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The Journal of Neuroscience, October 15, 2000, 20(20):7579-7586
The Excitatory Neuronal Network of Rat Layer 4 Barrel Cortex
Carl C. H.
Petersen and
Bert
Sakmann
Department of Cell Physiology, Max-Planck-Institute for Medical
Research, Heidelberg D-69120, Germany
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ABSTRACT |
Sensory whiskers are mapped to rodent layer 4 somatosensory cortex
as discrete units termed barrels, which can be visualized at high
resolution in living brain slices. Both anatomical and physiological
properties of the layer 4 neuronal network can thus be investigated in
the context of the functional boundaries of this sensory map.
Large-scale confinement of neuronal arbors to single barrels was
suggested by restricted lateral diffusion of DiI across septa between
barrels. Morphological analysis of dendritic and axonal arborizations
of individual excitatory neurons showed that neuronal processes remain
within the barrel of origin through polarization toward the center of
the barrel. Functionally, the large-scale properties of the neuronal
network were investigated through mapping the spatial extent of field
EPSPs, which were found to attenuate at barrel borders. This ensemble
property of a layer 4 barrel was further investigated by analyzing the
connectivity of pairs of excitatory neurons with respect to the
locations of the somata. Approximately one-third of the excitatory
neurons within the same barrel were synaptically coupled. At the septum between adjacent barrels the connectivity dropped rapidly, and very few
connections were found between neurons located in adjacent barrels.
Each layer 4 barrel is thus composed of an excitatory neuronal network,
which to a first order approximation, acts independently of its neighbors.
Key words:
neocortex; somatosensory cortex; barrel cortex; layer 4; synaptic transmission; EPSP; glutamate; neuronal network; dendritic
morphology; axonal morphology
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INTRODUCTION |
Sensory input is mapped to the
neocortex such that closely related sensory stimuli are also close to
each other spatially in the cortical representation. How individual
neurons and neuronal circuits are functionally arranged within these
maps is not well understood. This is in large part attributable to the
difficulties of simultaneously studying both synaptic transmission
between individual identified neurons and locating the neurons within the sensory map. The barrel field of the rodent somatosensory cortex
provides a unique neocortical region for such investigations. Each
sensory whisker is represented somatotopically in the large-scale anatomical structure of a barrel in layer 4 of the neocortex (Woolsey and Van der Loos, 1970 ). These anatomically defined structures have
also been investigated electrophysiologically by extracellular unit
recordings, and the location of the recording electrode within the
barrel field is found to be homologous to the physiologically defined
principal whisker (Welker, 1976; Simons, 1978; Armstrong-James and Fox,
1987). Thus, it is possible to relate the barrel pattern of the
neocortex directly to the somatosensory map. Furthermore, barrel-like
structures are visible in the living brain slice preparation of
somatosensory cortex (Agmon and Connors, 1991). The neuronal circuitry
can thus be investigated at the level of individual neurons and their
synaptic connections in the context of the functional boundaries of
this sensory map.
The analysis of the excitatory neuronal network of layer 4 rat barrel
cortex investigated in this study is thus a step to further our
understanding of how the first layer of the neocortical circuitry may
respond to input. In a recent study, Feldmeyer et al. (1999) described
highly reliable local synaptic connections with large NMDA receptor
components between excitatory neurons of layer 4 barrel cortex. In this
study, we combine the investigation of the ensemble properties of
barrels with the anatomy and physiology of individual neurons and pairs
of neurons with the aim of understanding the collective properties of
the underlying neuronal network. Whereas neurons lying inside the same
barrel are often synaptically connected, neurons from neighboring
barrels show low connectivity. The functional data are reflected also
in the underlying anatomy of the axonal and dendritic arbors, which are
largely confined to individual barrels. Neighboring barrels thus appear
to be only weakly connected to each other, and as a first order
approximation we may consider each barrel as an isolated neuronal
network. Such functionally independent neuronal networks of defined
numbers of neurons and defined connectivity offer an opportunity to
study the activity of physiologically relevant neuronal circuits in quantitative detail. As well as providing data on how sensory information is processed in the input layer of the neocortex, such a
description of the functional architecture is also a prerequisite for
understanding how reorganization of cortical representation occurs at a
cellular level, for example after deprivation of sensory input (Diamond
et al., 1994; Glazewski and Fox, 1996; Finnerty et al., 1999; Polley et
al., 1999).
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MATERIALS AND METHODS |
Slice preparation. Thalamocortical slices of 250-300
µm thickness were prepared from anesthetized Wistar rats (13- to
15-d-old) following the description of Agmon and Connors (1991) with
modifications by Feld-meyer et al. (1999). Slices were cut by a
vibratome in ice-cold extracellular medium and were subsequently
incubated at 35°C for 15-30 min after slicing. The slices were then
transferred to room temperature (20-23°C) until required for
analysis. Throughout the procedure slices were maintained in
extracellular medium containing (in mM): 125 NaCl, 25 NaHCO3, 25 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, and 1 MgCl2 bubbled
with 95% O2 and 5%
CO2.
