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The Journal of Neuroscience, May 15, 2001, 21(10):3600-3608
Layer-Specific Input to Distinct Cell Types in Layer 6 of Monkey
Primary Visual Cortex
Farran
Briggs and
Edward M.
Callaway
Systems Neurobiology Laboratories, Salk Institute for Biological
Sciences and Department of Biology, University of California, San
Diego, La Jolla, California 92037
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ABSTRACT |
Layer 6 of monkey V1 contains a physiologically and anatomically
diverse population of excitatory pyramidal neurons. Distinctive arborization patterns of axons and dendrites within the functionally specialized cortical layers define eight types of layer 6 pyramidal neurons and suggest unique information processing roles for each cell
type. To address how input sources contribute to cellular function, we
examined the laminar sources of functional excitatory input onto
individual layer 6 pyramidal neurons using scanning laser
photostimulation. We find that excitatory input sources correlate with
cell type. Class I neurons with axonal arbors selectively targeting
magnocellular (M) recipient layer 4C receive input from M-dominated
layer 4B, whereas class I neurons whose axonal arbors target
parvocellular (P) recipient layer 4C receive input from P-dominated
layer 2/3. Surprisingly, these neuronal types do not differ
significantly in the inputs they receive directly from layers 4C or
4C. Class II cells, which lack dense axonal arbors within layer 4C,
receive excitatory input from layers targeted by their local axons.
Specifically, type IIA cells project axons to and receive input from
the deep but not superficial layers. Type IIB neurons project to and
receive input from the deepest and most superficial, but not middle
layers. Type IIC neurons arborize throughout the cortical layers and
tend to receive inputs from all cortical layers. These observations
have implications for the functional roles of different layer 6 cell
types in visual information processing.
Key words:
macaque; visual cortex; V1; layer 6; photostimulation; excitatory input; local circuits; caged glutamate
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INTRODUCTION |
Layer 6 of monkey primary visual
cortex, or V1, contains a morphologically and functionally diverse
population of pyramidal neurons (Hubel and Wiesel, 1968 ; Lund and
Boothe, 1975; Hawken et al., 1988; Wiser and Callaway, 1996). Each
distinct, anatomically defined cell type is hypothesized to play a
unique role in visual information processing (Wiser and Callaway, 1996,
1997; Callaway, 1998). Intracellular labeling studies reveal two
classes of layer 6 pyramidal neuron (Wiser and Callaway, 1996). Class I
neurons are characterized by dense axonal and dendritic arborizations within the lateral geniculate nucleus (LGN) recipient layer 4C. Five
class I cell types are defined by distinct axonal/dendritic locations
within the magnocellular (M) and parvocellular (P) LGN-recipient subdivisions of layer 4C (Fig. 1,
top row). Two cell types, I and Im, arborize specifically
in the M-recipient layer 4C (Wiser and Callaway, 1996), suggesting
roles in circuits related to visual motion processing (Lund and Boothe,
1975) (for review, see Livingstone and Hubel, 1988; Merigan and
Maunsell, 1993; Callaway, 1998). I and IA cell types arborize in
the P-recipient layer 4C, suggesting roles related to shape or color
processing. Type IC neurons arborize in both M- and P-recipient layers.
Anatomical relationships between the dendrites of class I cells and
axons of cells in other layers suggest that class I cells should
receive input from the same subdivision or subdivisions of layer 4C
targeted by their local axons, consistent with participation in direct
feedback circuits (Callaway, 1998).
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Figure 1.
Eight anatomical types of layer 6 pyramidal
neurons in monkey V1. Cell type is labeled beneath each reconstruction.
Dendrites are black, and axons are gray.
Axons extending below layer 6 indicate cell types that sometimes
(dashed lines) or always (solid lines)
project out of V1. Laminar borders are depicted by the
horizontal lines and labeled to the left
of each row. Class I neurons are in the top row, and
class II neurons are in the bottom row. Most cells shown
are from Wiser and Callaway (1996), their Fig. 12. Camera lucida
drawings were modified to allow accurate depictions of laminar
specificity in the context of idealized, width-invariant cortical
layers.
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Class II neurons avoid arborizing in layer 4C and instead extend axons
to deep and/or more superficial cortical layers (Fig. 1, bottom
row). They also differ from class I cells by having more extensive
dendritic arbors in layer 5, suggesting they are more likely to receive
input from superficial layer neurons (Blasdel et al., 1985; Fitzpatrick
et al., 1985; Lachica et al., 1992; Callaway and Wiser, 1996). These
anatomical relationships suggest that class II neurons play roles in
feedback circuits involving the deepest and most superficial layers
rather than layer 4C (Callaway, 1998).
Close examination of the anatomy of each layer 6 cell type reveals that
each layer 6 pyramidal neuron could receive input from any cortical
layer. This observation illustrates the necessity of an assay of
functional connections to discriminate the individual input patterns
for each cell type (Sawatari and Callaway, 1996, 2000; Dantzker and
Callaway, 2000; Yabuta et al., 2001). To identify functional
connectivity, we used scanning laser photostimulation and whole-cell
voltage-clamp recording to assay the laminar sources of functional
excitatory input onto each type of layer 6 pyramidal neuron (Callaway
and Katz, 1993; Katz and Dalva, 1994; Sawatari and Callaway, 1996,
2000; Dantzker and Callaway, 2000; Yabuta et al., 2001).
We discovered that different layer 6 pyramidal cell types receive
different patterns of laminar input. Class II cells receive excitatory
input from the layers targeted by their axons, consistent with the
prediction that these cells provide direct feedback to those layers
providing their input. Unexpectedly, class I cells did not receive
specific inputs from the layer 4C subdivisions targeted by their axons.
Specific inputs arose instead from superficial cortical layers.
