1 Anatomisches Institut der Albert-Ludwigs-Universität Freiburg, Albertstrasse 17, D-79104 Freiburg and , 2 Abteilung Zellphysiologie, Max-Planck-Institut für medizinische Forschung, Jahnstrasse 29, D-69120 Heidelberg, Germany, 3 All authors have contributed equally to this work
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Abstract |
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Introduction |
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The electrical representation of a single whisker deflection in layer 2/3 is multi-columnar spreading almost across the entire barrel field (Orbach et al., 1985; Masino and Frostig, 1996
; Chen-Bee and Frostig, 1996
; Moore and Nelson, 1998
; Zhu and Connors, 1999
; Brett-Green et al., 2001
; Petersen et al., 2003
) (M. Brecht, A. Roth and B. Sakmann, unpublished observations). Functional connections between barrels are sparse (Feldmeyer et al., 1999
; Petersen and Sakmann, 2000
), consistent with the fact that L4 spiny neuron excitation following stimulation of one barrel is restricted to a single barrel-column (Petersen and Sakmann, 2001
) and reflecting the predominantly vertical organization of their axons (Lübke et al., 2000
). With respect to the anatomical basis of the cortical representation of a whisker deflection the questions arise where the divergence from single-column to multi-columnar excitation occurs and what the exact dimensions of the L4-to-L2/3 projection in supra- and infragranular layers with respect to the barrel borders are.
The functional architecture of connections in the barrel cortex can be altered by modifying sensory input from the whisker pad, often referred to as plasticity of sensory maps (Armstrong-James et al., 1992; Keller and Carlson, 1999
; Kossut and Juliano, 1999
; Wallace and Fox, 1999
) [for a review see Woolsey (Woolsey, 1990
)]. Such alterations in the representational areas could be mainly functional, e.g. by changing synaptic efficacy in existing circuits. Alternatively, they could include morphological changes in the density of axon collaterals, synaptic boutons and/or dendritic spines. Sensory deprivation experiments suggest that such plastic changes of representational maps may occur in the vertical connections between excitatory L4 and L2/3 neurons as well as in the horizontal connections within layer 2/3 (Shepherd et al., 2003
). To interpret such changes a quantitative anatomy of connections within a barrel-column must be established. For this we developed a method to average the dendritic and axonal domains of neurons.
Here we describe the dimensions of axonal arbors of L4 spiny neurons and their overlap with L2/3 pyramidal cell dendrites as well as the spread of L2/3 axons to delineate the morphological substrate for the vertical and horizontal spread of excitation. For this purpose we analysed the anatomical dimensions of monosynaptically connected pairs of neurons in layers 4 and 2/3, respectively, using reconstructions in relation to barrel borders; the locations of putative synaptic contacts established by the L4 spiny neuron axons on L2/3 pyramidal cells were determined and the average spread of axon collaterals of L2/3 pyramidal cells was measured.
The dimensions of the vertical axonal projections of L4 spiny neurons and of the dendritic arbor of L2/3 pyramidal cells are almost perfectly matched. They form a barrel-column-restricted innervation domain and, together with the projections to infragranular layers, a cytoarchitectonic barrel-column while the long-range horizontal axons of L2/3 pyramidal cells project to adjacent columns. We suggest that the major anatomical substrate for the spread of weak afferent excitation in layer 2/3 is the axonal arbor of L4 spiny cells whereas for strong excitation it is the axonal arbor of barrel-related L2/3 pyramidal cells.
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Materials and Methods |
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The anatomical data are based on 16 pairs of synaptically connected L4 spiny neurons and L2/3 pyramidal cells that were reconstructed after biocytin filling via the recording pipette in acute thalamocortical brain slices. In addition three individual L2/3 pyramidal cells were reconstructed. The morphometric data were derived from 14 reconstructed pairs, the 2D density maps were derived from nine reconstructed pairs. Two pairs of cells were analysed in parallel by camera lucida (2D) drawings and by 3D Neurolucida (MicroBrightField, Colchester, VT) reconstruction for comparison. In two cell pairs the light-microscopically identified synaptic contacts were confirmed by serial EM sectioning. In addition, five pairs of synaptically coupled L4 spiny neurons were analysed morphometrically.
Electrophysiological Recordings
All experiments were carried out in accordance with the animal welfare guidelines of the Max Planck Society and the University of Freiburg. Wistar rats (1925 days old) were anaesthetized with halothane, decapitated and slices through the somatosensory cortex (barrel cortex) were cut in cold extracellular solution using a vibrating microslicer (DTK-1000, Dosaka Co. Ltd, Kyoto, Japan) and prepared according to methods described elsewhere (Agmon and Connors, 1991; Feldmeyer et al., 1999
, 2002
). Whole-cell voltage recordings from pre- and postsynaptic neurons were made as in detail described elsewhere (Feldmeyer et al., 1999
, 2002
). In brief, a postsynaptic cell was recorded from with one pipette and subsequently synaptic connections to this cell were searched with a second pipette in the loose-patch configuration. After establishing a loose seal on a presumed presynaptic cell, action potentials were elicited by applying 10 ms current pulses through the membrane. The extracellularly recorded action potential was visible as a small deflection on the voltage response. When this stimulation resulted in an EPSP in the post-synaptic neuron, the presynaptic cell was patched with a new pipette filled with biocytin-containing intracellular solution and action potentials were elicited in the whole-cell (voltage recording) mode. At the end of each experiment, low power micrographs were taken with the recording pipettes in place to later orient the reconstructions with respect to barrel borders (Feldmeyer et al., 2002
). All pairs of neurons used in this study were located in the dorsal end of the posterior medial barrel subfield of the primary somatosensory cortex as indicated by the relatively large barrel size (see Table 3
).