Identification of barrels in living slices. The barrel
subfield of somatosensory cortex is easily recognized in the
thalamocortical slice preparation even by the naked eye. Under
low-power light microscopy (Zeiss Axioskop fitted with a 2.5× lens)
the dark band of layer 4 separates into barrels separated by lighter
septal regions. To identify barrels at higher magnifications (Zeiss
water-immersion lenses, 10× and 40×), it is essential to increase
contrast by closing the aperture. In this study only barrels that could
be clearly distinguished at 40× with high-contrast apertures and further enhanced with a camera image processor (C2400; Hamamatsu, Tokyo, Japan) were used for further study. This selects for the most clearly delineated barrels and allows the detailed study of
neurons in well defined regions of a single barrel. The barrels selected by these criteria did not necessarily lie in the posterior medial barrel subfield, and thus barrels with a wide range of diameters
were investigated.
Identification of barrels by cytochrome C stain. Fixed
slices were washed five times with PBS (100 mM
sodium phosphate, pH 7.2) over a period of 2 hr. Subsequently they were
incubated at 35°C until clear staining of barrels were observed
(0.5-5 hr) in PBS containing (in mg/ml): 0.3 cytochrome C, 0.3 catalase, and 0.5 diaminobenzidine. Slices were subsequently washed
five times with PBS over a period of 2 hr.
Quantification of barrel boundaries. Digital photographs of
barrels in living or stained slices were taken using either a monochromatic camera (C2400; Hamamatsu) or a color CCD camera (Seescan
LC100; INTAS, Göttingen, Germany). The photographs
were converted into grayscale images and imported into IgorPro
(Wavemetrics, Lake Oswego, OR) for quantitative analysis. Pixel
intensities were measured as a function of position within layer 4. Maxima (bright) in this intensity function identify septal regions,
which separate the low-intensity (dark) barrels. The intensity function was normalized such that the lowest brightness was represented by a
value of zero and the maximal brightness by one. For quantification of
the barrel boundary sigmoidal functions of the form (a + b/ (1 + exp(cx + d))) were fitted,
and the half-maximal brightness was chosen to indicate the edge of a barrel.
DiI injections. Barrels in layer 4 of somatosensory cortex
of living slices were identified using high-contrast video microscopy. 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes, Eugene, OR) was dissolved in dimethyl formamide at 0.25%. Small volumes of DiI were pressure-injected into
the center of a barrel, and the amount injected was estimated by
measuring the DiI signal within 1 min after injection. The mean
diameter of the injections was found to be 43 ± 6 µm. The slices were allowed to recover for several hours in extracellular medium before overnight fixation in 4% paraformaldehyde in PBS at
4°C. Slices were subsequently washed five times in PBS and then
incubated for 3 weeks at 35°C. No obvious change in DiI fluorescence was observed between 2 and 3 weeks incubation, suggesting that the DiI
diffusion had reached equilibrium within this period.
DiI labeling was subsequently analyzed both by standard epifluorescence
and confocal fluorescence microscopy. Both methods gave equivalent
results, however it was not possible to simultaneously observe barrel
boundaries and DiI fluorescence under confocal microscopy, and thus
only the epifluorescent measurements were used for further analysis.
Epifluorescence was recorded by a CCD camera (Seescan LC100; INTAS)
operating in a linear range allowing quantitation of the fluorescence
signal. The fluorescence intensity distributions were analyzed by
custom-written routines in IgorPro. Linear measurements of the spatial
distribution of DiI fluorescence were made either parallel to layer 4 to quantify horizontal diffusion or vertically to quantify diffusion
toward layer 2/3 and the pia. These fluorescence distributions were
fitted by sigmoidal functions to quantify the half-maximal fluorescence
values and also the 20-80% transition length as a measure of the
steepness of the drop in fluorescence.
Biocytin staining and reconstruction. After staining of
barrels by cytochrome C the slices were washed in PBS five times over a
period of 2 hr. Endogenous peroxidases were then quenched by a 5 min
incubation with 1% H2O2.
The slices were subsequently rinsed in PBS five times over a period of
2 hr. Slices were conjugated with avidin-biotinylated horseradish
peroxidase following the manufacturer's instructions (ABC-Elite;
Vector Laboratories). Slices were then washed five times over a period
of 2 hr with PBS, and subsequently biocytin-stained neurons were
visualized under a reaction with 0.5 mg/ml DAB and 0.01%
H2O2. When the neuronal processes were clearly visible, the reaction was stopped by washing with PBS. Finally the slices were mounted on slides using mowiol. Axonal and dendritic processes were subsequently reconstructed using
Neurolucida software (Microbrightfield, Colchester, VT), and
quantitative analysis of neuronal processes in relationship to barrel
borders was performed by custom-written routines in IgorPro.