M-dominated layer 4B, which receives its strongest input from layer
4C (Fitzpatrick et al., 1985; Lachica et al., 1992; Yoshioka et al.,
1994; Yabuta and Callaway, 1998b), provides strong excitatory
input to class I cells whose axons target layer 4C. P-dominated
layer 2/3, which receives its strongest input from layer 4C (Blasdel
et al., 1985; Fitzpatrick et al., 1985; Lachica et al., 1992; Yoshioka
et al., 1994; Yabuta and Callaway, 1998b), provides strong
excitatory input to class I cells whose axons target layer 4C.
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MATERIALS AND METHODS |
Laser-scanning photostimulation was used to map laminar sources
of excitatory input to layer 6 pyramidal neurons in living V1 brain
slices prepared from macaque monkeys. V1 tissue was collected from the
opercular cortical surface and brain slices were prepared from seven
juvenile (19-24 months old) macaque monkeys (four Macaca mulatta, three Macaca radiata) of both sexes. For some
animals, tissue was collected on two separate days. In this case, a
recovery surgery was performed to collect tissue from one cortical
hemisphere on the first experimental day, followed 5-10 d later with
an identical nonrecovery surgery to collect tissue from the second
hemisphere (Sawatari and Callaway, 2000). On average, 10 cells were
recorded per animal, of which an average of six yielded data for layer 6 cells analyzed in this study. Additional brain slices from the same
animals were used for other studies. Details of the preparation of
living brain slices from monkeys have been described previously (Callaway and Wiser, 1996; Wiser and Callaway, 1996; Sawatari and
Callaway, 2000). All procedures were approved by the Institutional Animal Care and Use Committee.
Local stimulation of presynaptic input neurons by light-evoked
conversion of caged glutamate to glutamate (photostimulation) was used
to map laminar sources of functional connections onto individual
recorded neurons (Callaway and Katz, 1993; Katz and Dalva, 1994;
Sawatari and Callaway, 1996, 2000; Dantzker and Callaway, 2000). The
methods used to collect data in this study were described in detail by
Sawatari and Callaway (2000). Briefly, a 400-µm-thick coronal or
sagittal brain slice was transferred from an interface holding chamber
and submerged in a recording chamber containing room temperature,
oxygenated ACSF with 150 µM -CNB caged
glutamate [ -(2-carboxy-2-nitrobenzyl) ester, trifluoroacetic acid
salt; G-7055; Molecular Probes, Eugene, OR]. Using an 8-12 M
resistance glass microelectrode filled with potassium gluconate-based
intracellular solution containing 0.5% biocytin, a single neuron in
layer 6 of V1 was whole-cell patched, voltage-clamped at  65 mV, and
inward, EPSCs were measured. UV light from an argon-ion laser,
focused through a 40× microscope objective into the brain slice, was
used to photostimulate discrete sites in the slice. Two different
microscope set-ups were used for photostimulation in this study. In the
first set-up, laser light was focused through an oil-immersion
objective mounted on a set of motorized stages below the recording
chamber (Sawatari and Callaway, 1996). The second set-up used a DIC
microscope with the UV light focused through a water-immersion
objective onto the top of the brain slice (Sawatari and Callaway,
2000). The laser power output was adjusted such that the amount of UV light reaching the brain slice was the same for both set-ups. Photostimulation involved flashing the UV light for 10 msec, causing uncaging of glutamate at the focal point of the objective. Control experiments (photostimulation during current-clamp recording) reveal
that these stimulation parameters result in action potential generation
only in neurons with somata within 50-75 µm of the stimulation site
(Sawatari and Callaway, 2000). Control experiments using identical
procedures were performed on four additional cells (data not shown),
and these results agreed with those of Sawatari and Callaway
(2000).
This spatial resolution allows mapping of laminar-specific excitatory
input in monkey V1. Photostimulation experiments performed in parallel
with those described here, using identical methods and equipment,
revealed specificity of inputs from cortical layers in close proximity.
These studies identified cell type specificity of inputs from layer
4C versus 4C onto layer 4B neurons (Yabuta et al., 2001) and from
layer 4C onto layer 3B neurons (Sawatari and Callaway, 2000).
Therefore, photostimulation is suitable for assaying laminar-specific
connections in primate V1.
One exception, however, is layer 4A. Because layer 4A is typically only
~50- to 75-µm-thick, photostimulation within layer 4A is likely to
activate neighboring neurons in layers 4B or 3B. Additionally, only a
small number of stimulation sites can be made in layer 4A. For these
reasons, we rarely detected significant input from layer 4A (4 of 45 cells in our sample) (Table 1). Most
cells receiving layer 4A input also received significant layer 4B input
(three of four cells), and the single cell receiving layer 4A but not
4B input was a type that often received layer 4B input (types I and
Im; see Results). Furthermore, layer 4A contains a heterogeneous
population of neurons, some of which are morphologically similar to
layer 4B neurons with substantial dendritic spread in layer 4B and/or
axonal projections to thick stripes in V2 (Levitt et al., 1994). These
observations suggest that inputs detected after stimulation in the
layer 4A region may have actually originated from neurons in layer 4B
or from functionally equivalent layer 4A cell types. Because
significant input from layer 4A was rare, difficult to interpret and
usually corresponded to layer 4B input, it is not considered in detail in our analyses below. For the same reasons just described,
photostimulation of areas at laminar borders could result in activation
of neurons in the adjacent cortical layer. However, even if a
photostimulated site encroached slightly into the next layer, the
majority of activated cells would most likely be in the stimulated
layer.
While recording EPSCs from each layer 6 neuron, hundreds of sites in
each brain slice were photostimulated, allowing generation of maps of
laminar locations of cells functionally connected to the layer 6 neurons (Dantzker and Callaway, 2000; Sawatari and Callaway, 2000).
Stimulation sites were located throughout a rectangular area
surrounding the recorded neuron, typically extending ~300 µm
laterally on either side of the cell and vertically from the white
matter to layer 1 (Fig. 2). Stimulation
trials were interspersed with no-stimulation trials to sample
spontaneous EPSCs. Custom data-acquisition software digitized and
recorded currents during each trial. The digitized records were
analyzed to identify number and amplitudes of EPSCs occurring in each
trial (Dantzker and Callaway, 2000).
