Download Distribution of GABAergic neurons and axon terminals in the

THE JOURNAL OF COMPARATIVE NEUROLOGY 264:73-91(1987)
Distribution of GABAergic Neurons and
Axon Terminals in the Macaque Striate
Cortex
D. FITZPATRICK, J. S. LUND, D.E. SCHMECHEL, AND A.C. TOWLES
Department of Anatomy and Psychology (D.F.) and VA Medical Center and Department
of Medicine (D.E.S.), Duke University, Durham, North Carolina 27710; Department of
Psychiatry (J.S.L.), University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 15261; and Department of Neurology (A.C.T.), Cornell University Medical
Center, New York, New York 10021
ABSTRACT
Antisera to glutamic acid decarboxylase (GAD) and y-aminobutyric acid
(GABA) have been used to characterize the morphology and distribution of
presumed GABAergic neurons and axon terminals within the macaque
striate cortex. Despite some differences in the relative sensitivity of these
antisera for detecting cell bodies and terminals, the overall patterns of
labeling appear quite similar. GABAergic axon terminals are particularIy
prominent in zones known to receive the bulk of the projections from the
lateral geniculate nucleus; laminae 4C, 4A, and the cytochrome-rich patches
of lamina 3. In lamina 4A, GABAergic terminals are distributed in a honeycomb pattern which appears to match closely the spatial pattern of geniculate terminations in this region. Quantitative analysis of axon terminals
that contain flat vesicles and form symmetric synaptic contacts (FS terminals) in lamina 4C, and in lamina 5 suggest that the prominence of GAD
and GABA axon terminal labeling in the geniculate recipient zones is due,
at least in part, to the presence of larger GABAergic axon terminals in these
regions.
GABAergic cell bodies and their initial dendritic segments display morphological features characteristic of nonpyramidal neurons and are found in
all layers of striate cortex. The density of GAD and GABA immunoreactive
neurons is greatest in laminae 2-3A, 4A, and 4C,. The distribution of
GABAergic neurons within lamina 3 does not appear to be correlated with
the patchy distribution of cytochrome oxidase in this region; i.e., there is no
significant difference in the density of GAD and GABA immunoreactive
neurons in cytochrome-rich and cytochrome-poor regions of lamina 3.
Counts of labeled and unlabeled neurons indicate that GABA immunoreactive neurons make up at least 15% of the neurons in striate cortex.
Layer 1 is distinct from the other cortical layers by virtue of its high
percentage (77-81%) of GABAergic neurons. Among the other layers, the
proportion of GABAergic neurons varies from roughly 20% in laminae 2-3A
to 12% in laminae 5 and 6.
Finally, there are conspicuous laminar differences in the size and dendritic arrangement of GAD and GABA immunoreactive neurons. Lamina
4C, and lamina 6 are distinguished from the other layers by the presence of
populations of large GABAergic neurons, some of which have horizontally
spreading dendritic processes. GABAergic neurons within the superficial
layers are significantly smaller and the majority appear to have vertically
oriented dendritic processes. These results provide support for the idea that
GABAergic neurons make up a significant proportion of the neurons within
Accepted April 8,1987.
0 1987 ALAN R. LISS, INC.
D. FITZPATRICK ET AL.
74
the macaque striate cortex and that there are laminar differences in the
number and the types of GABAergic neurons-differences that may be relevant for understanding the contributions of GABA-mediated inhibition to
striate cortex function.
Key words: GABA, GAD, inhibition, aspinous neurons, cytochrome oxidase,
immunocytochemistry
Neurons that utilize the neurotransmitter y-aminobutyric acid (GABA) appear to play an important role in
generating many of the receptive field properties that distinguish neurons in the striate cortex from those in the
lateral geniculate nucleus. When the inhibitory effects of
GABA are blocked by the administration of the potent
GABA antagonist bicuculline, the selective responses of
cortical neurons for the orientation, length, direction of
movement and velocity of a visual stimulus are abolished
or diminished (Sillito, '75, '77a,b, '79; Sillito and Versiani,
'77; Pate1 and Sillito, '78; Tsumoto et al., '79).
GABAergic neurotransmission within the striate cortex,
as in other regions of neocortex, is thought to be mediated
largely by a morphologically diverse class of non-pyramidal
neurons that share the feature of having relatively spinefree dendritic processes. Evidence for the selective role of
aspinous neurons in cortical inhibition is derived from the
observations that neurons with this morphology (1)accumulate JH-GABA (Chronwall and Wolff, '78; Hamos et al.,
'83; Hendry and Jones, '81; Somogyi et al., '84b); (2) are
hmunoreactive for glutamic acid decarboxylase (GAD),
the synthesizing enzyme for GABA (Ribak, '78; Peters and
Fairen, '78; Freund et al., '83; Houser et al., '83; Somogyi
et al., '83b); (3) are immunoreactive for GABA itself (Ottersen and Storm-Mathisen, '84; Somogyi and Hodgson, '85);
and (4)have terminals that contain flat vesicles and make
symmetric synaptic contacts, characteristics thought to be
indicative of inhibitory transmission (Uchizono, '65; Peters
and Fairen, '78; Somogyi and Cowey, '81).
While a general consensus has been reached on the morphological class of neurons responsible for GABAergic
transmission in the striate cortex, considerably less is
known about the numbers of these neurons and their distribution within the macaque striate cortex. The existing information on the precise laminar and columnar segregation
of cell types and connections within the monkey striate
cortex provides a valuable structural and functional framework within which to examine the distribution of GABAergic neurons. For example, the projections from the
magnocellular and parvicellular layers of the lateral geniculate nucleus (LGN) to the striate cortex terminate in separate tiers of lamina 4C (4C,, and 4C,, respectively; Hubel
and Wiesel, '72; Hendrickson et al., '78). Differences in the
number and types of GABAergic neurons in these layers
may have important implications for understanding the
way striate cortex processes the information derived from
these two pathways. The discovery that lamina 3 has distinct cytochrome-oxidase-rich patches that are distinguished from the surrounding regions in their response
properties and in their connections (Horton and Hubel, '81;
Livingstone and Hubel, '82, '84; Fitzpatrick et al., '83a)
provides another set of functional landmarks for examining
the distribution of GABAergic neurons. Indeed, there is
already some evidence that the cytochrome-oxidase-rich re-
gions of lamina 3 are particularly rich in GABAergic neurons and terminals (Hendrickson et al., '81; Carroll and
Wong-Riley, '85).
In the present study, immunocytochemical markers for
GAD and GABA were used to characterize the morphology,
number, and distribution of presumed GABAergic neurons
and axon terminals in the macaque striate cortex. The
main purpose of this study was to establish how the normal
distribution pattern of GABAergic neurons and terminals
relates to the well-defined laminar and columnar subdivisions of the monkey striate cortex. It is our view that this
information constitutes a useful first step in understanding
how neurons and terminals involved with GABAergic neurotransmission are arranged with respect to the terminal
fields of functionally different geniculocortical pathways
and to the cortical cells that give rise to extrastriate and
brainstem projections.