Field stimulation and recording. A glass microelectrode
filled with extracellular solution with resistance of 3-4 M was
placed in the middle of a barrel identified under high-contrast video microscopy. Brief stimulating currents of 0.5 msec duration were applied every 5 sec. The amplitude of the current injection (range, 15-50 µA) was chosen to give a clearly identifiable field EPSP (fEPSP) (amplitude range, 0.3-0.7 mV) as measured at a
recording site within the same barrel as the stimulating electrode. The recording electrode was also a glass microelectrode filled with extracellular solution with resistance of 5-6 M, and voltage changes were monitored using an Axopatch 200B amplifier. The recording electrode was subsequently moved over a range of positions crossing the
barrel boundary between the stimulated barrel and the nearest neighbor
several times, and fEPSPs were compared at different locations evoked
by identical stimuli.
Whole-cell recordings from identified layer 4 neurons. Layer
4 neurons were identified using infrared differential interference contrast microscopy. Excitatory neurons of layer 4 have round somata of
~10 µm diameter and often appear to be clustered. Slices were
continuously perfused with extracellular medium bubbled with 95%
O2 and 5% CO2. All
experiments were performed at 35°C. Whole-cell recordings were
established using pipettes with resistances of 5 M filled with a
solution containing (mM): 105 potassium gluconate, 30 KCl,
10 HEPES, 10 phosphocreatine, 4 MgATP, and 0.3 Na3GTP (adjusted to pH 7.3 with KOH). Whole-cell
electrophysiological measurements were made with Axoclamp 2A, Axopatch
200A, Axopatch 200B, or EPC-7 amplifiers. Biocytin (2 mg/ml) was
routinely included in the intracellular solution to allow the
morphology of the neurons to be more closely analyzed. Excitatory
neurons were further identified by regular action potential firing
pattern in response to continuous current injection; broad action
potentials (half-widths of over 1 msec) and initial axonal segments
directed toward deeper cortical layers.
Loose-patch recordings. Using the same pipettes as for
whole-cell recordings except filled with extracellular solution and without the need to change pipette for each cell, it is possible by
gentle suction (10 mbar) to form electrically tight seals of ~50 M
resistance between pipette and an identified neuron. In such a
configuration it is possible to evoke action potentials by injection of
5 msec duration current pulses with a threshold of ~5 nA. The evoked
action potentials are recorded as short duration depolarizations with
amplitudes of ~10 mV. Because of the requirements of strong current
injections, only the Axoclamp 2A amplifier was used for this recording
configuration. The loose-patch configuration does not allow the
analysis of the action potential firing pattern, giving rise to some
uncertainty concerning the neurotransmitter identity of a presynaptic
neuron, although this can often be judged from the appearance of the
cell under IR-DIC. Thus, to isolate EPSPs, 10 µM
bicuculline was included in the extracellular medium. To test whether
the loose-patch technique provides equivalent results to whole-cell
recordings, we compared EPSPs evoked by loose-patch stimulation to
EPSPs evoked by stimulation from the whole-cell recording configuration
for the same cell pairs. In each case where loose-patch stimulation
evoked responses in the postsynaptic neuron so did the whole-cell
configuration. The amplitude of connections identified in loose-patch
mode did not change significantly in the whole-cell configuration
(ratio of EPSP evoked by loose-patch vs whole-cell action potentials
was 1.1 ± 0.2; n = 9 cell pairs). Thus,
loose-patch stimulation correctly identifies connected neurons and
quantitatively provides results comparable to whole-cell recordings for
the EPSP amplitude.
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RESULTS |
Neurons can be identified within a specific region of a barrel
Each whisker is uniquely represented in the somatosensory cortex
by a barrel-like structure as described by Woolsey and Van der Loos
(1970). Although originally identified by histological methods, the
barrel cortex of rats is equally obvious in living brain slices under
bright-field illumination (Agmon and Connors, 1991). Regions of the
neocortex that appear dark in bright-field microscopy are also heavily
stained for cytochrome C oxidase (Fig. 1), which is one of the staining methods
to identify barrels (Wong-Riley and Welt, 1980). To make a quantitative
comparison of the structures visualized by the two different
approaches, photographs of a slice under bright-field illumination were
taken first, and then the slice was stained for cytochrome C oxidase.
The color images (Fig. 1A2, B2) were converted to
gray scale, and the changes in the brightness of pixels parallel to
layer 4 were plotted across the same region identified in both images.
The maxima in the light intensity function represent the bright regions
separating the barrels. These septa occur at identical positions in
both techniques for identifying barrels. The structure visible by
bright-field microscopy thus appears to be entirely equivalent to the
one identified by cytochrome C staining.
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Figure 1.
Barrels in layer 4 of somatosensory cortex
identified by bright-field microscopy correspond to barrels defined by
cytochrome C staining. A, Barrels can be visualized in
brain slices of rat somatosensory neocortex using bright-field
microscopy. A subset of the barrels are shown at higher magnification
(A2), and the normalized brightness of pixels is
quantified across the barrels in layer 4. The dark barrels are observed
to be clearly separated by light septa. B, The same
slice as shown above was subsequently stained for cytochrome C, which
has been used to define barrels in previous work. A very similar
pattern of dark regions separated by light septa within layer 4 of the
neocortex is observed. The quantitative analysis of the normalized
pixel brightness across the barrels (B2) indicates that
there is an excellent correspondence between barrels defined by
bright-field microscopy and barrels defined by cytochrome C
staining.