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Figure 2.
Functional excitatory input maps for individual
class I (A, B, C) and class II (D, E,
F) neurons. The colors at each location indicate
linearly interpolated sum of EPSC amplitudes (minus spontaneous EPSCs)
collected at photostimulation sites for each cell. Colors are scaled
according to the scale bars between the plots. Laminar borders are
represented by (near) horizontal lines and labeled to
the left of the plots. Mean ± SEM of EPSC
amplitude sums (in picoamperes; spontaneous EPSCs were not
subtracted out, but are shown at the bottom right of
each plot) for each layer are shown to the right of each
plot. Asterisks next to mean ± SEM values indicate
layers providing statistically significant input based on sum of EPSC
amplitudes (single asterisk) or EPSC amplitude only
(double asterisks) compared with spontaneous EPSCs.
Camera lucida drawings of axons (thin white lines) and
dendrites (thick) are overlaid onto plots. Gray
areas are present so that neuronal processes can be seen
against the otherwise white background; no
photostimulation occurred within these areas. A, A type
I neuron (cell number B48c11) (Table 1) received significant input
from layers 4B, 4C, and 4C, 5, and 6, but not from layer 2/3.
B, A type IC neuron (A48c1) (Table 1) received
significant input from layers 2/3, 5, and 6. C, A type
I neuron (B48c2) (Table 1) received significant input from layers
2/3, 4C, and 4C, 5 and 6, but not from layer 4B.
D, A type IIA neuron (A48c12) (Table 1) received
significant input from layers 4C, 5, and 6 but not from layers above
4C. E, A type IIB neuron (B45c7) (Table 1) received
significant input from layer 2/3, but not from the middle layers.
F, A type IIC neuron (A43c4) (Table 1) received
significant input from layers 2/3, 4A, 4B, 4C, and 5. The axons of the
type IIC neuron shown leave the plane of the slice at the layer 4C/5
border. Scale bars, 100 µm (shown at the top, right
corner of each plot).
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After photostimulation and spontaneous-trial data were collected for an
individual cell, the cell was iontophoresed with biocytin. Next, five
sites of cytochrome oxidase photobleaching (4 sec laser stimulation)
were made adjacent to the rectangular area of stimulation sites
(Dantzker and Callaway, 2000). These sites were later used to align the
laminar borders and the anatomical reconstruction of the cell
with the photostimulation sites. Slices were then fixed with 4%
paraformaldehyde in 0.1 M PBS, resectioned, and stained for cytochrome oxidase and biocytin to reveal laminar borders
and neuronal morphology using methods previously described (Yabuta and
Callaway, 1998a,b). After staining, labeled axonal and dendritic
processes were reconstructed using a camera lucida. Sections were
counterstained for thionin to visualize borders not well delineated
with the cytochrome oxidase stain alone (namely the layer 4C/4C
border and the layer 1/layer 2 border) (cf. Yabuta and Callaway,
1998b).
Each neuronal reconstruction, showing the morphology of the cell, the
laminar borders, and the alignment sites, was aligned with the
coordinate map of stimulation sites using Adobe Illustrator (Adobe
Systems, San Jose, CA). Because the alignment sites were made in the
live tissue, before fixing, sectioning, and staining, any shrinkage of
the slice after those procedures was corrected by scaling the
coordinate map to the reconstruction. Only linear scaling was used and
invariably all five alignment sites matched the coordinate sites within
25 µm. Thus, any errors were small relative to the spatial resolution
of the stimulation (Dantzker and Callaway, 2000, their Fig. 1).
Individual intracellular recordings were analyzed using a mini-analysis
program (Synaptosoft Inc., Leonia, NJ), then grouped according to the
laminar locations of stimulation sites and further analyzed for
specific EPSC attributes using custom Matlab programs. The number,
amplitude, and sum of amplitudes of EPSCs for each stimulation and
spontaneous trial were calculated, including all events occurring
within 300 msec of the light flash. Inward currents recorded as a
result of direct stimulation of the cell were easily differentiated
from EPSCs and were excluded from all analyses (Dantzker and Callaway,
2000). Laminar groupings of EPSC attributes (amplitude, number, and sum
of amplitudes) were compared with spontaneous EPSCs to identify
statistically significant differences in EPSC amplitude, number, or sum
of amplitudes using Mann-Whitney U tests (Table 1). Cells
were then grouped according to their morphological type (defined by
Wiser and Callaway, 1996).
Two different analyses were conducted on the photostimulation data.
First, comparisons between EPSC attributes from stimulation and
spontaneous trials were used to determine the percentages of cells in
each anatomical group that received statistically significant input
from each cortical layer (Figs.
3A,
4A). The second
analysis measured the strength of laminar input. We calculated the
normalized evoked input (NEI) from each layer for each cell as follows:
[(average experimental sum of amplitudes) (average spontaneous
sum of amplitudes)]/(average spontaneous amplitude). Subtracting the
spontaneous activity allowed for an estimate of evoked activity.
Division by spontaneous EPSC amplitude normalized for variations in
recording conditions between cells, such as different recording access
resistances, which produced different ranges of EPSC amplitudes. These
normalization procedures facilitated quantitative comparisons
among cells (Dantzker and Callaway, 2000). NEIs for each layer
were tabulated for cells in the same anatomical group. Significant
differences in layer-specific NEIs between cell types were determined
using Rank Sum Tests. In addition, we examined the locations of cells
with respect to blobs in layer 2/3 and whether or not cells projected
axons into the white matter to assess possible correlations with
functional laminar input patterns.
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Figure 3.
Laminar input to class I neurons.