MATERIALS AND METHODS
Immunocytochemistry
A total of five macaque monkeys (Mucaca fasciculuris)
were used in these experiments. Animals were deeply anaesthetized and perfused through the heart with a 0.9%
saline solution followed by one of the following fixatives:
4% paraformaldehyde, 0.1%glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (two animals); 0.5% zinc salicylate
dissolved in 10% formalin in water as described by Mugnaini and Dahl('83) (one animal); or a mixture of paraformaldehyde, lysine, and sodium periodate (PLP) made according to the protocol of McLean and Nakane ('74) (two
animals). Except for the animals perfused with the PLP
mixture, the fixative was flushed out with a solution of 10%
sucrose in 0.1 M phosphate buffer, pH 7.4.
Sections from the striate cortex were cut at 15-40 pm on
an Oxford vibratome and collected in 0.01 M phosphatebuffered saline (PBS), pH 7.6. Selected sections were processed using GAD or GABA immunocytochemistry, cytochrome oxidase histochemistry (Wong-Riley, '79) or Nissl
stains (either thionin or toluidine blue).
The antiserum developed by Oertel et al. ('81a) was used
for the demonstration of GAD immunoreactivity. Details of
the purification of GAD and the preparation of the antiserum have been published (Oertel et al., '81a,b). The PLP
fixative was superior to other fixatives for labeling cortical
cell bodies with this antiserum, but labeled terminals were
found with all three fixatives. In some cases, prior to immunocytochemical processing, sections were run through
an increasing and decreasing series of alcohol solutions (10,
25, 50%) for 3 minutes each. This procedure significantly
increased the penetration of the antiserum; in the thinner
sections, terminals appeared through the depth of the section. However, immersion in alcohol solutions significantly
75
GABAERGIC NEURONS IN STRIATE CORTEX
reduced the intensity and number of labeled cell bodies, so
this method was of limited value in assessing the distribution of GAD immunoreactive neurons.
Sections were incubated in primary antiserum diluted
1:1,500-1:2,000 in PBS containing 1% normal rabbit serum
for 2-24 hours. Linking antiserum (biotinylated antigoat)
was used a t a dilution of 1 5 0 and the avidin-biotin complex
(ABC, Vector Laboratories) was localized with 0.1%, 3,3'diaminobenzidine tetrahydrochloride (DAB) in 0.1 M phosphate buffer, pH 7.4, containing 0.001% H202. For controls,
adjacent sections were incubated in preimmune serum at a
dilution of 1:1,000 and processed in the same way as the
sections incubated in the anti-GAD serum. Control sections
showed no labeling of cell bodies or terminals.
The antiserum produced by Lauder et al. ('86) was used
for the demonstration of GABA immunoreactivity. This
antiserum was raised in rats against GABA conjugated to
keyhole limpet hemocyanin with glutaraldehyde and it
shows no cross-reactivity with glutamate, aspartate, 0-alanine, taurine, or glycine. Immunoreactive staining was completely abolished by absorption with the GABA conjugate.
The most robust staining of cell bodies and dendrites was
achieved by fixation with the 4% paraformaldehyde, 0.1%
glutaraldehyde mixture. The intensity of GABA immunoreactivity in cell bodies was not affected by alcohol pretreatment, and this was routinely used to enhance penetration.
Sections were incubated in primary antiserum diluted at
1:2,000 in PBS containing 1%normal rat serum (NRS) for
12-24 hours. Linking antiserum (biotinylated antirat) was
used at a dilution of 1 5 0 and the ABC complex was localized with DAB as described above. In some cases, the silver
intensification procedure described by Liposits et al. ('84)
was used to enhance the staining of GABA immunoreactive
terminals.
Electron microscopy
One macaque monkey was deeply anaesthetized and perfused with 0.9% saline followed by a solution of 2.0% paraformaldehyde, 1.0% glutaraldehyde in 0.1 M phosphate
buffer, pH 7.4. The brain was postfixed in the same solution
overnight. Blocks of tissue (1-2 mm) from area 17 were
rinsed in phosphate buffer and placed in 2% osmium tetroxide in 0.1 M phosphate buffer for 1hour. Following a brief
rinse in phosphate buffer, the tissue was dehydrated and
embedded in Polybed 812.
In order to compare the size of axon terminals that contained flat vesicles and made symmetric synaptic contacts
(FS terminals) in lamina 4C and in lamina 5 , 1-2-pm-thick
sections were cut, stained with toluidine blue, and viewed
with the light microscope. Drawings were made to illustrate the 4C-5 border, and blocks were trimmed to include
4C, and lamina 5 . Rows of thin sections were cut, placed on
0.25% Formvar-coated 2-mm slot grids, and stained with
uranyl acetate and lead citrate.
Measurements of the size of FS terminal profiles from
lamina 4C, and lamina 5B were made from electron micrographs taken at a magnification of 15,000 with the aid of a
graphics tablet and computer.
the white matter at a magnification of 540 x with a camera
lucida. This strip of cells was then divided into sequential
65- or 70-pm horizontal sectors and the number of cells in
each of these rectangular sectors (500 x 65 or 70 pm) was
counted. Multiple nonoverlapping strips were drawn from
nearby areas of the same section and the cell counts from
all the sectors of a given depth were added together to
achieve a reasonable sample size from each depth. Sample
collection continued until the bin with the smallest number
of cells (usually from the vicinity of lamina 4B) exceeded 20
neurons. Only those neurons in which a nucleus could be
discerned were counted.
The proportion of GABAergic neurons as a function of
depth was determined by counting GABA immunoreactive
and non-immunoreactive neurons from 15-20-pm counterstained sections by using procedures identical to those described above. This was done using only the sections that
had undergone alcohol pretreatment to enhance antiserum
penetration. Since alcohol pretreatment is incompatible
with GAD immunoreactive labeling of cell bodies, this issue
was not addressed with the GAD antiserum.
The relationship between the distribution of GABA and
GAD immunoreactive neurons and the cytochrome-rich
patches of lamina 3 was analyzed with the aid of a Videometrics M-100 graphics system attached to an IBM PC-XT.
A custom-designed program superimposed a square box
(0.08 pm2 in area) on a video image of the tissue section.
This box served as the sample window in which cell counts
were made and it could be positioned to include the cytochrome-rich or cytochrome-poor regions of the tissue section. The size of the sample window was automatically
adjusted to the objective magnification so that it enclosed
0.08 pm of tissue area regardless of magnification. This
allowed the borders of the cytochrome-rich regions to be
seen at low power (where the boundaries appear sharper),
a sample region to be chosen, and counts to be made of the
number of GAD or GABA immunoreactive neurons in the
sample region at higher power (so that cell nuclei could be
detected). Typically, the process consisted of (1)choosing a
sample site on a section treated for cytochrome oxidase with
a 4 x objective and drawing both the borders of the region
to be sampled ana the pattern of blood vessels on the video
image with the graphics tablet; (2) replacing the cytochrome oxidase section with an adjacent section treated for
GAD or GABA immunocytochemistry and aligning the
blood vessel patterns; (3) changing the objective to 20 x and
enlarging the sample window proportionally; and (4)counting the number of cells within the borders of the sample
region. Cells falling on the border of the sample region
were included in the sample.
The cross-sectional area of the neurons within a given
layer was also measured with the computer graphics system. The borders of the neurons were drawn at a magnification of 1,050x on the video monitor. To ensure an accurate
representation of the size distribution of cells within a
layer, samples were taken throughout the depth of each
layer. Statistical methods for comparison of samples were
taken from Hays and Winkler ('71).