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Slices were continuously superfused with extracellular solution containing (in mM): 125 NaCl, 2.5 KCl, 25 glucose, 25 NaHCO3, 1.25 NaH2PO4, 2 CaCl2 and 1 MgCl2 bubbled with 95% O2 and 5% CO2. The intracellular pipette solution contained (in mM): 105 K-gluconate, 30 KCl, 10 HEPES, 10 phosphocreatine, 4 ATP-Mg, 0.3 GTP (adjusted to pH 7.3 with KOH). The osmolarity of this solution was 300 mOsm. For morphological analysis, 23 mg/ml biocytin (Sigma, Munich, Germany) was added to the internal solution and neurons were filled during 0.52.5 h of recording.
Histological Procedures
Following recording and intracellular filling with biocytin, brain slices were fixed in 100 mM phosphate-buffered solution (PB, pH 7.4) containing 1% paraformaldehyde and 2.5% glutaraldehyde at 4°C for at least 24 h. Selected pairs were processed for light- and electron microscopy to perform a detailed morphological analysis and to confirm putative light-microscopically identified synaptic contacts as described elsewhere (Lübke et al., 2000). For light-microscopic reconstruction (see below) slices were embedded in a water-based medium (Mowiol; Clariant, Sulzbach i. Taunus, Germany) to reduce tissue shrinkage. For electron microscopy, slices were post-fixed in 0.5% OsO4 (3045 min), then dehydrated and embedded in Durcupan (Fluka, Deisenhofen, Germany). Ultrathin sections were counterstained and examined with a Philips CM 100 electron microscope (Philips, Eindhoven, The Netherlands).
Morphological Reconstructions
Pairs of synaptically coupled neurons were examined under the light microscope at high magnification (x1200). Only pairs for which a complete physiological analysis was made and that had no obvious truncation of their dendritic and axonal profiles were used for qualitative and quantitative analysis of their morphology. Representative pairs of neurons were photographed at different magnifications to document dendritic morphology, axonal projection and location of synaptic contacts. Neurons were then drawn with the aid of a camera lucida attached to an Olympus BX50 microscope (Olympus, Hamburg, Germany) at a final magnification of x720 or x1200. For some pairs of neurons, three-dimensional reconstructions were also made using a x100, 1.4 NA oil-immersion objective (Zeiss) and Neurolucida software. These reconstructions provided the basis for further quantitative morphological analysis of the following parameters: (i) maximal horizontal field span of the dendrites and axons of the pre- and postsynaptic neurons, (ii) total number and dendritic location of putative synaptic contacts per neuron, and (iii) dendritic and axonal length density and total number and density of synaptic boutons counted for the axons of L4 spiny neurons and L2/3 pyramidal cells. Measurements were not corrected for shrinkage. For all data, means ± S.D. were calculated.
Axonal and Dendritic Density Maps
We developed a method to obtain two-dimensional (2D) maps of axonal and dendritic length density or axonal bouton density from 2D reconstructions. First, the length of all axonal branches was measured or all boutons were counted and the length of all dendritic branches was measured manually in a 25 µm x 25 µm (L4L4 connections) or 50 µm x 50 µm (L4L2/3 connections) Cartesian grid superimposed on the drawing, yielding a raw density map. For alignment of these maps with respect to the barrel centre, barrel borders were identified in the low power (x2.5 objective) bright field micrographs made from the acute brain slice (Lübke et al., 2000; Petersen and Sakmann, 2000
; Feldmeyer et al., 2002
). Spatial low-pass filtering of these maps was performed by 2D convolution with a Gaussian kernel (
= 25 µm or 50 µm) and continuous 2D density functions were constructed using bicubic interpolation in Mathematica 4.1 (Wolfram Research, Champaign, IL).
Two-dimensional maps constructed from 2D reconstructions involve two projection steps: first, the z component of dendritic and axonal lengths is neglected, and second, an essentially three-dimensional (3D) density map is projected onto a 2D plane. To estimate possible errors in the widths of regions containing 80% of the integrated density (Table 3) due to the use of 2D reconstructions and 2D projections of 3D density maps, two pairs of cells were fully reconstructed in Neurolucida (Micro-brightfield, Colchester, VT). The generation of 2D reconstructions and 2D maps from these 3D reconstructions was then simulated in Mathematica using the same spatial low-pass filtering and interpolation as described above. Horizontal and vertical widths (in a plane containing the soma) of regions containing 80% of the integrated density were measured. Widths of 80% regions (bounded by isosurfaces) in the full 3D maps were 9% to 24% larger than in the corresponding 2D projections, while the additional effect of neglecting the z component of dendritic and axonal lengths in 2D reconstructions was less than 2% change in widths. Furthermore, care was taken to minimize shrinkage in the xy plane by using water-based embedding media (see above).
For the L2/3 pyramidal cell axons the dimensions of the density map may be underestimated when the axonal arbor is not rotationally symmetric. However the axonal spread of L2/3 pyramidal cells described here for 2D projections is comparable to that for 3D reconstructions of identified L2/3 pyramidal cells recorded and filled during in vivo experiments (Petersen et al., 2003) (M. Brecht, A. Roth and B. Sakmann, unpublished observation). Nevertheless the axonal arbor outline of L2/3 pyramidal cells in the 2D map represents only a lower estimate of its width in a projection plane almost parallel to the row of barrels.
