized in others
(Fig. 7D). Thus activities evoked by common inputs to electrically connected FS cells were transmitted to nearby cells in
a lateral inhibition manner in the depolarized state, while in the
hyperpolarized state either sub- or suprathreshold synchronous
inputs produced excitatory potentials in surrounding cells both
in the coupled and decoupled cases (Fig. 7, C, bottom, and D).
These results suggest that the impact of common inputs on the
FS cell network depends on the network state and input
patterns to FS cells.
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1
*
200
500
400
(µm)
E + Chem
connections
E connections
400
300
(µm)
(µm)
Hyperpolarized State Depolarized State
Chem connections
500
(µm)
(µm)
E connections
B
500
(µm)
A
803
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
In the above simulations, we applied EPSC-like currents to
three pairs of FS cells (n ⫽ 6). To examine the dependence of
activity spread among FS cells on the number of FS cells
receiving synchronous inputs, we quantified voltage responses
in the model circuit in response to synchronous inputs to
varying numbers of cells. Synchronous inputs were applied to
2–10 electrically connected cells (1–5 cell pairs). In both
depolarized and hyperpolarized states, increase in the number of synchronous input-recipient cells produced stronger
inhibitions and excitations in surrounding cells, respectively
(Fig. 8, A and B). Depending on the number of receiving
cells, excitatory and inhibitory effects in surrounding cells
were linearly increased (Fig. 8, C and D).
DISCUSSION
In the present study, we investigated excitatory synaptic
input patterns to electrically connected FS cell networks. In the
visual cortex, FS cells showed broad tuning responses to visual
stimuli (Kerlin et al. 2010), indicating that FS cells receive
synaptic inputs from several distinct pyramidal cells with
different selectivity for orientation tunings (Hofer et al. 2011).
Consistently, we have shown that FS cells equally receive
excitatory synaptic inputs from different pyramidal subtypes
that project to distinct brain areas in a nonselective manner. To
our knowledge, this is the first report of input convergence on
individual FS cells from various projection subtypes of pyramidal cells. At the network level, however, we found that
pyramidal cells frequently coinnervated FS cells when they
connected electrically. Our morphological analysis suggests
that electrical coupling itself is important for coinnervations to
FS cells by pyramidal cells. Electrical couplings among FS
cells enhance synchronous spike discharges when they receive
excitatory common inputs (Fig. 7) (Hestrin and Galarreta
2005). Spike timing-dependent plasticity, the so-called HebE connections
C
E + Chem connections
1
Area (mV*msec)
0.8
2
4
6
8
10 cells
0.6
0.4
0.2
Depolarized state
E
E + Chem
-10
-20
-30
Depolarized state
-40
0
B
0
Depolarized state
0
Cumulative probability
Fig. 8. Dependence of the voltage spread
pattern on the number of cells with synchronous inputs. A and B: cumulative histograms
of FS model cell voltage responses in the
depolarized and hyperpolarized states. Receiving cells were excluded from histograms. Left: responses in the electrically connected network. Right: responses in the network including both electrical and chemical
connections. In the hyperpolarized state, synchronous sub (50% amplitude of supra)- and
supra-spike threshold EPSC-like currents
were applied to a set of electrically coupled
cells in the network model that included
electrical and chemical synapses. C and
D: voltage responses of FS model cells in the
depolarized and hyperpolarized states. The
area of voltage responses was averaged.
EPSC-like current-receiving cells were excluded. In both states, synchronous EPSClike currents were applied to 2–10 cells (1–5
cell pairs) connected electrically. In each
EPSC-like current injection, 5 different sets
of stimulated cell pairs were examined. Data
are means ⫾ SD.