Analysis of the distribution of GABAergic neurons
RESULTS
GABAergic axon terminals
The laminar distribution of GAD and GABA immunoreactive neurons was analyzed by drawing all of the labeled
neurons in a 500-pm-wide strip extending from the pia to
Sections processed for GAD immunoreactivity and those
processed for GABA immunoreactivity show a distinct
punctate pattern of labeling that is distributed throughout
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4c
5
Fig. 1. Darkfield photomicrograph showing the distribution of GAD immunoreactive axon terminals in a coronal section through macaque striate cortex. GAD immunoreactive axon terminals are
prominent in zones that receive direct projections from the LGN: laminae 4C, 4A and the patches in
lamina 3. Scale bar = 150 pm.
the neuropil or directly apposed to cell bodies. While some
of these immunoreactive elements may be dendritic processes or fibers cut in cross section, the vast majority are
likely to be axon terminals, and we have chosen to apply
this term to the punctate immunoreactive staining. In general, the GAD antiserum produced more robust staining of
axon terminals than the GABA antiserum, but both antisera produced comparable patterns of labeled terminals
throughout the cortex.
GAD and GABA immunoreactive axon terminals are
found in all layers of striate cortex from layer 1 to the deep
aspect of layer 6, but are particularly striking in the zones
that have been shown to receive the bulk of the projection
from the LGN; laminae 4C and 4A (Fig. 1). GAD and GABA
immunoreactive terminals are also more prominent in the
geniculate recipient, cytochrome-rich patches of lamina 3, a
finding first reported by Hendrickson et al. ('81).The coincidence between the cytochrome-rich and GAD immunoreactive patches can be seen in Figure 2, which shows photomicrographs from adjacent tangential sections through
lamina 3A that have been processed for cytochrome ox<dase
and for GAD immunoreactivity.
The precision with which the prominent GAD immunoreactive terminals match the pattern of geniculate terminations is exemplified by the intricate pattern of terminations that is found in lamina 4A. The projections from
the LGN to lamina 4A have been shown to terminate in a
weblike or honeycomb pattern when viewed in tangential
sections (Hendrickson et al., '78; Blasdel and Lund, '82),
and this pattern is reflected in the distribution of cytochrome oxidase staining in this region. Tangential sections
through lamina 4A show that GAD immunoreactive terminals are also distributed in a honeycomb pattern and
that this pattern is strikingly similar to the pattern of
cytochrome oxidase staining (Fig. 3). While the photomicrographs of Figure 3 are from adjacent sections, the honeycomb pattern of cytochrome oxidase staining appears to be
shifted to the left of the prominent GAD terminal distribution. This shift is due to the difficulty in achieving perfectly
tangential sections through a layer as thin as 4A. Never-
GABAERGIC NEURONS IN STRIATE CORTEX
77
Fig. 2. Comparison of the distribution of prominent GAD immunoreactive terminal staining and
cytochrome oxidase staining in adjacent tangential sections through lamina 3 of macaque striate
cortex. a: GAD immunocytochemistry (darkfield illumination). b: Cytochrome oxidase histochemistry
(brightfield illumination). Cytochrome-oxidase-richpatches are distingushed by prominent GAD immunoreactive terminal staining. Scale bar = 300 pm.
theless, the similarity in the size and shape of the two
patterns provides strong evidence that they are coincident.
The phrase “prominence of terminal staining” has been
used to refer to the difference in the quality of terminal
staining that distinguishes the geniculate recipient regions
from the others. At first glance, it might be assumed that
this difference is simply a reflection of the density of GABAergic terminals. But when viewed at higher-power magnification, GABAergic axon terminals appear to vary in
size, and many of the GAD and GABA immunoreactive
terminals in the geniculate-recipient regions appear larger
in caliber than those in regions that do not receive geniculate terminals (Fig. 4). This apparent difference can be seen
between lamina 4C and the immediately adjacent laminae
(4B and 5 ) and between the cytochrome-rich and cytochrome-poor regions of laminae 3 and 4A. A difference in
size of this magnitude would be difficult to quantify at the
light microscopic level, so the size of putative GABAergic
terminals was examined with the electron microscope.
It is generally accepted that GAD and GABA immunoreactive terminals in the cortex have a distinctive morphology. They contain flattened or pleomorphic vesicles and are
associated with symmetric synaptic thickenings (Ribak, ’78;
Hendry et al., ’83; Somogyi et al., ’83b).For the sake of con-
venience, profiles with this morphology will be referred to
as FS (flat-symmetrical) profiles. If the population of GABAergic axon terminals in the geniculate-recipient regions
are larger than those in other regions, there should be a
detectable difference in the cross-sectional area of the FS
profiles in these regions. To test this idea, the cross-sectional area of FS profiles in lamina 4C, were measured and
compared with the cross-sectional areas of FS profiles in
lamina 5 . Typical electron micrographs of FS profiles and
profiles with round vesicles and asymmetric contacts (RA
profiles) from lamina 4C, of macaque striate cortex are
shown in Figure 5 .
The distributions of area measurements for 158 FS profiles from lamina 4C, and 124 FS profiles from lamina 5B
are shown in Figure 6 (a and b). Large profiles (> 0.6 pm2)
appear to make up a greater proportion of the FS profiles
in lamina 4C, than their counterparts in lamina 5, and
conversely, small profiles (< 0.4 pm2) make up a larger
proportion of the FS profiles in lamina 5 than their counterparts in lamina 4C,. The difference between the two distributions is best seen in Figure 6c, where the values for each
bin in the lamina 5 distribution have been subtracted from
the corresponding bins in the lamina 4C, distribution. Statistical analysis supports the interpretation that the profiles in lamina 4C, are significantly larger than their
D. FITZPATRICK ET AL.
78
Fig. 3. Comparison of the distribution of prominent GAD immunoreactive terminal staining and
cytochrome oxidase staining in adjacent tangential sections through lamina 4A of macaque striate
cortex. a: GAD immunocytochcmistry (darkfield illumination). b: Cytochrome oxidase histochemistry
(brightfield illurnination). Note the similarity in the honeycomb distribution of GAD immunoreactive
terminals and the pattern of cytochronie oxidase activity. Scale bar = 150 pm.
counterparts in lamina 5 (lamina 4C,: mean = 0.49 pm2,
SD = 0.3; lamina 5: mean = 0.35 pm2, SD = 0.2; Kolmogorov-Smirnov two-sample test: P < .001). These distributions probably underestimate the actual size of FS boutons
since many of the profiles with small cross-sectional areas
are parts of larger boutons that have not been sectioned
through their centers. Despite this limitation, the results
show that FS profiles in lamina 4C, are, on average, 40%
larger in cross-sectional area than their counterparts in
lamina 5 .
GABAergic cell bodies
Laminar differences in size and morphology. Both antisera labeled neurons that were distributed across all layers
of striate cortex. However, the staining for GABA was
much more robust and this staining often continued into
the proximal portions of dendritic processes. Examples of
the morphology of the GABA immunoreactive neurons from
different layers of striate cortex are shown in Figures 7 and
8. GABA immunoreactive neurons have smooth or occasionally beaded dendrites that are displayed in multipolar
or bitufted dendritic arrangements. Each layer contains a
morphologically heterogeneous population of GABAergic
neurons that differ in cell size as well as dendritic configuration. In general, the GABA immunoreactive neurons in
the superficial layers appear smaller in size than those in
other layers and have predominantly vertically oriented
dendritic fields. Larger GABA immunoreactive neurons
with horizontally oriented dendritic processes are most
common in lamina 4C, and in lamina 6.