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Identification of barrels in bright-field microscopy raises the
possibility that we can make measurements from a specific region of an
individual barrel in layer 4 of living brain slices. Through use of
video-enhanced high-contrast microscopy, barrel borders can be
identified at high magnification using 10× (Fig. 2A) and 40×
water-immersion lenses (Fig. 2B). This allows
whole-cell recordings to be made from neurons very close to barrel
borders (Fig. 2A,B). Excitatory neurons were
identified by small round somata often appearing in clusters and
regular firing patterns with broad action potentials. Biocytin was
included in the pipette solution, allowing the structure of the
neuronal processes to be reconstructed later (Fig. 2C,D).
Double staining for biocytin and cytochrome C reveals that the neuron,
from which we recorded, indeed was located at the edge of the barrel
(Fig. 2C,D). The data in Figures 1 and 2 indicate that the
functional architecture of barrels can be studied at high resolution
and correlated with the physiological and anatomical analysis of
individual neurons.
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Figure 2.
Whole-cell recordings from identified neurons in a
specific region of a barrel. Barrels within layer 4 can by visualized
at high magnification by enhancing contrast through video microscopy,
allowing whole-cell recordings to be made from a given region of a
barrel. The neuron in this example is located on the right-hand of the
barrel as seen with 10× (A) and 40×
(B) water immersion lenses. During the
electrophysiological recording the cell is filled with biocytin,
allowing the neuron to be visualized later in conjunction with a
cytochrome C stain (C). These stains confirm the
location of the neuron within the barrel and allow the axonal
(green) and dendritic (black)
arborizations to be reconstructed (D) in
relationship to the barrel boundaries (cyan). Scale bar:
A, C, D, 100 µm;
B, 50 µm.
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Axonal and dendritic arbors are confined to a single barrel
To define the anatomy of layer 4 barrels we investigated the
extent of the axonal and dendritic arborizations at a large-scale level
and at the level of individual neurons. One method for tracing overall
membrane structure is to measure the diffusion of fluorescent lipophilic molecules. We made microinjections of DiI into individual barrels of 10 slices and allowed the compound to diffuse in the cell
membranes (Fig. 3A). Strong
DiI labeling was observed to spread throughout the injected barrel and
the associated vertical column of neocortex, but even after 3 weeks of
diffusion, fluorescence signals were only weak in neighboring barrels.
There was a clear correlation between the width of the barrel and the
spread of the DiI signal (Fig. 3B), suggesting that the DiI
diffusion is laterally restricted to a single barrel. Equally the
fluorescence intensity showed a sharp decrease at the barrel boundary
compared to the longer vertical spread into layer 2/3 (Fig.
3C). These results are in complete agreement with a recent
study of biocytin uptake and diffusion after small extracellular
injections into layer 4 barrel cortex in vivo, which also
demonstrated labeling confined to the injected barrel (Kim and Ebner,
1999).
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Figure 3.
DiI diffusion is confined to the injected barrel.
A, The layer 4 barrels are still visible in these
bright-field micrographs (A1, A2) of a
slice that has been microinjected with DiI, fixed in paraformaldehyde,
and incubated at 35°C for 3 weeks to allow DiI diffusion. The DiI
injection was confined to a diameter of 50 µm in the center of the
middle barrel outlined in cyan (A2). DiI
fluorescence images of the same slice (A3, A4)
indicate that although the DiI has spread vertically toward the pia,
the diffusion of DiI in the horizontal direction is confined to remain
within the barrel boundary. Scale bar, 100 µm. B, The
width at half-maximal intensity of the DiI fluorescence is well
correlated to the width of the injected barrel (black
line shows best linear fit by least squares method).
C, The fluorescence intensity versus distance in the
horizontal (within layer 4) or vertical (up toward the pia) was fitted
by a sigmoidal function and the transition distance from 80% maximal
to 20% maximal intensity is shown. At the barrel/septal boundary there
is an abrupt decrease in DiI fluorescence, whereas the vertical spread
decays slowly.
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These DiI fluorescence results suggest that axonal and dendritic arbors
in layer 4 should be confined to individual barrels. To investigate
this at a cellular level, excitatory neurons located within a barrel
were filled with biocytin. The axonal and dendritic structure of the
labeled neurons was then reconstructed and correlated to the
large-scale barrel morphology revealed by cytochrome C stains (Fig.
4A). The axons and
dendrites of these neurons are indeed largely confined to the home
barrel the barrel where the soma is located. The analysis of 19 reconstructed excitatory neurons (some of which were in the middle of a
barrel and some near the edge) show that 91% of axonal and 97% of
dendritic length in layer 4 remains within the home barrel (Fig.