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Results |
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Infrared video microscopy facilitated the selection of pairs of neurons according to shape, size and location of somata within a single barrel or above the barrel throughout layer 2/3 as described (Feldmeyer et al., 2002). Paired recordings were made exclusively from neurons in the somatosensory cortex. The mean geometric distance of the postsynaptic pyramidal cell from the presynaptic neuron varied between 204 and 403 µm and they were all located above barrels (barrel-related pyramidal cells). Pre- and postsynaptic neurons were defined as excitatory by their regular action potential firing pattern and the sensitivity of the postsynaptic response to glutamate receptor antagonists [AP5 and NBQX; see Feldmeyer et al. (Feldmeyer et al., 2002
)].
Spiny Layer 4 Neurons
From the sample of synaptically coupled pairs of neurons, ~80% of the presynaptic neurons were identified as spiny stellate neurons, the remainder as star pyramidal cells (Feldmeyer et al., 2002). A clear distinction between the two classes of cells was not always possible because of the variability in the morphology of the apical dendrite. Almost all spiny stellate neurons had dendrites with an asymmetric dendritic arrangement oriented towards the centre of a barrel (Figs 1A
and 2AC
). The somata and dendrites of L4 star pyramidal cells were also exclusively located within layer 4 with the exception of the distal apical dendrite that extends in layer 2/3 without forming a terminal tuft (Fig. 2D
). In contrast to spiny stellate cells, star pyramidal cells were located always towards the centre of a barrel and their basal dendrites showed no asymmetry (Feldmeyer et al., 1999
; Lübke et al., 2000
) (V. Egger and B. Sakmann, unpublished observation). However, the dendritic field of L4 spiny neurons was always restricted to the borders of the home column in which the somata of the presynaptic neurons were located.
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The axonal collaterals of all synaptically coupled L4 spiny stellate and star pyramidal cells project throughout all cortical laminae from layer 1 to the white matter, but were largely confined to their home column (Fig. 2). The majority of axonal collaterals branch off in layer 4 or upper layer 5 and ascend towards layer 2/3 forming a dense axonal projection with a high degree of collateralization. The axonal collaterals of L4 spiny neurons were densely covered with synaptic boutons suggesting a high probability of innervation of target neurons. Morphological characteristics of the presynaptic neurons, the pattern of their dendritic and axonal arborization are summarized quantitatively in Table 1
.
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L2/3 pyramidal cells constitute a more heterogeneous population within the class of neocortical pyramidal cells with respect to their dendritic configuration (Gilbert and Wiesel, 1979; Valverde, 1986
; Burkhalter, 1989
; Gottlieb and Keller, 1997
) [for review see DeFelipe and Farinas (DeFelipe and Farinas, 1992
)]. The primary basal dendrites emerging from the soma give rise to numerous secondary, tertiary and higher-order basal dendrites of different length (Table 1
) that form an almost symmetric receptive field (Fig. 2
). The basal dendrites of pyramidal cells monosynaptically innervated by L4 spiny neurons were also confined to the same barrel-column (Fig. 2
) with one exception (Fig. 2C
). The shape of the apical dendrite was, however, highly variable with respect to its length, bifurcation pattern, number of apical oblique dendrites and field size of the terminal tuft in layer 1 (compare pyramidal cells in Fig. 2
; see also Table 1
). Pyramidal cells in the upper to middle part of layer 2/3 have rather short main apical trunks that often bifurcate in twinned dendrites relatively close to the soma (~4080 µm) giving rise to an extensive terminal tuft with a field span of ~300 µm (compare postsynaptic neurons in Fig. 2
). Pyramidal cells in the lower part of layer 2/3 have a long (150 µm) and more prominent apical dendrite with several oblique dendrites of various length and distance from the soma (Figs 1A
and 2D
; Table 1
).
In one paired recording an unusual pattern of dendrites and axonal collaterals of pre- and postsynaptic neurons was observed (Fig. 2C). The postsynaptic L2/3 pyramidal cell was located at the border of the barrel-column close to the pial surface and had an apical dendrite and a terminal tuft that were slightly tangentially oriented. The soma of the L4 spiny stellate neuron was not located directly underneath the postsynaptic neuron, however its axonal projection was largely vertically oriented as shown for other L4 spiny neurons. In this pair of neurons two synaptic contacts were located in the terminal tuft of the target neuron (Fig. 2C
).
In Figure 2D the L4 spiny neuron was a star pyramidal cell. Star pyramidal cells had an axonal projection similar to that of spiny stellate neurons (Lübke et al., 2000
).
Patch-clamping under visual control involves the selection of neurons to record from and may therefore lead to biased sampling. We tried to minimize this problem by attempting to use a fair sampling strategy, recording from postsynaptic neurons throughout layer 2/3 as well as to the left and right of the barrel-column. Similarly, presynaptic neurons were searched for in the entire barrel. Figures 2 and 3
illustrate the range of positions of both pre- and postsynaptic neurons recorded from.