General impressions about the size distribution of GABAergic neurons were confirmed by measurements of the
cross-sectional area of GAD and GABA immunoreactive
cell bodies in different layers (Table 1). The GABA immunoreactive population as a whole shows a considerable variation in size, from 25 to 200 pm2 in cross-sectional area.
While each layer has a range of cell sizes, the largest GAD
and GABA immunoreactive neurons are found in lamina
4C, and in lamina 6. Indeed, these are the only two layers
that contain immunoreactive neurons with soma areas
greater than 140 pm2. At the same time, neurons in laminae 2 and 3A are distinctly smaller than those in the other
layers; in our sample of 214 neurons from 2-3A, none were
found to exceed 110 pm2 in area.
The sizes of GABA and GAD immunoreactive neurons in
the portions of lamina 4C that receive their projections
from the magnocellular and parvicellular layers of the LGN
were examined in more detail. Figure 9 shows histograms
of the soma area of GABA and GAD immunoreactive neurons in 4C, and 4C, from two different macaque monkeys
GABAERGIC NEURONS IN STRIATE CORTEX
79
Fig. 4. Comparison of G A D and GABA immunoreactive axon terminals in lamina 4C, and lamina
5. a: G A D immunoreactive axon terminals in lamina 4C,. b: GABA immunoreactive axon terminals
in lamina 4C,. c: G A D immunoreactive axon terminals in lamina 5. d: GABA immunoreactive axon
terminals in lamina 5. Many of the immunoreactive axon terminals in lamina 4C, appear larger than
those in lamina 5. The staining of the GABA immunoreactive terminals in the material used for this
comparison was enhanced by the silver intensification procedure described by Liposits et al. ('84).
Scale bar = 5 pm.
perfused with fixatives that were optimal for each antiserum. In both cases, the distribution of GABAergic neurons in 4C, has a broader range than that of 4C, and
includes large neurons that are not found within 4C,. These
differences are statistically significant (Kolmogorov-Smirnov two-sample test P < .001)
The largest GAD and GABA immunoreactive neurons in
lamina 4C, are not distributed evenly throughout the layer
but appear more frequently near the lamina 4B border. The
size of the neurons in 4C was plotted as a function of their
distance from the top of 4C, (the top of 4C being defined as
the upper boundary of the zone of prominent GABA immunoreactive axon terminals), and the results of this analysis
are presented in Figure 10 (total 316 neurons). This plot
shows that a population of large GABA immunoreactive
neurons (> 120 pm2) is clustered in the upper 20% of
lamina 4C (or the upper half of 4CJ. These large GABA
immunoreactive neurons make up a small percentage of
the total population of GABA immunoreactive neurons in
lamina 4C, (roughly 7%;nine out of 135), but their restriction to the upper half of 4C, is significant since there are
other lines of evidence that the upper part of 4C, is distinct
from the lower part (see Discussion).
Density andproportion of GABAergic neurons across cortical layers. While GABA and GAD immunoreactive neurons are found in all layers of striate cortex, there are
obvious differences in their density across the cortical depth.
GAD and GABA immunoreactive neurons are more dense
in laminae 2-3,4A, and 4C, than in the other laminae. The
density of GABA and GAD immunoreactive neurons as a
function depth is depicted in the histograms of Figures l l a
and 12. A low-power photomicrograph showing the laminar
distribution of GABA immunoreactive neurons is shown in
Figure 13.
The fluctuation in the density of GABAergic neurons
across cortical layers resembles the fluctuation in the density of unlabeled neurons and this relationship is depicted
in Figure l l b . In general, regions with a high density of
D.
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FITZPATRICK ET AL.
Fig. 5. Examples of profiles with flat vesicles and symmetric synaptic contacts (FS)and those with
round vesicles and asymmetric synaptic contacts @A) from lamina 4C,. Arrows point to sites of
synaptic contact. Scale bar = 0.2 pm.
GABA immunoreactive neurons also appear to have a high
density of unlabeled neurons (4C,, 4A, 2-3A) while zones
with a low density of GABA immunoreactive neurons (4B
and 5 ) contain fewer unlabeled neurons. The similarity in
the fluctuations in the density of labeled and unlabeled
populations across the cortical depth is reflected in the fact
that layers that differ markedly in their total density of
neurons have similar percentages of GABA immunoreactive neurons. For example, lamina 4C,, which contains the
greatest density of neurons, has a percentage of GABA
immunoreactive neurons that is not significantly different
from lamina 4B or 4Ccy- layers that contain far fewer total
neurons (Fig. llc, Table 2).
However, the fluctuation in the density of the GABA
immunoreactive population is not a simple function of the
density of the unlabeled population. If this were true, then
the percentage of GABA immunoreactive neurons should
be constant across all cortical layers, and, as shown in
Figure 8c and Table 2, this was not the case. GABA immunoreactive neurons make up approximately 15% of all the
neurons in striate cortex, but layer 1is clearly distinct from
the other layers in containing a very high proportion (>
75%) of GABA immunoreactive neurons. This finding is
consistent with previous reports in other species and in
other cortical areas (Schmechel et al., '84; Gabbott and
Somogyi, '86; Lauder et al., '86; Lin et al., '85).
Differences between other layers in the proportion of
GABA immunoreactive neurons are modest by comparison
with lamina 1. In general the proportion of GABA immunoreactive neurons is highest in the superficial layers, decreases through the middle layers, and is lowest in the deep
layers. In both monkeys that were examined, lamina 3A
had the greatest proportion of GABA immunoreactive neurons (roughly 20%), and this value was almost twice that
found in lamina 5 and lamina 6. These differences are
statistically significant (lamina 3 vs. lamina 5 : x2 = 10.9,
d.f. = 1,P < .Ol); lamina 3 vs. lamina 6: x2 = 8.8, d.f. = 1,
P < .01).
Areal distribution of GABAergic neurons within lamina
3. One question that remains to be addressed is whether
the patchy distribution of GABergic axon terminals in lamina 3 is associated with a patchy distribution of GABAergic
cell bodies. This issue was examined by comparing adjacent
tangential sections through lamina 3 that had been reacting for cytochrome oxidase and for GABA immunocytochemistry (Fig. 14). There are no obvious signs of a clustered
or patchy distribution of GABA immunoreactive neurons
that might correspond to the pattern of cytochrome-oxidaserich patches. A similar result was found with the GAD
antiserum, and an example of the relationship between
GAD immunoreactive cell bodies and the cytochrome-oxidase-rich patches is shown in Figure 15. From the photo-
81
GABAERGIC NEURONS IN STRIATE CORTEX
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Fig. 6. Cross-sectional area of FS profiles from lamina 4C, (a)and lamina
5 (b) of macaque striate cortex, The distributions are significantly different
and the nature of this difference is best seen in c, where the bins from the
lamina 5 distribution have been subtracted from the lamina 4C, distribution. Lamina 4C, has a greater percentage of FS terminals that are larger
than 0.5 pm2 in cross-sectional area.
Fig. 7. Camera lucida drawings of GABA immunoreactive neurons from
different layers of macaque striate cortex. All neurons were drawn a t the
same magnification but the widths of the layers are not drawn to scale.