Cumulative probability
A
-20
-40
-60
0
Area (mV*msec)
-20
-40
0
-60
Area (mV*msec)
0.8
2
4
6
8
10 cells
0.6
0.4
0.2
50% supra
0
0
5
Hyperpolarized
state
10
15
Area (mV*msec)
20 0
supra
Hyperpolarized
state
4
6
8
10
12
number of cell
D 25
1
2
50% supra
supra
20
15
10
Hyperpolarized
state
5
0
20
40
60
Area (mV*msec)
J Neurophysiol • doi:10.1152/jn.00071.2013 • www.jn.org
80
0
2
4
6
8
10
Number of cell
12
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bian rule (Caporale and Dan 2008), may be one of the mechanisms underlying the selective innervation patterns of excitatory synapses onto electrically connected FS cell networks.
Cortical cells including FS interneurons take different membrane potential states depending on behavioral state (Gentet et
al. 2010; Steriade et al. 2001). Membrane potentials of cortical
neurons alternatively change between depolarization and hyperpolarization during wakefulness and sleep. In particular,
during deep sleep membrane potentials of cortical cells show
synchronous slow wave oscillations composed of Up (depolarized) and Down (hyperpolarized) states. Our experimental
and computer simulation results showed that electrical communication between FS cells highly depends on membrane
state. When FS cells were in the depolarized state, spike
discharges in FS cells produced inhibitory effects on surrounding electrically connected FS cells. By contrast, in the hyperpolarized state, either sub- or suprathreshold excitatory inputs
induced excitatory potentials in nearby FS cells. Similar to
these bidirectional changes in activity transmission between
electrical connections, chemical synapses between FS cells can
act as either excitatory or inhibitory inputs depending on the
membrane potential state, because FS cells have relatively
depolarized reversal potentials for GABA (Martina et al.
2001). Consistently, it has been reported that reversal potentials of GABAergic currents in hippocampal FS cells were
relatively depolarized potentials (Vida et al. 2006). If reversal
potentials of GABAergic currents in FS cells are more hyperpolarized potentials, inhibitory effects of spike transmission
between FS cells connected by both electrical and chemical
synapses would be further enhanced in the depolarized state.
By contrast, the depolarized effect by chemical synapses in
spike transmission would become small or less in the hyperpolarized state. However, the excitatory effect through electrical synapses is still preserved.
Area (mV*msec)
804
COMMON INPUTS TO ELECTRICALLY CONNECTED FS CELLS
ner (Fig. 3). Similarly, FS cells frequently innervate nearby
pyramidal cells (Packer and Yuste 2011). Moreover, pyramidal
and FS cells often form reciprocal connections among them
(Otsuka and Kawaguchi 2009; Yoshimura and Callaway
2005). These observations suggest that individual FS cells
regulate several distinct pyramidal subnetworks. Cortical cells
show synchronous spiking activities during specific phases of
a task (Baker et al. 2001). Common inputs from a specific set
of pyramidal cells to electrically connected FS cells would
induce synchronous spikes in FS cells. Synchronous recurrent
feedback inhibitions from FS cells would further enhance the
synchronization of spiking activities in the pyramidal subnetwork by strong somatic inhibition. On the other hand, activities
in one pyramidal subnetwork would induce feedforward inhibition in other pyramidal subnetworks. Moreover, some cortical cells show persistent activities related to working memory
(Goldman-Rakic 1995). Cortical cells are likely to encode
several sets of information and transform online information to
sustained spiking activities, according to a given behavioral
context (Genovesio et al. 2009; Katori et al. 2011; Mushiake et
al. 2006; Romo et al. 1999). Theoretical study has shown that
switching of sustained activities between excitatory subnetworks can be well regulated in the attractor network model, in
which inhibitory cells are reciprocally connected with multiple
excitatory subnetworks (Katori et al. 2011). Thus FS cells may
play important roles in the communication between pyramidal
subnetworks.
GRANTS
This work was supported by Japan Science and Technology Agency, Core
Research for Evolutional Science and Technology and Grant-in-Aids for
Scientific Research from the Ministry of Education, Culture, Sports, Science,
and Technology.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: T.O. conception and design of research; T.O. performed experiments; T.O. analyzed data; T.O. interpreted results of experiments; T.O. prepared figures; T.O. and Y.K. drafted manuscript; T.O. and Y.K.
edited and revised manuscript; T.O. and Y.K. approved final version of
manuscript.
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