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Overlap of Projection and Receptive Fields in the L4-to-L2/3 Connection
When all reconstructions of L4-to-L2/3 neuron pairs (n = 9) are superimposed and aligned with respect to the barrel centre (Fig. 3) it is clearly evident that within a barrel-column the projection zone of L4 spiny neuron axons overlaps considerably with the dendritic receptive field of L2/3 pyramidal cells. To quantify this overlap the axonal length of L4 spiny neurons was measured using a 50 µm x 50 µm grid superimposed on 2D reconstructions of the cell pairs (n = 9). We then constructed a 2D map of the axonal length density of L4 axons using bicubic interpolation of the original grid points yielding an average axonal projection of L4 spiny neurons. The reference point for alignment of the reconstructions was either the centre of the barrel (Fig. 4A1
), the soma of the L4 spiny neuron (not shown) or the soma of the L2/3 pyramidal cell (Fig. 4B1
). The map of the L4 axonal length density clearly shows that the largest fraction of the L4 axon is restricted to a barrel-column. Comparing the outline of the average barrel-column to the contour line including 80% of the L4 axonal length density shows that its majority is restricted to the barrel-column throughout all cortical layers (Table 3
). However in layer 2/3 the width of the 2D map is somewhat larger than in L4 and L5 (Fig. 4A1
, 4B1
, Table 3
).
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Assuming that synaptic connections between axons and dendrites in a given region are formed in a random process, we calculated the predicted innervation domain by multiplying the axonal length density and the dendritic length density (Fig. 4A3); for discussion, see also Stepanyants et al. and Kalisman et al. (Stepanyants et al., 2002
; Kalisman et al., 2003
). The extent of this predicted innervation domain is limited by the extent of the dendritic arborization. The sharp delineation of this innervation domain of L2/3 pyramidal cells by L4 spiny neurons is particularly clear when L4 axonal and L2/3 dendritic length maps are normalized with respect to the somata of pyramidal cells. The 2D projected dimensions of the innervation domain are ~240 µm (horizontal) and 330 µm (vertical; see Table 3
). Only the basal dendritic field is overlapping with axonal arbors of L4 cells (Fig. 4B3
). Within this innervation domain, the axonal length of a single L4 spiny neuron was 5519 µm and 3936 µm, with a bouton density of 0.405 ± 0.01 per µm, corresponding to 2235 and 1594 synaptic boutons in the innervation domain. The L2/3 dendritic length was 2724 µm and 2730 µm for the barrel-centred and the soma-centred density maps, respectively, with a dendritic spine density on the basal dendrites of 0.97 ± 0.1 spines/µm (n = 3 cells). From these numbers the fraction of synaptic contacts established by the axon collaterals of L4 spiny neurons can be estimated (see Discussion).
Synaptic Contacts Between L4 Spiny Neurons and L2/3 Pyramidal Cells
In 14 pairs of synaptically coupled L4 spiny neurons and L2/3 pyramidal cells the number and dendritic location of putative synaptic contacts was analysed light- and electron-microscopically (Fig. 1BE, insets in Fig. 2
; see also Table 2
). The total number of light-microscopically identified synaptic contacts in these cell pairs was 65. Synaptic contacts were exclusively found on the dendrites of L2/3 pyramidal cells and were always located within the home column of the presynaptic neuron. For individual connections, their number varied between 4 and 6 [mean: 4.8 ± 0.6; see also Table 2
and Feldmeyer et al. (Feldmeyer et al., 2002
)]. Synaptic contacts were preferentially located on the basal dendrites (86.2%, n = 56; Table 2
). Of these, 89.3% were en passant synaptic contacts predominantly on second- and third-order basal dendrites. The remaining contacts (10.7%) were found on fourth-order basal dendrites. Synaptic contacts on basal dendrites were located at a distance of 15130 µm from the soma (see also Table 2
). Only a smaller fraction (13.8%) was located on second- and third-order apical oblique dendrites at a distance of 115200 µm from the soma (Fig. 2C
; see also Table 2
). Out of 65 light-microscopically identified synaptic contacts only 5 (7.7%) were found on the same dendrite within a distance of 50 µm to each other.
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Innervation Domain and Location of Synaptic Contacts
To estimate whether the extent of the innervation domain of axonal collaterals of L4 spiny neurons and L2/3 pyramidal cell dendrites corresponds to the actual density of innervation, the location of synaptic contacts was marked in the innervation domain (Figs 5 and 7
; blue dots). Most contacts are indeed located within the borders of the innervation domain. This is the case irrespective of whether the reconstructions were centred with respect to barrels (Fig. 5A
) or to L2/3 pyramidal cell somata (Fig. 5B
). Only three or four of 32 contacts (913%; n = 7 connections) were located outside the predicted innervation domain.
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To identify possible anatomical determinants of the lateral spread of excitation between barrel columns in layer 2/3 the axonal projection of L2/3 pyramidal cells receiving monosynaptic input from L4 spiny neurons was also reconstructed and quantitatively analysed. The extent of the axonal arborization of L2/3 pyramidal cells was variable between different pairs of neurons. However, the axonal arbor always displayed two distinct domains: firstly, a vertically oriented domain of collaterals that emerged from the main axon ascending towards layer 1 where they terminated; and secondly long-range horizontal collaterals (Fig. 8A) in layers 2/3 and 5.
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Projection Density Map
To obtain a quantitative estimate of the outline of L2/3 axonal projections reconstructions of L2/3 pyramidal cells were aligned with respect to the barrel centres (Fig. 8B). A 2D map of L2/3 axonal length was obtained with the same grid of 50 µm x 50 mm as was used for the quantification of L4 axonal arborizations. The map generated when reconstructions where aligned with respect to the barrel centres illustrates the large horizontal spread of L2/3 pyramidal cell axons across the borders of adjacent barrel-columns (Fig. 8C
). In the horizontal plane in layer 2/3 the contour line including 80% of the axonal length density extends across the adjacent barrel-columns with a maximal width of ~1.2 mm. A second projection field in layer 5 is narrower (maximal width is ~800 µm), whereas in layer 4 the axonal length density is restricted to the width of the barrel (Table 3
).