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D. FITZPATRICK ET AL.
Fig. 8. Photomicrographs of GABA immunoreactive neurons in different laminae of macaque
striate cortex. a-e: Lamina 2-3. f,g: Lamina 4C,,. h,i: Lamina 4C,. j-I: Lamina 6. Scale bar = 20 pm.
GABAERGIC NEURONS IN STRIATE CORTEX
83
TABLE 1. Cross-Sectional Area Measurements of GABA and GAD
Immunoreactive Neurons in Macaque Striate Cortex'
Laver
GABA neurons
2-3A
3B-4A
4B
4C,"
4C6
5
6
GADneurons
2-3A
3B-4A
4B
4c
4c;;
5
6
No.
Mean
SD
Range
214
196
175
219
249
176
191
53.0
73.6
64.7
87.2
73.8
56.9
72.7
19.3
16.6
18.6
34.1
18.0
27.5
26.8
24.3-107.7
36.6-132.7
27.6-134.8
21.0-183.3
27.4-126.3
24.1-131.8
33.2-160.5
100
97
107
176
196
60.8
62.0
64.3
98.7
72.0
61.3
70.3
16.3
16.5
19.2
34.7
17.5
22.5
33.4
28.6-104.3
31.8-118.6
33.6-117.3
32.1-197.2
35.6-132.5
30.3-122.7
26.2-193.1
99
105
'Measurements (pm2) are from two different macaque monkeys perfused with fixative
optimal for each antiserum.
DISTRIBUTION
micrographs and the plots, it becomes apparent that if there
is any difference in the density of GABAergic neurons in
the cytochrome-rich and cytochrome-poor regions of lamina
3, this difference must be small. To test this possibility, the
number of GABA immunoreactive neurons in equal-size
samples of cytochrome-rich and c tochrome-poor regions
was determined. A total of 1.3 mm of the cytochrome-rich
patches and 1.3 mm2 of the cytochrome-poor zones were
examined and 1,595 GABA immunoreactive neurons were
counted. There was considerable variation in the number
of GABA immunoreactive neurons found in the sampIes
from both regions. The average density of GABA immunoreactive neurons was 586 neurons /mm2 (SD = 90) in the
cytochrome-rich regions and 624 neurons/mm2 (SD = 107)
in the cytochrome-poor zones. The slightly greater number
of GABA immunoreactive neurons found in the cytochrome-poor regions (6%) is not statistically significant
(Kolmogorov-Smirnovtwo-sample test: P = .49).Thus, while
there is a difference in the GABAergic axon terminals in
x
OF SOMA SIZE IN LAMINA 4C
15
15
GAD Neurons in 4Ca
GABA Neurons in 4Ca
N = 219
10
10
-X = 87.2
c
C
N=176
-
X = 98.7
SD = 34.7
*
C
SD = 34.1
?
2
n
a
5
5
0
0
0
25
50
75
100
125
150
175
200
GAD Neurons in 4 c p
GABA Neurons in 4Cp
N = 249
10
10
C
SD=18.0
a,
2
N=196
K=72.0
SD = 17.5
c
C
0
L
2
n
a
5
5
0
0
0
25
50
75
100
125
Soma Area vm2
150
175
200
0
25
50
75
100
125
Soma Area pm2
Fig. 9. Distribution of cross-sectional area measurements for GAD and GABA immunoreactive
neurons in laminae 4C,, and 4C,, of macaque striate cortex.
150
175
200
D. FITZPATRICK ET AL.
84
DISTRIBUTION OF GAEA IMMUNOREACTIVE NEURONS
11
2-3A
I
38
1
4A
I
48
1
1
t
4C0 ] 4 C p
1
I
5
6
I
I
70.
6 0.
=0
.:I .. ..._. ... . .. ... :. -- . .
*-
100
E
*.
- .
50.
0
40-
U
30.20.
. - ... . .
~
4
v
.
m
.
. . . . , a .
:... . -
10.
*
0-
0
20
40
60
80
100
DlSTRlEUTlON OF GAEA AND UNLABELED NEURONS
Percent Distance from Top of 4C
Fig. 10. Cross-sectional area of GABA immunoreactive neurons as a
function of their depth from the top of lamina 4C,,.
b
320
300
280
260
the cytochrome-rich and cytochrome-poor regions, this difference is not correlated with a difference in the density of
GABAergic cell bodies.
DISCUSSION
In this study antisera to GAD and GABA have been used
to show that GABAergic neurons constitute a significant
proportion of the neurons in macaque striate cortex and
that there are differences between laminae in the number
and sizes of GABAergic cell bodies and axon terminals.
While GAD and GABA immunoreactivity was not examined in tissue from the same animal, the laminar distribution of labeled neurons was found to be quite similar in
tissue that was fixed optimally for each antiserum (compare
Figs. l l a and 12). Both antisera also showed similar laminar differences in the size of labeled neurons and in the size
and/or density of immunoreactive terminals.
However, there were differences in the total density of
labeled cell bodies and terminals stained by these antisera;
the number of cell bodies labeled and the intensity of cell
body labeling seemed greater with the GABA antiserum
than with the GAD antiserum. Conversely, axon terminal
labeling generally appeared more robust with the GAD
antiserum than with the GABA antiserum. These differences in staining intensity do not appear to be unique to
the particular antisera that were used in this study. Somogyi et al. ('84a) used a different set of GAD and GABA
antisera in the study of GABAergic neurons in cat striate
cortex and also noted the increased effectiveness of GABA
antiserum for labeling cell bodies. Hendry and Jones ('86)
applied still-another GABA antiserum to macaque striate
cortex, and their photomicrographs show cell body and terminal staining levels comparable to the GABA antiserum
used in the present study. Whatever the reasons for these
differences, the overall similarities in the pattern of GAD
and GABA immunoreactivity strengthen the argument that
the labeled cells and terminals are involved in GABAergic
neurotransmission.
GABAergic axon terminals
In the present report we have noted the coincidence in
the distribution of prominent GABAergic axon terminals
g
240
g
I
f
E
=
2
220
200
180
160
140
120
100
80
60
40
20
0
PERCENTAGE OF GABA IMMUNOREACTIVE NEURONS
G A B A I I G A B A +Unlabeledl
30
C
0
ff
20
10
0
Depth in 65 pm
Intervals
Fig. 11. a: Distribution of GABA immunoreactive neurons across the
depth of macaque striate cortex. The number of neurons in lamina 1 has
been collected in a single bin. The remaining bins reflect the number of
neurons in successive 65pm steps through the cortex. Approximate laminar
boundaries are indicated a t the top. The number of neurons in each bin is
expressed a s a percentage of the total population of GABA immunoreactive
neurons. The actual number of GABA immunoreactive neurons in each bin
is indicated in Figure llb. b: Comparison of the distribution of GABA
immunoreactive neurons (white) and unlabeled neurons (black) across the
depth of macaque striate cortex. The height of each bar represents the total
number of neurons in each sample (GABA + unlabeled). c: Percentage of
neurons that are GABA immunoreactive a s a function of depth. The number
of GABA immunoreactive neurons in each bin was divided by the total
number of neurons in the bin to arrive a t a percentage value.