4B). To account for such a high degree of axonal and
dendritic confinement to a single barrel, neurons located close to the
border of a barrel have polarized axonal and dendritic arbors directed
toward the center of the barrel, whereas neurons located in the middle
of a barrel are less polarized (Fig. 4C). These results are
in excellent agreement with previous descriptions of Golgi-impregnated
layer 4 neurons, which showed that only 5% of neurons with somata
located in a particular barrel have dendrites spanning additional
barrels (Woolsey et al., 1975). Previous studies have also suggested
that the axonal arborizations within layer 4 of barrel neurons are
largely restricted to single barrels (Harris and Woolsey, 1983;
Lübke et al., 2000).
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Figure 4.
Axonal and dendritic processes are confined to the
home barrel. A, An example of a reconstructed axon
(green) and dendrite
(black) of an excitatory spiny stellate neuron found at
the right edge of a layer 4 barrel (outlined in cyan).
Neuronal process are largely confined to the home barrel. Neurons with
somata close to the edge of a barrel have axonal and dendritic trees
oriented toward the center of the barrel. Scale bar, 100 µm.
B, Quantification of the fraction of axonal
(hatched bars) and dendritic (open bars)
length within layer 4 found in the barrel in which the soma is also
located (the home barrel), in the septa between barrels and in the
adjacent barrels. Within layer 4 very little axonal and dendritic
length is found outside of the home barrel. C, The
polarization of axonal (open circles) and dendritic
(filled squares) arbors within layer 4 is related
to its location in the barrel. The y-axis is normalized
so that a neuron that had its entire arbor to the left of the soma
would be plotted at zero, whereas a neuron possessing only
arborizations to its right would be plotted at unity. The
x-axis is normalized for each barrel such that the left
border of the barrel is represented as the left extreme, and the right
border of the barrel is also the right extreme of the graph. The graph
shows quantitatively that neurons are polarized with respect to their
position in the barrel such that their processes avoid entering
neighboring barrels through polarization toward the center of the home
barrel.
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Excitatory neuronal circuits within layer 4 are functionally
confined to a single barrel
To gain an appreciation of the functional connectivity of the
large-scale neuronal network we investigated the spatial extent of
fEPSPs in layer 4 evoked by local stimulation within a single barrel
(Fig. 5). The recording pipette was moved
to different locations, both within the barrel being stimulated and in
adjacent barrels, while stimulation was kept constant. Evoked fEPSPs
were recorded and plotted as a function of the recording position (Fig. 5A-C). As the recording pipette was moved away from the
stimulation electrode, the amplitude of the evoked fEPSP gradually
decreased. However, an abrupt decrease in the fEPSP amplitude was
observed as the position of recording pipette traversed a barrel
boundary entering the neighboring barrel. To quantify this effect, the edge of a barrel was defined at the half-maximal intensity of the
bright-field image of the barrel being stimulated with the peak
representing the septum dividing neighboring barrels (Fig. 5D). Alignment of normalized field potentials as a function
of distance from the barrel edge allows the pooling of data from different experiments (n = 7). At the edge of the
barrel there is a sharp drop in evoked fEPSPs, which is detected by a
fit of a combined sigmoidal and linear function with all parameters
free for optimization by a least squares fitting routine (Fig.
5E). The half-maximal point of the fitted sigmoidal function
was 11 µm outside the stimulated barrel with a sharp transition.
Synaptic transmission under these conditions thus appears to be
restricted to a single barrel.
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Figure 5.
fEPSPs are attenuated at the barrel
border. A, Photograph of layer 4 barrels with glass
stimulation pipette in the central barrel. The various positions of the
recording electrode are indicated by the white dots. Scale
bar, 100 µm. B, The curve is a plot of pixel
brightness across the barrels in the photograph above and indicates
quantitatively how the barrels are separated by lighter regions of
septum. The black dots again represent the locations
where fEPSPs were recorded. C, Evoked fEPSPs recorded at
the different positions shown above with the left to right order
preserved. Relatively small responses are recorded in positions outside
of the barrel where the stimulation electrode is located.
D, The average brightness of pixels across the region of
layer 4 where the field recordings were made defines the barrel border
and septum for the entire set of experiments. The border of the barrel
is defined as the location where the brightness is half-maximal.
E, The amplitudes of field responses recorded from many
experiments at many positions were normalized to the value at the edge
of the barrel and plotted against distance from the border of the
barrel. The superimposed curve is the best fit (least squares method)
of a summation of a linear and a sigmoidal function with all parameters
held free. The fit suggests that fEPSPs are attenuated at the barrel
boundary.
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From the anatomical data and the spatial extent of the fEPSP responses,
it would thus seem reasonable to expect that neurons with somata in
different barrels would not be synaptically connected. To address this,
many pairs of excitatory neurons were recorded from, some with somata
in neighboring barrels and some with somata in the same barrel.
Attention was paid to ensure that the distance between the somata of
the neurons being recorded from was always <200 µm with
approximately equal distances between the pairs of neurons in the same
barrel and those in neighboring barrels (somata of neurons recorded in
the same barrel were separated by 98 ± 47 µm, whereas somata of
neurons in adjacent barrels were separated by 137 ± 30 µm).