This density map illustrates the average projection of axon collaterals of L2/3 pyramidal cells receiving monosynaptic excitatory input from layer 4. Clearly, the main axonal projection of the L2/3 pyramidal cell extends far into the neighbouring barrel-columns both in layers 2/3 and 5. However, it is possible that in slices a fraction of the long-range axon collaterals are truncated and that their maximal horizontal spread is sub-stantially wider.
For a subset of cell pairs (n = 7) bouton counts for both L4 spiny neurons and L2/3 pyramidal cells were determined using the same 50 µm x 50 µm grid as for the axonal length density measurements. The L4 and L2/3 bouton density maps thus obtained were very similar to the axonal length density maps described above (not shown). Likewise, the resulting innervation domains had comparable dimensions using either bouton density or axonal length density maps.
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Discussion |
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Columnar Organization of the L4-to-L2/3 Connection
In the somatosensory cortex the restriction of L4 spiny cell dendrites to barrel column and laminar borders is well documented (Lorente de Nó, 1922; Woolsey and van der Loos, 1970
; Simons and Woolsey, 1984
; Lübke et al., 2000
). In addition, the axonal domain of L4 spiny neurons shows also a high degree of columnarity (Harris and Woolsey, 1983
; Bernardo et al., 1990
; Lübke et al., 2000
). Its dimensions are comparable to those of functional barrel columns identified either by 2-deoxyglucose autoradiography (Chmielowska et al., 1986
; McCasland and Woolsey, 1988
), imaging of cerebral blood flow (Woolsey et al., 1996
) or voltage sensitive dye imaging (Laaris et al., 2000
; Petersen and Sakmann, 2001
; Petersen et al., 2003
). However, inter-barrel projections were occasionally observed (Brecht and Sakmann, 2002
). The axonal collaterals of barrel-related L2/3 pyramidal cells project across the borders of a barrel column, extending horizontally in layer 2/3 and 5 but largely sparing layer 4. On the other hand, the hourglass structure apparent after extracellular dye injection into a single barrel (Bernardo et al., 1990
; Miller et al., 2001
) may represent axon collaterals of L4 spiny neurons and L2/3 pyramidal cells and possibly also of inhibitory interneurons. The orientation of these axon collaterals is highly asymmetrical in the SI barrel cortex, as barrel-related columns within a row appear more strongly interconnected than those in different rows. This was not found outside the SI cortex (Bernardo et al., 1990
). In summary, the morphology of neurons connecting L4 with L2/3 seems to be markedly more stereotyped in the barrel cortex than in other cortices.
Two Innervation Domains in a Barrel-column
The innervation domain of L2/3 pyramidal cells by L4 spiny neurons is located above a barrel, with similar horizontal dimensions. It is largely co-extensive with the distribution of light-microscopically identified synaptic contacts (Figs 5 and 7
; blue dots). The same holds true for the L4-to-L4 innervation domain (Fig. 7
; purple dots; see below). Whether such co-extension of the predicted innervation domains and the actual distribution of synaptic contacts exists in other cortical connections remains to be determined. An estimate of the number of L2/3 pyramidal cells innervated by the axon collaterals of a single L4 spiny neuron can be obtained from the number of boutons in the L4-to-L2/3 innervation domain which was 2235 and 1594, for the barrel-centred and the soma-centred density maps, respectively. When divided by the mean number of contacts per connection [4.5 (Feldmeyer et al., 2002
)] this yields 497 and 354 as the number of L2/3 pyramidal cells innervated by a single L4 spiny neuron. However, since ~1520% of cortical neurons are GABAergic interneurons, this number has to be reduced to 397 and 283 (assuming for simplicity that connections between L4 spiny neurons and L2/3 interneurons have a roughly similar number of contacts per connection). On the other hand, synaptic connections are also made outside the innervation domain containing 80% of the predicted synaptic density, suggesting that the number of postsynaptic targets is actually higher. In the adult animal, a barrel-column with the dimensions: width, 350 µm; layer 4 height, 300 µm; layer 2/3 height, ~500 µm (Gottlieb and Keller, 1997
) and neuron densities of 111 830 per mm3 in layer 4 and 69 290 per mm3 in layer 2/3 (Keller and Carlson, 1999
) contains 4110 and 4244 neurons in layers 4 and 2/3 [see also Bruno and Simons (Bruno and Simons, 2002
)], respectively, of which 80% are excitatory. As the ratio between layer 4 and layer 2/3 neurons is close to unity, it follows by symmetry that ~300400 L4 spiny neurons innervate a single L2/3 pyramidal cell. Another way to calculate the L4-to-L2/3 convergence is based on the spine density of L2/3 pyramidal cells [0.97 per µm, comparable to the value of 1.12 per mm reported by Trevelyan and Jack (Trevelyan and Jack, 2002
) and the total dendritic length of a neuron in the innervation domain (~2730 µm)]. As the number of contacts per connection is 4.5 (Feldmeyer et al., 2002
) and since ~50% of these are made onto spines (Lübke et al., 2000
) the number of L4 spiny neurons per L2/3 pyramidal cell would be (0.97 per µm x 2730 µm)/(0.5 x 4.5) = 1177. The difference between the two values for the convergence (300400 vs 1177) indicates that between one quarter to one-third of all synaptic contacts onto L2/3 pyramidal cells in the L4-to-L2/3 innervation domain are made by vertical inputs from L4. About two-thirds are made by other neurons, most likely by other L2/3 pyramidal cells (Reyes and Sakmann, 1999
; Egger et al., 1999
; Yoshimura et al., 2000
) located in the same and adjacent columns.