A, Percentages of class I neurons of each type receiving
significant increases in EPSC amplitude, number, or sum of amplitudes
compared with spontaneous trials for each cortical layer. *A smaller
percentage of type I and Im neurons received significant layer 2/3
input compared with the other class I types
(p = 0.03; Fisher's Exact test). **A larger
percentage of type I and Im neurons received significant layer 4B
input than the other class I types (p = 0.01). B, Mean ± SEM of NEIs (see Materials and
Methods) for each layer and for each cell type. ***Type I and Im
neurons received significantly weaker layer 2/3 input than other class
I neurons (p = 0.02; Rank Sum test; type
I and IA vs type I and Im, p = 0.04; type
IC vs type I and Im, p = 0.07). ****Type IC
neurons received stronger layer 6 input than type I and IA
neurons (p = 0.045). Gray
bars represent type I and Im cells, black
bars represent type IC cells, and open bars
represent type I and IA neurons.
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Figure 4.
Laminar input to class II neurons.
A, Percentage of class II neurons of each cell type
receiving significant input from each layer. *A smaller percentage of
type IIA neurons received significant layer 2/3 input than type IIB and
IIC neurons (p = 0.045; Fisher's Exact
test). **A smaller percentage type IIB neurons received significant
layer 4C input than the other types (p = 0.02; IIA vs IIB, p = 0.04; IIC vs IIB,
p = 0.04). B, Mean ± SEM of
NEIs from each cortical layer for each cell type. ***Type IIC neurons
received stronger layer 2/3 input than type IIA neurons
(p = 0.046; Rank Sum test). ****Type IIB
neurons received weaker layer 4C input than type IIA or IIC neurons
(p = 0.01 for both comparisons). Gray
bars represent type IIA neurons, black bars
represent type IIB neurons, and open bars represent type
IIC neurons. Conventions are the same as for Figure 3.
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Disparities between the percentages of cells receiving significant
laminar input and the strengths of input from the same layers were
sometimes observed (see Results). In such cases, the proportion of
cells receiving significant input usually suggested stronger
connectivity than the corresponding NEI value. This occurred because
detectable increases in EPSC amplitude were generally smaller (e.g.,
20-30%) than detectable increases in EPSC number (e.g., twofold to
threefold). Thus, when significant laminar inputs were based on
increased EPSC amplitude only (see Table 1), these inputs could make
important contributions to the percentages of cells receiving
significant input but relatively weak contributions to NEIs.
Cells with poor biocytin labeling, such that their anatomical type
could not be determined, poor electrical recording access (>50M),
high levels of electrical noise, or <10 stimulation sites in any
layer, were excluded from the analysis. The only exceptions were two
cells that lacked sufficient stimulation sites in layer 6 because of
inaccurate estimation of the white matter/layer 6 border during
photostimulation. These cells were analyzed for laminar input from
layers 5 and above and are indicated by "not stim" in the layer 6 column in Table 1.
To obtain clearer illustrations of input patterns for individual cells,
smoothed graphs of excitatory input (linear interpolations of sum of
EPSC amplitude data) were generated using custom Matlab programs (Fig.
2). These plots illustrate estimated evoked activity measured in a
given cell (mean sum of EPSC amplitudes for simulated trials minus mean
sum of EPSC amplitudes for spontaneous trials) after stimulation at
various locations.
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RESULTS |
Laser-scanning photostimulation was used to map laminar sources of
functional excitatory input onto 45 individual pyramidal neurons in
layer 6 of living brain slices from monkey V1. This method uses focal
uncaging of glutamate to generate action potentials in a spatially
localized population of neurons. Sources of excitatory inputs to
individual neurons were mapped by recording EPSCs in single neurons
while photostimulating hundreds of discrete sites. Trials measuring
spontaneous activity, in which currents were recorded under identical
conditions but without photostimulation, were interspersed between
stimulation trials. These trials enabled the determination of sizes and
frequencies of spontaneous EPSCs for each cell. We conducted two
different analyses of input data for each cell. The first involved a
statistical comparison between stimulated and spontaneous EPSC inputs
to determine which layers provided significant input to each cell. The
second analysis involved calculating the NEI to each cell to determine
the strength of laminar input (see Materials and Methods).
Control experiments done during this study and in previous
photostimulation studies of monkey V1 (Sawatari and Callaway, 2000; Materials and Methods) revealed that neurons fired action potentials only if photostimulation occurred within a ~50-75 µm radius of the
cell body. Therefore, postsynaptic currents resulting from photostimulation reflect direct monosynaptic input from a neuron or
neurons located at or near a stimulation site. Disynaptic or polysynaptic activation of a recorded neuron via neurons distant from
the stimulation site would require the generation of action potentials
at distant sites. The control experiments showed, however, that distant
activation did not occur.
The 45 layer 6 pyramidal neurons sampled in this study included at
least two of each of the previously described anatomical types: class I
types I, Im, I, IA, and IC and class II types IIA and IIB
(Wiser and Callaway, 1996) (Fig. 1), in addition to a newly defined
cell type, IIC. Although individual neurons, even within the same
anatomically defined group, sometimes received different patterns of
input, consistent laminar input trends were observed in certain
anatomically defined groups. Anatomical features of new, more precisely
defined class II cell types are first described followed by analysis of
the laminar excitatory input sources to each layer 6 class I and class
II cell type.
Anatomy of type IIA and IIC cells
The type IIA neurons described by Wiser and Callaway (1996) were
not a distinct group but displayed diverse morphological features.
During the course of this study, a third type of class II neuron was
defined, separating type IIA neurons into two cell types, IIA and IIC.
Two invariably correlated anatomical attributes separated type IIA from
IIC cells: type IIA neurons always projected their main descending axon
into the white matter and never projected local axons above layer
5, whereas type IIC neurons never projected axons into the white matter
and always above layer 5. A Fisher's Exact test indicated that these
are separate populations, with the chances of misclassification being 2 in 10,000 for a cell with only one of the two parameters measured (0 of
5 vs 11 of 11 or 5 of 5 vs 0 of 11 cells; p = 0.0002).