Action potentials were evoked to test for connections (Fig.
6). Approximately one-fifth of the neuron
pairs tested were synaptically connected (21 of 103 cell pairs). Except
in one case, all of these neurons had their somata located in the same
barrel as identified by cytochrome C staining and/or bright-field
microscopy. The mean unitary EPSP (uEPSP) amplitude of the
connections was 0.93 ± 0.2 mV with a range from 0.12 to 3.3 mV.
The single connection found between two neighboring barrels was well
within the normal range, although smaller than the average with an
amplitude of 0.43 mV. In general, the axonal and dendritic trees of
neurons with somata located in different barrels showed little overlap and at a functional level were not synaptically connected.
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Figure 6.
Synaptically connected neurons are located in the
same barrel. A, An example of a synaptically unconnected
pair of excitatory layer 4 neurons, with somata lying in different
barrels. The reconstructed axonal (green) and
dendritic (black) processes are shown in relationship to
the barrel borders (cyan) (A1). The
regular firing pattern (A2) and morphology define these
cells as excitatory spiny stellate neurons. Action potentials evoked in
the left- or the right-hand cell do not evoke responses in the other
cell, showing that they are not synaptically connected.
B, An example of a connected pair of excitatory layer 4 spiny stellate neurons with soma in the same barrel (B1)
with regular firing patterns (B2). The neurons are
bidirectionally coupled, such that an action potential in either neuron
evokes an EPSP in the other (B3). Scale bars:
A1, B1, 100 µm. Calibration:
A2, B2, 50 mV, 100 msec; A3,
B3, 50 mV (top trace) or 1 mV (bottom
trace), 10 msec.
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To investigate the connectivity of the excitatory neuronal network in a
more systematic way, we tested for synaptic connections between many
different presynaptic neurons and a single postsynaptic target cell.
Whole-cell recordings were established with a postsynaptic target
neuron close to the border of a barrel, with quantification of barrel
boundaries as described earlier using the half-maximal pixel
intensities. Using loose-patch stimulation, candidate presynaptic neurons were tested, and the location of each cell relative to the
target cell and the barrel boundaries were measured. In a single
experiment between 20 and 30 presynaptic cells were tested, with care
being taken to test equal numbers of neurons at similar distances on
either side of the postsynaptic target cell (Fig. 7A,B). To evaluate whether a
pair of cells was connected, 30 action potentials were evoked in the
presynaptic cell, and the average EPSP in the postsynaptic cell was
obtained. A pair of neurons was classified as connected if an average
peak response of >0.1 mV was evoked with a short latency (beginning of
response within the 5 msec presynaptic current injection period).
Correlating the position of the tested presynaptic neurons with the
barrel showed that most connected neurons are located within the same barrel as the postsynaptic neuron, and very few connections were found
between neighboring barrels (Fig. 7A,B). Alignment of data from all experiments (n = 11) such that the origin
occurs at the barrel border indicates that this is a robust observation
(Fig. 7C). There is a sharp drop in connectivity as
presynaptic neurons with somata located in the septum are tested, and
very few connections can be found between neurons in different adjacent
barrels. Within the barrel connectivity appears to be very high with
36% of the neurons (52 of 146) within 300 µm of the target neuron
being connected (Fig. 7D). This is in good agreement with
the estimate of 31% connectivity reported by Feldmeyer et al. (1999).
The vast majority of synaptically connected neurons are thus found to
be located within the same barrel with relatively little communication
between barrels at the level of layer 4, as schematically illustrated in Figure 7F.
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Figure 7.
Neurons are highly connected within a
barrel but not with adjacent barrels. A, Photograph of a
slice showing barrels divided by a septum. The postsynaptic whole-cell
recording pipette is visible, located close to the edge of the
left-hand barrel. Square symbols indicate the locations
of cells tested using the loose-patch technique that were found to be
synaptically connected to the postsynaptic neuron. Open
circles indicate the locations of cells that were not connected
to the postsynaptic cell. The seven cells that evoked responses in the
postsynaptic cell were located either in the same barrel as the
postsynaptic cell or in the near half of the septum. Scale bar, 100 µm. B, The responses recorded in the postsynaptic cell
in response to loose-patch evoked action potentials of the cells shown
above. Traces in bold show the EPSPs
evoked by action potentials of the connected neurons, whereas the
thin traces show the absence of responses evoked by
stimulation of neurons without connections to the postsynaptic cell. An
example of an action potential evoked by loose-patch stimulation of a
presynaptic cell is shown below. Calibration: 0.2 mV for postsynaptic
potentials, 10 mV for presynaptic potentials, 5 msec for horizontal
scale bar. C, Summary of locations of cells that
were connected (squares) or not (circles) to the
postsynaptic cells in the entire set of these experiments. Most of the
connected neurons are found to lie inside the same barrel as the
location of the postsynaptic cell, which on average was located 25 µm
left of the barrel border. D, The fraction of connected
cells is strongly dependent on location. Approximately one-third of the
cells tested within 300 µm of the postsynaptic cell within the same
barrel are connected to a given postsynaptic target cell. Cells from
the adjacent barrel make very few synaptic connections. The averaged
observed brightness curve defining the home barrel, septum and adjacent
barrel in these experiments is superimposed on the connectivity data
points. E, The frequency of connection strengths
observed for neurons in the same barrel. A wide distribution of EPSP
amplitudes is observed with many small amplitude connections and a few
large connections. Along with the spatial constraint imposed by
the confines of the barrel, this distribution of connection strengths
defines the excitatory neuronal network of layer 4. F, A
schematic diagram illustrating the connectivity observed in the
experiments. Each barrel is shown to have neurons that are synaptically
connected to each other within the barrel but not to adjacent
barrels.