L4 spiny neurons also innervate other L4 neurons in the same barrel. They also form a sharply column-restricted L4-to-L4 innervation domain extending just below the L4-to-L2/3 domain. What is the connectivity of these neurons in layer 4? To address this the number of L4 axonal boutons established by a single L4 spiny neuron in the L4-to-L4 innervation domain was calculated (see Results), yielding 913 boutons of which only 0.5 are forming an autaptic contact (Feldmeyer et al., 1999). Dividing this number by the mean number of contacts per connection [3.4 (Feldmeyer et al., 1999
)] gave a value of 268 L4 spiny neurons that are innervated by a single L4 spiny neuron. However, since 1520% of cortical neurons are GABAergic interneurons, this number is reduced to 215 (assuming for simplicity that connections between L4 spiny neurons and interneurons have a similar number of contacts per connection).
By symmetry, on average 215 spiny neurons in layer 4 converge onto this single L4 spiny neuron. Convergence can also be estimated from the spine density of L4 spiny neurons (0.45 ± 0.11 per µm) and the total dendritic length of a neuron in this innervation domain (1242 µm). The number of contacts in a connection between L4 spiny neurons is 3.4 (Feldmeyer et al., 1999) and ~50% of these are made onto spines (Lübke et al., 2000
) so that the number of L4 spiny neurons innervating a given L4 spiny neuron would be (0.45 per µm x 1242 µm)/(0.5 x 3.4) = 329. The difference between the two values obtained for the divergence (215 vs 329) indicates that ~65% of all synaptic contacts onto L4 spiny neurons in the L4-to-L4 innervation domain are made by other L4 spiny neurons.
Functional Connectivity
The above values then allow an order of magnitude estimate of the number of L4 spiny neurons and synaptic contacts on L2/3 pyramidal cells activated by a single whisker deflection in anaesthetized rats. In vivo imaging shows that the VSD signal evoked by principal whisker deflection is initially (15 ms) restricted to the cross-section of the PW barrel but later (>20 ms) spreads horizontally into surround whisker columns (Petersen et al., 2003
). Recent in vivo whole-cell recordings suggest that all L4 neurons in a PW column respond with subthreshold EPSPs. However, only 4% of the recorded cells generate APs following a single whisker deflection during this initial phase [(Brecht and Sakmann, 2002
); their Fig. 12D)]. Therefore, whisker stimulation excites ~140 neurons in layer 4 above threshold (4% of ~3400 excitatory neurons in an adult barrel). An individual L4 neuron innervates ~300400 pyramidal cells in layer 2/3 and an AP in a L4 neuron generates with high reliability near coincident EPSPs in L4 and L2/3 neurons within a column (Feldmeyer et al., 1999
, 2002
). On the other hand, ~300400 L4 spiny neurons innervate a single L2/3 pyramidal cell (see above). As 4% of these generate APs during the first 15 ms of a whisker deflection each L2/3 pyramidal cell in the PW column receives synaptic input from 12 to 16 L4 spiny neurons, on average. The probability of a L2/3 pyramidal cell receiving no synaptic input from layer 4 during this time is therefore small and consistent with in vivo whole-cell recordings from barrel-related L2/3 pyramidal cells which indicate that virtually all respond with a compound EPSP to principal whisker deflection (M. Brecht, A. Roth and B. Sakmann, unpublished). The average of these EPSPs is probably generating the initial column-restricted component of the single whisker-evoked VSD response in the neocortex (Petersen et al., 2003
). In layer 2/3, the column-restricted VSD signal thus reflects input from ~140 L4 spiny neurons that form 140 x 400 x 4.5 = 252 000 synaptic contacts located mostly on the basal dendrites of L2/3 pyramidal cells.
Relation Between Anatomical and Functional Topographic Maps
How can the overlapping maps of dendritic and axon length density be related to functional maps constructed from receptive fields mapped by EPSPs and/or APs that define the dynamic sub- or suprathreshold electrical representation (excitation map) of a whisker deflection? On the cortical surface subthreshold excitation evoked by deflection of a single whisker should be restricted initially to a relatively small area given by the blob-like L4-to-L2/3 innervation domain. Time resolved VSD imaging in acute slices of barrel cortex shows indeed that electrical stimulation of a single barrel elicits a wave of excitation largely delineated by the borders of a barrel-column (Laaris et al., 2000; Petersen and Sakmann, 2001
). However, when inhibition is blocked excitation remains confined to a barrel in layer 4 but spreads horizontally in layers 2/3 and 5 but not in layer 4 (Laaris et al., 2000
; Petersen and Sakmann, 2001
; Laaris and Keller, 2002
). Likewise in vivo the VSD signal evoked by a single whisker deflection on the cortex is initially restricted to the cross-section of the PW column and spreads later horizontally into surround whisker columns. This time dependent spread of excitation in the cortex may reflect the transition from sub- to suprathreshold excitation at L4-to-L2/3 synapses. The L4-to-L2/3 synapses are highly reliable (Feldmeyer et al., 2002
) and may thus serve as a gate for the horizontal spread of sensory stimulus-evoked excitation in supra- and to a smaller extent also in infragranular layers. The lateral borders of the representational map for a single whisker stimulus when the L4-to-L2/3 gate is open are largely determined by the long-range horizontal axons of L2/3 pyramidal cells receiving monosynaptic input from L4 spiny neurons. The representational area of a single whisker movement by subthreshold excitation at later times (>20 ms) after the stimulation is therefore several times larger than a single barrel-column in accordance with results obtained by intrinsic signal imaging (Masino and Frostig, 1996
; Chen-Bee and Frostig, 1996
; Kleinfeld and Delaney, 1996
; Brett-Green et al., 2001
). In addition, the horizontal spread of excitation is most likely also controlled by inhibitory connections (Laaris et al., 2000
; Petersen and Sakmann, 2001
; Laaris and Keller, 2002
).