GABAERGIC NEURONS IN STRIATE CORTEX
85
DISTRIBUTION OF GAD IMMUNOREACTIVE NEURONS
I
I 4C0
I
I
l
I
I
5
14Cp
l
n
I
6
1
Depth in 70 pm Intervals
Fig. 12. Distribution of GAD immunoreactive neurons in 70-pm steps
through macaque striate cortex.
and the pattern of termination of axons from the LGN. A
prominence of GABAergic axon terminals in the geniculate-recipient regions of the striate cortex has also been
found in the cat and squirrel monkey (Fitzpatrick et al.,
'83b; Bear et al., '85). At the very least, this suggests that
there is some particular specialization in the inhibitory
circuitry associated with the geniculate-recipient regions
that may play a crucial role in the transfer of information
from LGN axons to neurons within the striate cortex.
The specialized nature of the GAD immunoreactive terminals within the geniculate-recipient zones of macaque
striate cortex was first noted by Hendrickson et al. ('81)and
interpreted as an increase in their density. While the present results do not contradict the idea that GAD-immunoreactive axon terminals in these regions may be more
numerous, they do suggest that, as a population, they are
larger in size than the axon terminals in the regions that
do not receive geniculate afferents. A difference in the size
and density of the GAD immunoreactive terminals has also
been noted between lamina 4 and other layers of macaque
somatic cortex (Houser et al., '84).
Fig. 13. Photomicrograph showing the distribution of GABA immunoreactive neurons in a coronal
section through macaque striate cortex. Scale bar = 150 pm.
D. FITZPATRICK ET AL.
86
TABLE 2. Numbers of GABA Immunoreactive Neurons in Different
Layers of Macaque Striate Cortex'
Layer
No. of
GABA and
unlabeled
neurons
No. of
GABA
neurons
GABA
neurons
1
2
3A
3B
4A
4B
4c,,
4C,
5
6
Total
1
2
3A
3B
4A
4B
4C,.
4c,i
5
6
Total
21
330
445
516
409
231
343
614
335
473
3,717
17
175
314
381
299
167
254
393
217
323
2,540
17
43
86
79
77
33
59
92
39
57
582
13
31
67
55
47
24
33
52
29
28
379
81.0
13.0
19.3
15.3
18.8
14.3
17.2
15.0
11.6
12.1
(Avg.) 15.7
76.5
17.7
21.3
14.4
15.7
14.4
13.0
13.2
13.4
8.9
(Avg.) 14.9
%
'Figures are from two different macaque monkeys perfused with the optimum fixative
for GABA immunoreactive cell bodies (see Materials and Methods).
In the original description of GAD immunoreactivity in
attention
macaque striate cortex (Hendrickson et al., %1),
was focused on the similarity in the distribution of GAD
immunoreactivity and the patchy pattern of cytochrome
oxidase staining in lamina 3. However, it is not clear from
that report whether the increased GAD immunoreactivity
in the cytochrome-rich patches consisted of GAD immunoreactive terminals alone or an increase in both labeled cell
bodies as well as terminals. Another study using the uptake
of 3H-GABAas a marker for GABAergic neurons presented
evidence for an increased density of GABA-accumulating
neurons within the cytochrome-rich patches (Carroll and
Wong-Riley, '85). The present report demonstrates that the
cytochrome-rich regions of lamina 3 are distinguished by
an increased size and, perhaps, density of GAD immunoreactive terminals. However, these regions do not differ
from the rest of lamina 3 in their density of GABAergic
neurons. These results are in agreement with a previous
report of the distribution of GAD immunoreactive neurons
in lamina 3 of squirrel monkey striate cortex (Fitzpatrick
et al., '83b) and the recent description of GABA immunoreactive neurons in macaque striate cortex (Jones et al.,
'86). Since the cytochrome-rich patches in lamina 3 are the
targets of a direct projection from the LGN (Livingstone
and Hubel, '82; Fitzpatrick et al., '83a), it seems reasonable
to view the distinctive pattern of GAD immunoreactive
terminals as another example of the prominent role that
Fig. 14. Photomicrographs showing the distribution of cytochrome-oxidase-rich patches and GABA immunowactive neurons in adjacent tangential sections through lamina 3 of macaque striate cortex. a: Cytochrome
osidasc histochemistry. b: GABA immunocytochemistry. Scale bar = 200 pm.
GABAERGIC NEURONS IN STRIATE CORTEX
a
b
Fig. 15. a: Distribution of GAD immnnoreactive neurons in a tangential section through lamina 3 of macaque
striate cortex. Each dot represents a single labeled neuron. Circles represent blood vessels used as landmarks. b:
Comparison of the distribution of GAD immunoreactive neurons shown in Figure 15a with the distribution of
cytochrome oxidase activity in an adjacent section. Cytochrome-oxidase-rich regions are depicted with dark gray
stipple. Scale bar = 250 pm.
87
88
GABAergic inhibition plays in regions that receive direct
projections from the LGN.
The coincidence in the spatial distribution of large GABAergic axon terminals and terminals derived from the
LGN is further emphasized by their correspondence in the
honeycomb matrix of lamina 4A. The exact significance of
the honeycomb distribution of geniculate terminations in
lamina 4A remains unknown. Presumably it reflects some
form of segregation between different afferents to this region, such as those from the LGN and from lamina 4C
(Fitzpatrick et al., ’851, or some specialization in the arrangement of the dendrites that are postsynaptic to the
geniculate terminations. Whatever the basis for this pattern of terminations, the close spatial correspondence of
GABAergic axon terminals and LGN axon terminals raises
the question of whether some of the GAD immunoreactive
axon terminals might be derived from the LGN. Most lines
of evidence do not support the idea that GABAergic neurons in the LGN project to striate cortex. For example, GAD
or GABA immunoreactive neurons within the LGN are not
labeled following injections of retrograde tracers into cat
(Fitzpatrick et al., ’84; Montero and Zempel, ’85) or macaque (Montero, ’86) striate cortex, and GAD immunoreactive terminals within layer 4 are not noticeably diminished following the undercutting of striate cortex (Bear
et al., ’85). Furthermore, most studies report that LGN
axon terminals have round vesicles and establish asymmetic synaptic contacts (Garey and Powell, ’71; LeVay and
Gilbert, ’76; LeVay, ’86; Einstein et al., ’87), in contrast to
GAD immunoreactive profiles that have flat vesicles and
make symmetric synaptic contacts (Ribak, ’78; Hendry et
al., ’83; Somogyi et al., ’83b). While Einstein et al. (’87)
have recently reported that some of the profiles within
layer 4 of cat striate cortex that are labeled following injections of 3H-amino acids into the LGN have flattened vesicles and make symmetric synaptic contacts, there is, at
present, no evidence that these terminals are immunoreactive for GAD or GABA.