|
|
In addition to the spatial information regarding connectivity, these
experiments also yield functional data concerning the amplitude of the
connections. The distribution of connection amplitudes within a barrel
(Fig. 7E) is far from Gaussian, showing many small amplitude
connections with a few much larger connections in agreement with
previous data (Feldmeyer et al., 1999). There were no strong correlations between the separation of the presynaptic and the postsynaptic neurons with the uEPSP amplitude or the connection probability within a barrel over the first 300 µm investigated. This
suggests that a barrel may form an irreducible computational unit with
little spatial segregation.
| |
DISCUSSION |
The anatomical and functional data presented here at both a
large-scale level (DiI diffusion and fEPSP recordings) and at the level
of individual neurons (reconstruction of neuronal arbors and
electrophysiological analysis of synaptic connectivity) provide strong
evidence for functionally independent excitatory neuronal networks
within each barrel as defined by cytochrome C staining or bright-field microscopy.
Neuronal circuitry underlying the whisker-evoked
receptive field
The barrel cortex gains its physiological significance from the
observation that each barrel corresponds both functionally and
anatomically to an isomorphic sensory whisker. Extracellular recordings
of neuronal activity have revealed receptive fields dominated by a
single whisker isomorphic to the cortical barrel (Welker, 1976; Simons,
1978; Armstrong-James and Fox, 1987). Although a principal whisker can
often be clearly defined for cortical neurons it is also clear that
neurons in somatosensory cortex can respond to more than a single
whisker. Layer 4 neurons have the sharpest receptive fields, but even
for these neurons whiskers surrounding the principal whisker can evoke
EPSPs (Moore and Nelson, 1998; Zhu and Connors, 1999). In general these
surround whisker responses have reduced amplitude and increased latency
compared to the principal whisker (Armstrong-James et al., 1992). One
possible mechanism that has been suggested to underlie responses to
surround whiskers involves local cortical circuits. Although this may
well provide an important contribution to multiwhisker responses in layer 2/3 and layer 5, the data presented in this paper show that layer
4 barrels are anatomically and functionally independent. The excitatory
responses to surround whiskers recorded in layer 4 might thus be
attributed to earlier stages of the signaling pathway. In agreement
with this hypothesis, a recent in vivo study analyzing the
effects on vibrissa responses of ablating the neighboring neocortical
barrel found little reduction in the amplitude of responses to surround
whiskers (Goldreich et al., 1999). Furthermore, multiwhisker responses
have been detected in thalamocortical neurons of the ventral posterior
nucleus (Ito, 1988; Simons and Carvell, 1989; Armstrong-James and
Callahan, 1991; Diamond et al., 1992). Because these thalamocortical
neurons project into the barrels of layer 4, it is perhaps not
unexpected that large receptive fields can be recorded within layer 4, although at present it is unclear whether this is adequate to account
for the response properties quantitatively. In addition, layer 4 neurons receive substantial input from layer 6 (Stratford et al.,
1996), and it would interesting to investigate the underlying neuronal
circuitry determining whether the dendrites and axons of these neurons
respect barrel boundaries.
Determinants of connectivity
The boundaries of the barrel appear to provide a simple rule for
connectivity in layer 4 barrel cortex. Neurons within a barrel can be
synaptically connected, but because both dendritic and axonal arbors
are confined to the home barrel, there are few connections to
neighboring barrels. Within a barrel there is a high degree of
connectivity, which does not appear to depend strongly on distance between the neurons, at least over the first 300 µm investigated in
this study (Fig. 7D). Barrels of <300 µm diameter may
thus represent spatially homogenous processing units. Two further
pieces of evidence support this notion. First, there was no correlation between amplitudes of uEPSPs and the separation of the presynaptic and
postsynaptic neurons within the same barrel. Second, the horizontal span of both axonal and dendritic arborizations derived from our anatomical reconstructions extend across a large fraction of the barrel
diameter. The mean horizontal span within layer 4 of axonal processes
was 262 ± 86 µm and of dendritic processes was 181 ± 40 µm in barrels of mean diameter 240 ± 59 µm. It thus appears that there is no obvious spatial segregation of neuronal circuitry within a barrel, although further investigations should address the
matter specifically with respect to the large barrels of the posterior
medial subfield.