Based on these refined definitions, five type IIA and 11 type IIC
neurons were identified in this study. Figure 1 shows anatomical
reconstructions of a type IIA cell (far left of bottom row) and a type
IIC cell (far right of bottom row). Type IIA cells usually had cell
bodies located in or near the middle of layer 6. A primary axon always
projected into the white matter, and the local axons were limited
entirely to layers 5 and 6, extending laterally in the lower half of
layer 6 and occasionally diagonally or vertically into layer 5. Apical dendrites of type IIA neurons could reach as high as layer 2/3 with
branches primarily in layers 5 and 6 and their basal dendrites were
confined to lower layer 6. In contrast, type IIC neurons had cell
bodies located in the top two-thirds of layer 6. Instead of projecting
into the white matter, the axons of these cells extended laterally
within layers 5 and 6, diagonally through layer 5 into layer 4C, and
columnarly as high as layer 2/3. Each cell had at least one vertically
or diagonally rising axon extending above layer 5. Apical dendrites of
type IIC neurons could extend as far as layer 2/3 and tended to branch
throughout layers 5 and 6A. Their basal dendrites radiated below the
cell body, sometimes into lower layer 6, depending on the depth of the
cell body.
Excitatory input to class I neurons
Summary
Photostimulation data were collected from 21 class I neurons.
Class I neurons, characterized by dense axonal arbors within layer 4C,
were separated into three anatomical groupings according to the
sublaminar organization of their axons within layer 4C: type I and
Im cells have axons in layer 4C only; type IC cells have axons
throughout layer 4C; and type I and IA cells have axons in layer
4C, but not 4C. Based on their axonal specificity for these M- or
P-recipient layers, the class I cell types have been hypothesized to
participate in M- or P-dominant circuits (Wiser and Callaway, 1996;
Callaway, 1998). Although the present findings reveal that each class I
cell type did participate differently in one or the other pathway, this
difference was not manifested as distinct input from layers 4C or
4C, but instead from the more superficial layers. Cells with axonal
arbors in layer 4C only (types I and Im) (Fig.
2A) usually received significant input from layer 4B
but never from layer 2/3. Cells with axons in layer 4C (types IC,
I, and IA) (Fig. 2B,C) usually received layer
2/3 input but rarely received input from layer 4B. Neurons from all
class I cell types received input from layers 4C, 4C, 5, and 6 (Table 1). It is important to note that the input patterns represented
in Figure 2 are illustrations of the laminar input sources for
individual cells that are not always entirely representative of the
population. For example, the type I neuron in Figure 2A receives
stronger input from layer 4C than 4C, but overall, type I
neurons received stronger layer 4C input (see below). There were no
significant variations in input patterns among cells of the same
anatomical grouping depending on either their location relative to
blobs or interblobs or white matter-projection of axons. It should be
noted, however, that a larger population of cells might reveal more
subtle quantitative differences.
Superficial layer input
Type I and Im cells never received significant input from layer
2/3 (0 of 6 cells) compared with more than half (8 of 15 cells) of the
other (type IC, I, IA) class I cells (p = 0.03 Fisher's Exact test) (Fig. 3A). Based on NEI, type
I and Im cells received ninefold less evoked input from layer 2/3
than did type IC, I, and IA cells (p = 0.02; Rank Sum test) (Fig. 3B). Most type I and Im cells
received significant input from layer 4B (4 of 6 cells) compared with
only 7% (1 of 15 cells) of type IC, I, and IA cells
(p = 0.01) (Fig. 3A). As expected
based on the higher percentage of type I and Im cells receiving
significant layer 4B input, evoked layer 4B input to type I and Im
cells was fivefold stronger than layer 4B input to the other class I cell types (Fig. 3B). This difference was not statistically
significant because of the wide range of NEI values from layer 4B onto
type I and Im cells.
Layer 4C input
In contrast to the specific excitatory input patterns from the
superficial layers onto the class I types, inputs from layers 4C and
4C were not cell type-specific. Similar percentages of class I cells
of each type received significant input from layers 4C and 4C
(Fig. 3A, Table 1). Furthermore, there were no significant differences between cell types in the strengths of layer 4C or layer
4C inputs based on NEIs (Fig. 3B). To examine strengths of inputs from layer 4C versus 4C to each cell, we calculated ratios of NEIs from layer 4C versus 4C for each neuron. There were no differences in these ratios between cell types. There was,
however, an overall trend for layer 4C input to be stronger than
layer 4C input. Mean NEI from layer 4C for all class I cells
combined (0.79) was almost twice as large as mean NEI from layer 4C
(0.45; p = 0.046; paired t test).
Deep layer input
Class I neurons from all three anatomical groupings received
robust input from the deep layers 5 and 6. High percentages of cells in
each anatomical group received layer 5 input (Fig. 3A). The
strengths of layer 5 inputs to different cell types were not significantly different: NEIs to type IC cells were only 40% greater than to type I and Im cells and nearly equal to type I and IA cells (Fig. 3B). There were also no significant differences
between cell types in the percentage of cells receiving significant
input from layer 6. However, type IC neurons always received strong input from layer 6 (and layer 5), whereas strengths of deep layer input
were more varied to the other cell types. When comparing NEIs from
layer 6, type IC cells received twofold stronger input than did type
I and IA cells (p = 0.045) or type I
and Im cells (not significant).
Excitatory input to class II neurons
Summary
Photostimulation data were collected for 24 class II neurons.
Layer 6 pyramidal neurons of this class tend to lack dense axonal arborizations in layer 4C and extend dendritic branches within layer 5. Therefore, it has been suggested that these cells might receive more
input from layers 5 and 2/3 (pyramidal neurons in layer 2/3 project
axons into layer 5; Blasdel et al., 1985; Fitzpatrick et al., 1985;
Lachica et al., 1992; Callaway and Wiser, 1996) than from layer 4C.
Data presented here suggest that class II cell types tend instead to
receive inputs from the same layers targeted by their local axons. Type
IIA cells never extend axons above layer 5 and receive the weakest
inputs from superficial layers (2-4B) (Fig. 2D).