If one accepts the prevailing view that GABAergic terminals within cortex have a local origin, then it is natural
to wonder whether the large GABAergic terminals in the
geniculate-recipient zones originate from a particular type
of neuron. One possible source for the prominent endings
in the geniculate recipient regions is a class of small aspinous neurons that has been given a variety of names including “spider cell,” “clewed” cell, and more recently,
“clutch cell” or “B1 variety” (Ramon y Cajal, 1899; Valverde, ’71; Lund ’73, ’87; Lund et al., ”77; Mates and Lund,
’83; Kisvarday et al., ’85, ’86).This suggestion is consistent
with the fact that these neurons (1)appear to be restricted
to layers 4C and 4A, where they are the most frequently
impregnated smooth dendritic neurons; (2) have axons characterized by a dense, highly branched field close to the soma
and by the presence of conspicuously large boutons; and (3)
are immunoreactive for GABA. Kisvarday et al. (1986)have
also argued for a unique association between the axon
terminals of this cell class and those from the LGN, citing
the similarity in the sizes of the axonal fields of clutch cells
and LGN afferents and the correspondence in their postsynaptic targets within lamina 4C,. In the macaque the major
targets for both clutch cell axon terminals and LGN axon
terminals are spines and dendritic shafts, presumed to be
from spiny stellate neurons. It would be of some interest to
determine the spatial relationship between the synaptic
D. FITZPATRICK ET AL.
contacts of LGN terminals and clutch cell terminals along
the dendrites of individual postsynaptic neurons. Perhaps
the matching distribution of GABAergic and LGN terminals in lamina 4A signifies an underlying similarity in the
postsynaptic targets of these two populations of boutons.
Whether or not the large GABAergic axon terminals are
derived from a single cell type, the question remains as to
the functional significance of prominent terminal staining
in the geniculate-recipient regions. One major difference
between neurons in the LGN and those in striate cortex
lies in their level of spontaneous activity. Neurons in the
LGN have spontaneous activity levels at least three times
higher than neurons in the striate cortex (Creutzfield and
Ito, ’68).This difference that can be detected in the cortical
termination zones of geniculate afferents (low spontaneous
discharge rates characterize the neurons in lamina 4C and
lamina 4A in monkey striate cortex; Blasdel and Fitzpatrick, ’84).Presumably, this difference reflects the fact that
neurons within layer 4 are under the influence of tonic
inhibition derived from interneurons within the cortex. Perhaps the larger-caliber swellings of GABAergic neurons in
the geniculate-recipient regions are specializations that act
to “balance” the strong excitatory drive of the geniculate
afferents. For example, large-caliber endings provide a
larger surface area that could give rise to a greater number
of synaptic contacts. Against this suggestion is the evidence
from Beaulieu and Colonnier (’85) that type 2 synaptic
contacts are not more numerous in layers 4A and 4B in cat
striate cortex-layers also distingushed by large-caliber
GAD immunoreactive profiles. Another possibility is that
the more frequent release of neurotransmitters which would
be expected in a tonically active system might require a
larger pool of synaptic vesicles or a larger number of mitochondria-needs that could be met by a larger axon terminal. In this respect it is interesting to note that Kageyama
and Wong-Riley (’86)have described a distinct type of largecaliber axon terminal in layer 4 of cat striate cortex that
establishes symmetric synaptic contacts and contains a
large number of mitochondria that are rich in cytochrome
oxidase activity. Also, the recent observation that the axonal swellings of clutch cells contain “large groups of mitochondria” is consistent with this idea (Kisvarday et al.,
’85).
Distribution of GABAergic cell bodies
The results of this study support the idea that GABAergic
neurons constitute at least 15%of the neurons in monkey
striate cortex. Previous attempts to estimate the percentage of GABAergic neurons in macaque neocortex have produced a wide range of values. For example, Mates and Lund
(‘83) used the observation that Golgi-impregnated aspinous
neurons are distinguished from spiny neurons by the presence of both type 1 and type 2 contacts on their somata
(LeVay, ’73) to estimate that roughly 6% of the neurons in
lamina 4C, were aspiny stellate neurons. In contrast, Hendry and Jones (’81) suggested that up to 40% of the neurons
in monkey somatosensory and motor cortex might be GABAergic since they appeared to selectively accumulate 3HGABA. The present results are closer to the values obtained
with GABA immunocytochemistry by Gabbott and Somogyi (‘86) in cat striate cortex (20%) and recently by Jones et
al. (’86)in monkey striate cortex (20%).
While GABA immunocytochemistry appears to be a reliable method for identifying GABAergic neurons, it does
GABAERGIC NEURONS IN STRIATE CORTEX
89
in macaque striate cortex are non-pyramidal neurons with
smooth or beaded dendritic processes. In addition, these
results demonstrate conspicuous laminar differences in the
soma size and the orientation of the initial dendritic segments of GABAergic neurons. Perhaps the most distinctive
neurons observed in this study are the large GABA and
GAD immunoreactive neurons found in the upper part of
4C, and in lamina 6. Jones ('75) and DeFelipe et al. ('86)
have argued that the largest of the smooth dendritic neurons in somatic and motor cortex of macaque belong to the
class of cells referred to as basket cells. Basket cells are
distinguished from most other classes of smooth dendritic
neurons by axon arbors that form terminal nests around
the somata of pyramidal neurons and other cell types
(Marin-Padilla, '69; Szentagothai, '73; Jones '75; Somogyi
et al., '83~).
If this difference in size applies to the smooth dendritic
population in macaque striate cortex, then the large neurons in the upper part of 4C, and in lamina 6 would be good
candidates for basket cells. A recent Golgi study (Lund, '87)
has described a population of aspinous neurons restricted
to the upper part of 4C, that has horizontally spreading
dendrites much like the GABA immunoreactive neurons
described here. These neurons also have axon arbors that
extend horizontally for long distances (over 1 mm) within
upper 4C, and 4B and give rise to short vertical collaterals
resembling those identified as basket cell terminations by
Somogyi et al. ('83c) and DeFelipe et al. ('86). Long-distance
lateral connections are also characteristic of basket cells in
somatosensory and motor cortex (Marin-Padilla, '69; Jones,
'75).
Large aspinous neurons with horizontally displayed dendrites in lamina 6 have received only casual attention in
previous studies using the Golgi method. Jones ('75) noted
that basket cells in layer 6 of somatosensory cortex could
be extended in a horizontal form, and both Lund ('73) and
Tombol('78) mentioned the presence of large aspinous neurons with horizontally directed dendritic processes in lamina 6 of monkey striate cortex. Horizontally directed axonal
processes with basketlike terminations have been observed
in lamina 6 of macaque striate cortex, but the source of
these axon arbors remains to be determined (Lund, unpublished observations).
If the large GABA immunoreactive neurons in lamina
4C, and lamina 6 are, indeed, basket cells, then these
regions may be expected to have long-distance lateral connections that are, at least in part, inhibitory. Long-distance
lateral connections are also characteristic of the superficial
layers of striate cortex, but the bulk of the evidence suggests that these projections originate in pyramidal neurons
and that their effect is excitatory (Gilbert and Wiesel, '83;
Rockland and Lund, '83; Rockland, '85; Ts'o et al., '86).
Differences in the lateral extent of inhibitory connections
could obviously contribute to laminar differences in receptive field properties. In this context it is worth noting that
lamina 4C,, 4B and lamina 6 contain neurons with directionally selective receptive fields and that neurons in 4B
and 6 are the source of projections to the middle temporal
area (MT) which contains a high proportion of directionally
selective neurons (Dow, '74; Rockland and Pandya, '79;
Tigges et al., '81; Livingstone and Hubel, '84; Hawken and
Parker, personal communication). Whether long-distance
inhibitory connections contribute to the directionally selecMorphology of GABAergic neurons
tive responses of these neurons remains to be determined.