Sensitivity of L2/3 Pyramidal Cell Excitation to Coincident Vertical and Horizontal Inputs
When does the L4-to-L2/3 gate open? Presumably a strong whisker deflection is sufficient to reach threshold in L2/3 pyramidal cells. However the combination of a weaker stimulus with associative excitation from other cortical areas may also be sufficient. In L2/3 pyramidal cells, excitation might become suprathreshold when inputs arriving in layer 1 at the apical dendrite are coincident with inputs from layer 4 and 2/3 to basal dendrites. This was demonstrated for coincident inputs to L5 pyramidal cells (Larkum et al., 1999, 2001
). In consequence, the borders of the excitation area in the cortex evoked by a whisker deflection would also be very sensitive to associated synaptic input from afferents originating in other cortical regions and innervating the apical dendrites of L2/3 pyramidal cells.
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Notes |
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Address correspondence to Dirk Feldmeyer, Max-Planck-Institut für medizinische Forschung, Abteilung Zellphysiologie, Jahnstrasse 29, D-69120 Heidelberg, Germany. Email: feldmeyr{at}mpimf-heidelberg.mpg.de.
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References |
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Armstrong-James M, Fox K, Das-Gupta A (1992) Flow of excitation within rat barrel cortex on striking a single vibrissa. J Neurophysiol 68:13451358.
Bernardo KL, McCasland JS, Woolsey TA, Strominger RN (1990) Local intra- and interlaminar connections in mouse barrel cortex. J Comp Neurol 291:231255.[ISI][Medline]
Brecht M, Sakmann B (2002) Dynamic representation of whisker deflection by synaptic potentials in spiny stellate and pyramidal cells in the barrels and septa of layer 4 rat somatosensory cortex. J Physiol (Lond) 543:4970.
Brett-Green BA, Chen-Bee CH, Frostig RD (2001) Comparing the functional representations of central and border whiskers in rat primary somatosensory cortex. J Neurosci 15:99449954.
Bruno RM, Simons DJ (2002) Feedforward mechanisms of excitatory and inhibitory cortical receptive fields. J Neurosci 22:1096610975.
Burkhalter A (1989) Intrinsic connections of rat primary visual cortex: laminar organization of axonal projections. J Comp Neurol 279:171186.[ISI][Medline]
Chen-Bee CH, Frostig RD (1996) Variability and interhemispheric asymmetry of single-whisker functional representations in rat barrel cortex. J Neurophysiol 76:884894.
Chmielowska J, Kossut M, Chmielowski M (1986) Single vibrissal cortical column in the mouse labeled with 2-deoxyglucose. Exp Brain Res 63:607619.[ISI][Medline]
DeFelipe J, Farinas I (1992) The pyramidal neuron of the cerebral cortex: morphological and chemical characteristics of the synaptic inputs. Prog Neurobiol 39:563607.[CrossRef][ISI][Medline]
Egger V, Feldmeyer D, Sakmann B (1999) Coincidence detection and efficacy changes in synaptic connections between spiny stellate neurons of the rat barrel cortex. Nature Neurosci 2:10981105.[CrossRef][ISI][Medline]
Feldmeyer D, Egger V, Lübke J, Sakmann B (1999) Synaptic connections between excitatory layer 4 neurones in the barrel field of rat somatosensory cortex. J Physiol (Lond), 521:169190.
Feldmeyer D, Lübke J, Silver RA, Sakmann B (2002) 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 (Lond) 538:803822.
Gilbert CD, Wiesel TN (1979) Morphology and intracortical projections of functionally characterised neurones in the cat visual cortex. Nature 280:120125.[ISI][Medline]
Gottlieb JP, Keller A (1997) Intrinsic circuitry and physiological properties of pyramidal neurons in rat barrel cortex. Exp Brain Res 115:4760.[ISI][Medline]
Harris RM, Woolsey TA (1983) Computer-assisted analyses of barrel neuron axons and their putative synaptic contacts. J Comp Neurol 220:6379.[ISI][Medline]
Kalisman N, Silberberg G, Markram H (2003) Deriving physical connectivity from neuronal morphology. Biol Cybern 88:210218.[ISI][Medline]
Keller A, Carlson GC (1999) Neonatal whisker clipping alters intracortical, but not thalamocortical projections, in rat barrel cortex. J Comp Neurol 412:8394.[CrossRef][ISI][Medline]
Kleinfeld D, Delaney KR (1996) Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage-sensitive dyes. J Comp Neurol 375:89108.[CrossRef][ISI][Medline]
Kossut M, Juliano SL (1999) Anatomical correlates of representational map reorganization induced by partial vibrissectomy in the barrel cortex of adult mice. Neuroscience 92:807817.[CrossRef][ISI][Medline]
Laaris N, Keller A (2002) Functional independence of layer IV barrels. J Neurophysiol. 87:10281034.