Type IIB neurons receive deep layer and superficial layer inputs but
weak layer 4C inputs, consistent with their lack of axonal arbors
within layer 4C (Fig. 2E). Type IIC neurons receive
inputs from all layers, consistent with their diffuse local axonal
arbors (Fig. 2F). As with class I neurons, class II
neurons did not display any apparent variations in input patterns based
on locations of cells relative to blobs. (As noted above, a larger
sample size could reveal subtle differences.)
Superficial layer input
A higher proportion of type IIB and IIC cells received significant
input from layer 2/3 (14 of 19 cells) than did type IIA cells (1 of 5 cells; p = 0.045; Fisher's Exact test) (Fig.
4A). Based on NEI, type IIC cells received >100-fold
stronger input from layer 2/3 than type IIA neurons
(p = 0.046) (Fig. 4B). Type IIB cells also received 37-fold stronger layer 2/3 input based on NEI
than type IIA neurons (not significant) (Fig. 4B).
Although type IIC neurons received threefold stronger layer 2/3 input
than type IIB neurons, this difference was not statistically
significant (Fig. 4B). Layer 4B input was detected
for only ~20% of class II cells, regardless of cell type (Fig.
4A). Similar to layer 2/3 input, layer 4B input was
strongest to type IIC cells and weakest to type IIA cells, but these
differences were not statistically significant (Fig.
4B).
Layer 4C input
No type IIB cell (0 of 8 cells) received significant layer 4C
input, compared with 60% (3 of 5) of type IIA cells
(p = 0.04) and 46% (5 of 11) of type IIC cells
(p = 0.04) (Fig. 4A). The percentages of class II cells of each type receiving layer 4C input
were not significantly different (Fig. 4A).
Comparisons of NEIs from layer 4C onto the class II cell types revealed
that both type IIA and type IIC cells received threefold stronger
inputs from layer 4C than type IIB cells (p = 0.01 for both comparisons) (Fig. 4B). NEIs for layers
4C and 4C were combined into a single measure for layer 4C (Fig.
4B) because the relative strengths of inputs from
layer 4C versus 4C were similar for all three types of class II
cells (data not shown). [Significant layer 4C input to type IIB
neurons (Fig. 4A) was usually based solely on EPSC
amplitude (Table 1) and therefore contributed only weakly to NEI (see
Materials and Methods).] Although there were not cell type-specific
differences in the strength of inputs from layers 4C or 4C, there
was an overall trend for layer 4C to provide stronger input than
layer 4C, similar to the trend for class I cells. Mean NEI from
layer 4C to all class II cells combined (0.41) was almost twice the
mean NEI from layer 4C (0.24; p = 0.076; paired
t test). It is also noteworthy that these values are
approximately half the corresponding values for class I cells (0.41 vs
0.79 for layer 4C, p = 0.047; 0.24 vs 0.45 for layer 4C, p = 0.19). These differences are expected based
on the differences in dendritic arborization within layer 4C (Fig.
1).
Deep layer input
Similar percentages of class II cells of each type received
significant input from layer 5 (Fig. 4A), and layer 5 inputs onto cells of each type were of similar strengths (Fig.
4B). Likewise, similar percentages of cells received
significant layer 6 input (Fig. 4A), and this input
was similar in strength to cells of each type (Fig.
4B). Although there were sometimes up to twofold differences in NEI between cell types (e.g., type IIC cells received twofold greater NEI from layer 5 than type IIB cells and twofold greater NEI from layer 6 than type IIA cells), these differences were
not statistically significant.
| |
DISCUSSION |
Morphologically diverse neurons within a single cortical layer
have been shown previously to receive functional excitatory input from
different layers (Dantzker and Callaway, 2000; Sawatari and Callaway,
2000; Yabuta et al., 2001). We find that eight different types of
pyramidal neurons in layer 6 of primate V1 also receive distinct
laminar patterns of functional excitatory inputs. These input patterns
were not predicted based on anatomical observations of the spatial
overlap of dendrites and axons, thus demonstrating the necessity for an
assay of functional connections to distinguish the specific connections
and functional roles of various anatomical cell types.
We find evidence for both mixing and segregation of M and P input to
class I pyramidal neurons. Although each class I cell type receives
direct input from both layers 4C and 4C, input from the more
superficial layers (2-4B) is M or P stream-specific. Cell type
specificity of inputs therefore does not originate from the same layers
(4C and 4C) targeted by the axons of each cell type. In contrast,
functional input onto class II neurons correlates with the locations of
the axonal arbors of those neurons. The different input-output
relationships of class I and class II neurons suggest distinct
organizational principles for connectivity of these two basic cell classes.
Before discussing these results further, it is important to consider
relevant limitations of the methods used. First, although photostimulation has the advantage of limiting the spatial location of
cells firing action potentials to a small area around a stimulation site, stimulation probably activates all cells in that area,
indiscriminant of cell type. Therefore, a significant excitatory input
after stimulation in a given layer could have originated from any of the anatomically diverse excitatory cell types in that layer. We can
only predict identities of cell types providing input based on our
anatomical knowledge of the various cell types. Second, we must
consider the possibility of false negatives. Although photostimulation
may not reveal a connection, this does not absolutely exclude the
possibility that cells in the stimulated area make connections onto the
recorded neuron. For example, connections could have been missed
because they were shunted by inhibitory inputs from the same layer or
because processes were cut. Although some neuronal processes are cut
during brain slice preparation, this has a minimal impact on the
present studies of laminar input from within a home column (the single
vertical column containing the recorded neuron). Most vertical
connections between layers are not affected because in monkey V1, axons
of excitatory neurons ascend or descend vertically within the plane of
the brain slice before fanning out or branching laterally (Callaway and
Wiser, 1996; Wiser and Callaway, 1996; Yabuta and Callaway,
1998b) (Figs. 1, 2). Therefore, interlaminar connections remain
intact and detectable within the radial dimension from white matter to
the pial surface. In contrast, because horizontal connections are lost,
we did not attempt to stimulate locations distant from the home column.