The data suggest that there is a very broad range of uEPSP connection
strengths. The distribution of amplitudes indicates that there are a
few large connections, but the majority are under well under 2 mV in
peak amplitude. Such a distribution is unlikely to be formed by a
process of random connectivity, which would result in connectivity
distributions closer to Gaussian or decaying exponential. Such
functions are able to fit the observed connectivity for small
amplitudes (<2 mV), but they fail to account for the large amplitude
EPSP connections. That the distribution of connection amplitudes is far
from random suggests that rules may exist for determining the
connectivity of the excitatory neuronal network within a layer 4 barrel.
Recently, Egger et al. (1999) described a form of long-term depression
found between excitatory neurons of layer 4, which would decrease the
efficacy of a synaptic connection if both presynaptic and postsynaptic
neurons are highly active simultaneously. Such a process will act to
destabilize large connections and could account for the bias to small
connections. As yet no mechanism for increasing the synaptic strength
of connections between layer 4 neurons has been identified, and in
particular Egger et al. (1999) also reported that there does not appear
to be any NMDA receptor-dependent synaptic potentiation within the
mature layer 4. Perhaps, in analogy to the thalamocortical synapses,
there is a critical period for NMDA receptor-dependent potentiation such that rats >1-week-old do not express this type of plasticity (Crair and Malenka, 1995). There is thus a need to explore the development of layer 4 barrel cortex more closely to investigate what
determinants of connectivity are active as the neuronal circuitry is established.
Sensory deprivation provides a method for the analysis of
activity-dependent reorganization of the somatosensory signaling pathway. Although the developing layer 4 is clearly able to undergo plastic changes in response to deprivation of sensory input (Van der
Loos and Woolsey, 1973; Wong-Riley and Welt, 1980; Schlaggar et al.,
1993) it is at present unclear to what extent the neuronal network of
layer 4 in the adult can be rearranged (Diamond et al., 1994; Glazewski
and Fox, 1996). Our understanding of the determinants of connectivity
would benefit from a detailed analysis of what changes occur at both a
morphological and physiological level to the neuronal network after
sensory deprivation. The description of the undeprived excitatory
neuronal network presented here provides a reference point for studying
plastic changes induced by alteration in sensory input.
The simplest interpretation of the connectivity data of the layer 4 excitatory neuronal network within barrel would assume that the
connection between any two neurons can be chosen randomly from the
observed distribution. However, it is possible that the rules of
development and long-term plasticity lead to networks far from the
stochastic situation. Although there is no obvious spatial segregation
of neuronal networks within a barrel, there could in fact be functional
subnetworks of neurons, which the present set of experiments would not
have observed. Further experiments with simultaneous recordings from
many neurons will be necessary to address this important issue.
| |
FOOTNOTES |
Received May 8, 2000; revised July 17, 2000; accepted July 25, 2000.
C.C.H.P. was supported by a Marie Curie fellowship from the European
Commission. We thank Michael Brecht, Veronica Egger, and Dirk Feldmeyer
for helpful discussions and comments on this manuscript.
Correspondence should be addressed to Carl C. H. Petersen,
Department of Cell Physiology, Max-Planck-Institute for Medical Research, Jahnstrasse 29, Heidelberg D-69120, Germany. E-mail: petersen{at}mpimf-heidelberg.mpg.de.
| |
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C. C. H. Petersen
Short-Term Dynamics of Synaptic Transmission Within the Excitatory Neuronal Network of Rat Layer 4 Barrel Cortex
J Neurophysiol,
June 1, 2002;
87(6):
2904 - 2914.
[Abstract]
[Full Text]
[PDF]
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N. Laaris and A. Keller
Functional Independence of Layer IV Barrels
J Neurophysiol,
February 1, 2002;
87(2):
1028 - 1034.
[Abstract]
[Full Text]
[PDF]
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D. Feldmeyer, J. Lubke, R A. Silver, and B. Sakmann
Synaptic connections between layer 4 spiny neurone- layer 2/3 pyramidal cell pairs in juvenile rat barrel cortex: physiology and anatomy of interlaminar signalling within a cortical column
J. Physiol.,
February 1, 2002;
538(3):
803 - 822.
[Abstract]
[Full Text]
[PDF]
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M. Brecht and B. Sakmann
Whisker maps of neuronal subclasses of the rat ventral posterior medial thalamus, identified by whole-cell voltage recording and morphological reconstruction
J. Physiol.,
January 15, 2002;
538(2):
495 - 515.
[Abstract]
[Full Text]
[PDF]
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C. C. H. Petersen and B. Sakmann
Functionally Independent Columns of Rat Somatosensory Barrel Cortex Revealed with Voltage-Sensitive Dye Imaging
J. Neurosci.,
November 1, 2001;
21(21):
8435 - 8446.
[Abstract]
[Full Text]
[PDF]
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TOP
ABSTRACT
INTRODUCTION