Laaris N, Carlson GC, Keller A (2000) Thalamic-evoked synaptic interactions in barrel cortex revealed by optical imaging. J Neurosci 20:15291537.
Larkum ME, Zhu JJ, Sakmann B (1999) A new cellular mechanism for coupling inputs arriving at different cortical layers. Nature 398:338341.[CrossRef][ISI][Medline]
Larkum ME, Zhu JJ, Sakmann B (2001) Dendritic mechanisms underlying the coupling of the dendritic with the axonal action potential initiation zone of adult rat layer 5 pyramidal neurons. J Physiol (Lond) 533:447466.
Lorente de Nó R (1922) La corteza cerebral del ratón. Trab Lab Invest Biol (Madrid) 20:4178.
Lorente de Nó R (1943) Cerebral cortex: architecture, intracortical connections, motor projections. In: Physiology of the nervous system (Fulton JF, ed.), pp. 274313. London: OUP.
Lübke J, Egger V, Sakmann B, Feldmeyer D (2000) Columnar organization of single and synaptically coupled excitatory spiny neurons in layer 4 of the rat barrel cortex. J Neurosci 20:53005311.
Markram H, Lübke J, Frotscher M, Roth A, Sakmann B (1997) Physiology and anatomy of synaptic connections between thick tufted pyramidal neurones in the developing rat neocortex. J Physiol (Lond) 500:409440.[Abstract]
Masino SA, Frostig RD (1996) Quantitative long-term imaging of the functional representation of a whisker in rat barrel cortex. Proc Natl Acad Sci USA 93:49424947.
McCasland JS, Woolsey TA (1988) High resolution 2-deoxyglucose mapping of functional cortical columns in mouse barrel cortex. J Comp Neurol 278:555569.[ISI][Medline]
Miller B, Blake NMJ, Erinjeri JP, Reistad CE, Sexton T, Admire P, Woolsey TA (2001) Postnatal growth of intrinsic connections in mouse barrel cortex. J Comp Neurol 436:1731.[CrossRef][ISI][Medline]
Moore CI, Nelson SB (1998) Spatio-temporal subthreshold receptive fields in the vibrissa representation of rat primary somatosensory cortex. J Neurophysiol 80:28822892.
Orbach HS, Cohen LB, Grinvald A (1985) Optical mapping of electrical activity in rat somatosensory and visual cortex. J Neurosci 5:18861895.[Abstract]
Petersen CCH, Sakmann B (2000) The excitatory neuronal network of rat layer 4 barrel cortex. J Neurosci 20:75797586.
Petersen CCH, Sakmann B (2001) Functionally independent columns of rat somatosensory barrel cortex revealed with voltage-sensitive dye imaging. J Neurosci 21:84358446.
Petersen CCH, Grinvald, A, Sakmann B (2003) Spatiotemporal dynamics of sensory responses in layer 2/3 of rat barrel cortex measured in vivo by voltage-sensitive dye imaging combined with whole-cell voltage recordings and neuron reconstructions. J Neurosci 23, 12981309.
Reyes A, Sakmann B (1999) Developmental switch in the short-term modification of unitary EPSPs evoked in layer 2/3 and layer 5 pyramidal neurons of rat neocortex. J Neurosci 19:38273835.
Shepherd GMG, Pologruto TA, Svoboda K (2003) Circuit analysis of experience-dependent plasticity in the developing rat barrel cortex. Neuron 38:277289.[ISI][Medline]
Simons DJ, Woolsey TA (1984) Morphology of GolgiCox-impregnated barrel neurons in rat SmI cortex. J Comp Neurol 230:119132.[ISI][Medline]
Stepanyants A, Hof PR, Chklovskii DB (2002) Geometry and structural plasticity of synaptic connectivity. Neuron 34:275288.[ISI][Medline]
Trevelyan AJ, Jack J (2002) Detailed passive cable models of layer 2/3 pyramidal cells in rat visual cortex at different temperatures. J Physiol (Lond) 539:623636.
Valverde, F (1986) Intrinsic neocortical organisation: some comparative aspects. Neuroscience 18:123.[CrossRef][ISI][Medline]
Wallace H, Fox KD (1999) Local cortical interactions determine the form of cortical plasticity. J Neurobiol 41:5863.[CrossRef][ISI][Medline]
Woolsey TA (1990) Peripheral alteration and somatosensory development. In: Development of sensory systems in mammals (Coleman J, ed.), pp. 461516. New York: Wiley-Liss.
Woolsey TA, van der Loos H (1970) The description of a cortical field composed of discrete cytoarchitectonic units. Brain Res 17:205242.[CrossRef][ISI][Medline]
Woolsey TA, Rovainen CM, Cox SB, Henegar MH, Liang GE, Liu D, Moskalenko YE, Sui J, Wei L (1996) Neuronal units linked to microvascular modules in cerebral cortex: response elements for imaging the brain. Cereb Cortex 6:647660.[Abstract]
Yoshimura Y, Sato H, Imamura K, Watanabe Y (2000) Properties of horizontal and vertical inputs to pyramidal cells in the superficial layers of the cat visual cortex. J Neurosci 20:19311940.
Zhu JJ, Connors BW (1999) Intrinsic firing patterns and whisker-evoked synaptic responses of neurons in the rat barrel cortex. J Neurophysiol 81:11711183.