Finally, the sample sizes of neurons in some cell type groupings are
small. Increases in these sample sizes might reveal some smaller
quantitative differences that were missed (for example laminar input
patterns correlating with columnar architecture). However, because
laminar input strengths varied widely between cells in the same
grouping, even doubling the sample size may not reveal statistically
significant differences between cell types. We feel that these data are
more indicative of heterogeneity of input within cell classes rather than differences between classes.
Class I neurons
Based on their morphologies, most class I cell types are thought
to play functional roles related to either M or P pathways (Lund and
Boothe, 1975; Wiser and Callaway, 1996; Callaway, 1998). Type I and
Im neurons, which project axons specifically to upper or lower layer
4C, were expected to receive M-related inputs, whereas type I and
IA neurons, which project axons to layer 4C, were expected to
receive P-related inputs. Results presented here show that different
types of class I neuron do receive different M- or P-related inputs,
depending on the laminar specificity of their outputs. However, this
specificity did not come from layers 4C and 4C. Instead, class I
neurons received unique inputs from the superficial layers targeted by
layers 4C and 4C. M-dominated layer 4B provided input onto
neurons with axons limited to layer 4C (types I and Im).
P-dominated layer 2/3 provided input onto neurons with axons in layer
4C (types IC, I, and IA). Surprisingly, class I neurons tended
to receive similar layer 4C and 4C input regardless of cell type.
In light of the fact that class I neurons lack dendrites in lower layer
5, it was also surprising that all class I cell types received robust
input from layer 5 (Callaway and Wiser, 1996; Wiser and Callaway,
1996). All class I cell types also received input from layer 6, which
contains the densest dendritic arborizations of these cells.
Thalamocortical inputs to class I neurons
The function of each layer 6 cell type is dependent not only on
its local V1 inputs, but also on input from the LGN. Anatomical observations suggest that direct LGN input onto the class I cell types
is functional stream-specific. LGN afferents project to the main
recipient layers 4C and 4C, where each class I cell type could
receive P- or M-specific inputs onto their layer-specific apical
dendritic arbors. Thalamocortical afferents also tend to project
preferentially to lower (M input) versus upper (P input) layer 6 (Hendrickson et al., 1978; Blasdel and Lund, 1983) where they might
target type I and Im cells versus type I and IA cells,
respectively. Although any thalamocortical input onto apical dendrites
must be specific, it is possible that there is mixing of M and P LGN
afferent inputs onto basal dendrites, which are in positions to sample
input from both streams.
Functional implications of specific input patterns to class
I cells
Class I neurons receive M or P stream-specific inputs from the
same superficial cortical layers (2/3 and 4B) whose neurons project to
extrastriate cortical areas in the dorsal and ventral streams,
respectively (for review, see Livingstone and Hubel, 1988; Merigan and
Maunsell, 1993). This input specificity correlates with selective
axonal targeting of class I cell types for M recipient layer 4C or P
recipient layer 4C. Thus, class I neurons are sampling copies of M-
or P-related information that will be sent on to higher visual cortical
areas. In turn, class I neurons send their axons specifically to layers
4C or 4C, presumably to modify the inputs of these layers to
their superficial target layers. These correlations suggest that an
important role of class I neurons in layer 6 is to modify input to the
superficial neurons based on sampling of their outputs.
Whereas all class I neurons may be performing similar functions, there
are differences in the patterns of connectivity for different cell
types. M pathway-related type I and Im neurons never project axons
into the white matter (Wiser and Callaway, 1996) (Table 1) and
therefore cannot provide any feedback to the LGN. All layer
4C-projecting cell types (types I, IA, and IC) can project out
of V1, presumably back to the LGN P layers (type I and IA cells)
or M and P layers (type IC cells; Fitzpatrick et al., 1994; Wiser and
Callaway, 1996). Thus, the M pathway may use predominantly local V1
circuits to modify pathway-specific activity, whereas the P pathway may
use local and corticogeniculate circuitry. These observations suggest
that the circuitry involving type I and Im cells and layer 4C may
operate differently than the corresponding P pathway circuitry. Perhaps
faster modulation is achieved in the M pathway by using only local
connections and fewer synapses. This could be important for the M
pathway to process motion information at high temporal frequencies. The
P pathway, more concerned with visual acuity and form, may benefit from
an additional corticothalamic loop.
Class II neurons
Anatomy of type IIA and IIC cells
During the course of examining the anatomical features of layer 6 pyramidal neurons in this study, it became apparent that the previously
described type IIA category (Wiser and Callaway, 1996) actually
included two morphologically distinct types of class II neurons. Type
IIA neurons were more precisely defined as projecting axons into the
white matter and having local axons exclusively within the deepest
layers. These cells tend to be located in the middle of layer 6 and to
have long, laterally projecting axons within layer 6. Type IIC neurons
have only local axons which project diagonally and columnarly into
layer 2/3. The cell bodies of type IIC neurons tend to be located
mostly in the upper half of layer 6. We are confident that type IIA and
IIC neurons are indeed two distinct cell types since the two defining
characteristics of these cells (white matter projecting or not and
local axons above layer 5 or not) are binary criteria and invariably correspond.
Inputs to class II cells
Class II neurons, all of which have dense dendritic arbors in
layer 5, were expected, based on anatomical observations, to receive
inputs from layers 2/3 and 5. Results presented in this study show,
however, that the best predictor of laminar input onto the class II
cell types is the laminar organization of their axonal, not dendritic,
arbors. Type IIA cells lack axons above layer 5 and receive only very
weak input from the superficial layers. Type IIB cells arborize in the
deepest and most superficial layers, but not layer 4C and receive
inputs from the same layers. Type IIC neurons arborize throughout all
cortical layers and receive corresponding diffuse inputs. These
observations suggest that class II cells participate in more direct
interactions than class I cells, modifying the input-output of the
same neurons that provide their inputs.
| |
FOOTNOTES |
Received Dec. 8, 2000; revised March 5, 2001; accepted March 7, 2001.
This work was supported by National Institutes of Health Grant EY10742,
National Institutes of Health Training Grant T32 GM08107-15 (F.B.), and
the Chapman Charitable Trust (F.B.). We thank Jami Dantzker and Dr.