The results of this study are consistent with the evidence
Finally, the presence of a population of large GABA imderived from a variety of sources that GABAergic neurons munoreactive neurons in the upper portion of 4C, provides
have several limitations that may influence the accuracy of
quantitative analysis. For example, even though 15-20-pm
vibratome sections were used for cell counts, and alcohol
pretreatment was used to increase the depth of penetration,
it is still possible that our figures underestimate the total
number of GABA immunoreactive neurons because a certain percentage of small cell bodies that lie in the center of
the section were not detected. Likewise, it is possible that
some GABAergic neurons are not detected because the
levels of GABA within their somata fall below the detection
threshold of the antiserum. Recent evidence that the number of GABA immunoreactive neurons detected in layer 4
of macaque striate cortex is reduced by enucleation or visual deprivation (Hendry and Jones, '86) suggests that
GABA levels within neurons can vary and opens up the
possibility that at any given time, some percentage of the
GABA-containing population may be below the threshold
for detection. Given these considerations, it seems prudent
to view cell counts as minimal estimates since a variety of
factors, physiological and/or methodological, may affect the
number of cells labeled in any given animal.
Both the GAD and GABA antisera show consistent laminar differences in the density of labeled neurons. The greatest density of labeled neurons is found in laminae 4C,, 4A
and in laminae 2-3. The increased density of labeled neurons in the geniculate-recipient zones (4C, and 4A) is consistent with the observations made by Gabbott and Somogyi
('86) in cat visual cortex. In the cat, the greatest number of
GABAergic neurons is found in laminae 4 and 3B, and
since these regions are the major targets of geniculate afferents, they argued that this distribution reflects the allocation of a large share of cortical GABAergic inhibition to the
early stages of visual processing. However, it should be
pointed out that laminae 4 and 3B in the cat and lamina
4C, and 4A in the monkey also contain the greatest density
of non-GABAergic neurons. As a result, the ratio of GABA
immunoreactive neurons to the unlabeled population in
these layers is not greater (and may be less) than that found
at other levels within the superficial layers. In addition,
lamina 4C, and the cytochrome-rich patches of lamina 3 in
macaque are prominent geniculate-recipient regions, but
they do not have an increased density of GABAergic
neurons.
There does seem to be a significant difference in the
percentage of GABA immunoreactive neurons in the superficial and deep layers of striate cortex. The present study
suggests that almost 20% of the neurons in the upper part
of lamina 3 are GABAergic, while roughly 12% of the
neurons in laminae 5 and 6 are GABAergic. A difference in
the proportion of GABAergic neurons in the superficial and
deep layers has been reported also in the visual cortex of
the cat (Gabbott and Somogyi, '86). This difference may
reflect the fact that there is a greater number of potential
synaptic contact sites for the GABAergic neurons within
lamina 3 since they have access to the processes of neurons
whose somata lie within the layer as well as the apical
dendrites of pyramidal neurons whose somata lie in the
deeper layers. This seems unlikely to be the whole explanation, however, since at least some GABAergic neurons in
the superficial layers project to the deep layers and vice
versa (Valverde, '71; Lund, '73; Somogyi et al., '81, '83a).
D. FITZPATRICK ET AL.
90
further evidence for a functional subdivision within the Dow, B.M. (1974)Functional classes of cells and their laminar distribution
in monkey visual cortex. J. Neurophysiol. 37:927-946.
cortical target of the magnocellular layers of the LGN. The
original subdivision of lamina 4C into two tiers, a and 0, Einstein, G., T.L. Davis, and P. Sterling (1987) The ultrastructure of synapses from the A laminae of the lateral geniculate nucleus in layer IV
was based on differences in the dendritic morphology and
of cat striate cortex. J. Comp. Neurol. 260:63-75.
projections of spiny stellate neurons: those in 4C, were Fitzpatrick, D., K. Itoh, and LT. Diamond (1983a)The laminar organization
shown to send their axons into lamina 4B, while the spiny
of the lateral geniculate body and the striate cortex in the squirrel
stellate neurons in 4C3 were shown to send their axons
monkey (Saimiri sciurens). J. Neurosci. 3t673-702.
vertically through 4B, without arborization, to terminate Fitzpatrick, D., J.S. Lund, and D.E. Schmechel(1983b) Glutamic acid decarhoxylase immunoreactive neurons and terminals in the visual cortex of
in lamina 3B (Lund, '73). This distinction was reinforced by
monkey and cat. Soc. Neurosci. Abstr. 9:616.
the demonstration that the magnocellular and parvicelluFitzpatrick,
D., G.R. Penny, and D.E. Schmechel (1984) Glutamic acid delar layers of the LGN had largely separate terminal fields
carboxylase-immunoreactive neurons and terminals in the lateral genicwithin 4C,, and 4C,, respectively (Hubel and Wiesel, '72;
ulate nucleus of the cat. J. Neurosci. 4r1809-1829.
Hendrickson et al., '78). More recent evidence suggests that Fitzpatrick, D., J.S. Lund, and G.G. Blasdel (1985) Intrinsic connections of
4C, and 4C,, may not be uniform throughout their depth,
macaque striate cortex: Afferent and efferent connections of lamina 4C.
J. Neurosci. 53329-3349.
and that both contain sublaminae with different patterns
of connections (Blasdel et al., '85; Fitzpatrick et al., '85). Freund, T.G., K.A.C. Martin, A.D. Smith, and P. Somogyi (1983)Glutamate
decarboxylase-immunoreactive terminals of Golgi-impregnated axoaxThe upper part of 4C, appears to be distinguished from the
onic cells and of presumed basket cells in synaptic contact with pyralower part by (1)the termination of a distinct type of genimidal neurons of the cat's visual cortex. J. Comp. Neurol. 221:263-278.
culocortical axon (Blasdel and Lund, '82);(2) the presence Gahbott, P.L.A., and P. Somogyi (1986)Quantitative distribution of GABAof laterally spreading axon arbors that terminate in patches
immunoreactive neurons in the visual cortex (area 17) of the cat. Exp.
Brain Res. 61:323-331.
in umer 4C- and in 4B (Rockland and Lund., '83:, Blasdel et
al., '85); and (3) the presence of orientation-se~ective
neu- Garey, L.J., and T.P.S. Powell (1971) An experimental study of the termination of the lateral geniculo-cortical pathway in the cat and monkey.
rOnS (Blasdel and Fitzpatrick, >84).ourfinding of a class of
Roc. R. Soc. Lond. [Biol.] 179;41-63.
large GABAergic
in upper 4cm adds to the evi- Gilbert, C.D., and T.N. Wiesel(1983)Clustered intrinsic connections in cats
dence for further subdivision of 4C, and complements
~3;1116-1133,
~
~
~
~
~
visual cortex,J, ~
Lund'S observation that upper 4c, contains aspinous neu- Hamos, J.E., T.L. Davis, and P. Sterling (1983)Four types of neuron in layer
IVab of cat cortical area 17 accululate "H-GABA. J. Comp. Neurol.
rOnS that are distinct from those in lower 4C, and 4C,
21 7:449-457.
(Lund, '87).
I I
ACKNOWLEDGMENTS
This research was
by grants EY-06661 and EY05282 from the National Eye Institute, and VA Career
Development Award and National Institute on Aging
ADRC grant AG-05128. David Fitzpatrick is an Alfred P.
sloan research fellow. we thank ill ~ i ~for her
~ helpt ~
ful comments' Jay
for his
with the
quantitative
'Ox
and Tom Harper for
their help with the histology and Susan Havrilesky for
preparing the manuscript.
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