Atomu Sawatari for technical and programming assistance, Soumya
Chatterjee, and Drs. Amy Butler, Greg Horwitz, and Ed Lein for helpful
comments on the manuscript, and Sandra Tye for assistance with animals.
Correspondence should be addressed to Farran Briggs, Systems
Neurobiology Laboratories-C, Salk Institute for Biological Sciences, 10010 North Torrey Pines Road, La Jolla, CA 92037. E-mail:
fbriggs{at}biomail.ucsd.edu.
| |
REFERENCES |
-
Blasdel GG,
Lund JS
(1983)
Termination of afferent axons in macaque striate cortex.
J Neurosci
3:1389-1413[Abstract].
-
Blasdel GG,
Lund JS,
Fitzpatrick D
(1985)
Intrinsic connections of macaque striate cortex: axonal projections of cells outside lamina 4C.
J Neurosci
5:3350-3369[Abstract].
-
Callaway EM
(1998)
Local circuits in primary visual cortex of the macaque monkey.
Annu Rev Neurosci
21:47-74[ISI][Medline].
-
Callaway EM,
Katz LC
(1993)
Photostimulation using caged glutamate reveals functional circuitry in living brain slices.
Proc Natl Acad Sci USA
90:7661-7665[Abstract/Free Full Text].
-
Callaway EM,
Wiser AK
(1996)
Contributions of individual layer 2-5 spiny neurons to local circuits in macaque primary visual cortex.
Vis Neurosci
13:907-922[ISI][Medline].
-
Dantzker JL,
Callaway EM
(2000)
Laminar sources of synaptic input to cortical inhibitory interneurons and pyramidal neurons.
Nature Neurosci
3:701-707[ISI][Medline].
-
Fitzpatrick D,
Lund JS,
Blasdel GG
(1985)
Intrinsic connections of macaque striate cortex: afferent and efferent connections of lamina 4C.
J Neurosci
5:3329-3349[Abstract].
-
Fitzpatrick D,
Usrey WM,
Schofield BR,
Einstein G
(1994)
The sublaminar organization of corticogeniculate neurons in layer 6 of macaque striate cortex.
Vis Neurosci
11:307-315[ISI][Medline].
-
Hawken MJ,
Parker AJ,
Lund JS
(1988)
Laminar organization and contrast sensitivity of direction-selective cells in the striate cortex of the Old World monkey.
J Neurosci
8:3541-3548[Abstract].
-
Hendrickson AE,
Wilson JR,
Ogren MP
(1978)
The neuroanatomical organization of pathways between the dorsal lateral geniculate nucleus and visual cortex in Old World and New World primates.
J Comp Neurol
182:123-136[ISI][Medline].
-
Hubel DH,
Wiesel TN
(1968)
Receptive fields and functional architecture of monkey striate cortex.
J Physiol (Lond)
195:215-243[Abstract/Free Full Text].
-
Katz LC,
Dalva MB
(1994)
Scanning laser photostimulation: a new approach for analyzing brain circuits.
J Neurosci Methods
54:205-218[ISI][Medline].
-
Lachica EA,
Beck PD,
Casagrande VA
(1992)
Parallel pathways in macaque monkey striate cortex: anatomically defined columns in layer 3.
Proc Natl Acad Sci USA
89:3566-3570[Abstract/Free Full Text].
-
Levitt JB,
Yoshioka T,
Lund JS
(1994)
Intrinsic cortical connections in macaque visual area V2: evidence for interaction between different functional streams.
J Comp Neurol
342:551-570[ISI][Medline].
-
Livingstone M,
Hubel DH
(1988)
Segregation of form, color, movement, and depth: anatomy, physiology, and perception.
Science
240:740-749[Abstract/Free Full Text].
-
Lund JS,
Boothe RG
(1975)
Interlaminar connections and pyramidal neuron organization in the visual cortex, area 17, of the macaque monkey.
J Comp Neurol
159:305-334[ISI].
-
Merigan WH,
Maunsell JHR
(1993)
How parallel are the visual pathways?
Annu Rev Neurosci
16:369-402[ISI][Medline].
-
Sawatari A,
Callaway EM
(1996)
Convergence of magno- and parvocellular pathways in layer 4B of macaque primary visual cortex.
Nature
380:442-446[Medline].
-
Sawatari A,
Callaway EM
(2000)
Diversity and cell type specificity of local excitatory connections to neurons in layer 3B of monkey primary visual cortex.
Neuron
25:459-471[ISI][Medline].
-
Wiser AK,
Callaway EM
(1996)
Contributions of individual layer 6 pyramidal neurons to local circuitry in macaque primary visual cortex.
J Neurosci
16:2724-2739[Abstract/Free Full Text].
-
Wiser AK,
Callaway EM
(1997)
Ocular dominance columns and local projections of layer 6 pyramidal neurons in macaque primary visual cortex.
Vis Neurosci
14:241-251[ISI][Medline].
-
Yabuta NH,
Callaway EM
(1998a)
Cytochrome-oxidase blobs and intrinsic horizontal connections of layer 2/3 pyramidal neurons in primate V1.
Vis Neurosci
15:1007-1027[ISI][Medline].
-
Yabuta NH,
Callaway EM
(1998b)
Functional streams and local connections of layer 4C neurons in primary visual cortex of the macaque monkey.
J Neurosci
18:9489-9499[Abstract/Free Full Text].
-
Yabuta NH, Sawatari S, Callaway EM (2001) Two functional
channels from primary visual cortex to dorsal visual cortical areas.
Science, in press.
-
Yoshioka T,
Levitt JB,
Lund JS
(1994)
Independence and merger of thalamocortical channels within macaque monkey primary visual cortex: anatomy of interlaminar projections.
Vis Neurosci
11:467-489[ISI][Medline].
Copyright © 2001 Society for Neuroscience 0270-6474/01/21103600-09$05.00/0
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TOP
ABSTRACT
INTRODUCTION
