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The Journal of Neuroscience, November 15, 1999, 19(22):10082-10097
Total Number and Ratio of Excitatory and Inhibitory Synapses
Converging onto Single Interneurons of Different Types in the CA1 Area
of the Rat Hippocampus
Attila I.
Gulyás,
Manuel
Megías,
Zsuzsa
Emri, and
Tamás F.
Freund
Institute of Experimental Medicine, Hungarian Academy of Sciences,
Budapest, H-1450, Hungary
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ABSTRACT |
The least known aspect of the functional architecture of
hippocampal microcircuits is the quantitative distribution of synaptic inputs of identified cell classes. The complete dendritic trees of
functionally distinct interneuron types containing parvalbumin (PV),
calbindin D28k (CB), or calretinin (CR) were reconstructed at the light microscopic level to describe their geometry, total length, and laminar distribution. Serial electron microscopic reconstruction and postembedding GABA immunostaining was then used to
determine the density of GABA-negative asymmetrical (excitatory) and
GABA-positive symmetrical (inhibitory) synaptic inputs on their
dendrites, somata, and axon initial segments. The total convergence and
the distribution of excitatory and inhibitory inputs were then
calculated using the light and electron microscopic data sets.
The three populations showed characteristic differences in dendritic
morphology and in the density and distribution of afferent synapses. PV
cells possessed the most extensive dendritic tree (4300 µm) and the
thickest dendrites. CR cells had the smallest dendritic tree (2500 µm) and the thinnest shafts. The density of inputs as well as the
total number of excitatory plus inhibitory synapses was several times
higher on PV cells (on average, 16,294) than on CB (3839) or CR (2186)
cells. The ratio of GABAergic inputs was significantly higher on CB
(29.4%) and CR (20.71%) cells than on PV cells (6.4%). The density
of inhibitory terminals was higher in the perisomatic region than on
the distal dendrites.
These anatomical data are essential to understand the distinct behavior
and role of these interneuron types during hippocampal activity
patterns and represent fundamental information for modeling studies.
Key words:
inhibitory neurons; GABA; synaptic convergence; dendrite
geometry; serial reconstruction; 3D; electron microscopy; database for
modeling
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INTRODUCTION |
The functional architecture of
neuronal networks is defined by the building blocks and their
connectivity. Cortical networks consist of two basic elements:
excitatory principal cells and inhibitory interneurons. In the
hippocampus, interneurons are important regulators of electrical
activity patterns (Freund and Buzsaki, 1996 ). They control the output,
the plasticity of inputs, and the excitability of principal cells
(Miles et al., 1996), they are able to synchronize large cell
populations at different frequencies (Buzsaki and Chrobak, 1995; Cobb
et al., 1995), and, by being targets of subcortical pathways (Freund
and Antal, 1988; Freund et al., 1990; Miettinen and Freund, 1992), they
mediate motivational, emotional, and autonomic control of cortical
activity patterns. The types, distribution, and connectivity of
hippocampal interneurons have been described in detail.
Parvalbumin-containing (PV) cells are basket and axo-axonic cells
(Kosaka et al., 1987) which, by exerting strong perisomatic inhibition,
control the generation of Na+ spikes and
thus the output of cells (Miles et al., 1996). Calbindin D28K-containing (CB) cells, whose axonal arbor
overlaps with Schaffer collateral terminals in the dendritic region
(Gulyás and Freund, 1996) are involved in the control of
dendritic Ca2+ spikes (Miles et al.,
1996). Calretinin-containing (CR) cells form frequent dendrodendritic
and axodendritic contacts with each other and selectively innervate
other interneurons (Gulyás et al., 1996). Their connectivity
makes them a good candidate for generating rhythmic synchronous
activity patterns of local origin.
The distribution of postsynaptic targets of these and several other
identified interneuron types (Buhl et al., 1994; Gulyás et al.,
1996; Halasy et al., 1996) has been precisely characterized, allowing
the calculation of their divergence. In contrast, there is no
quantitative estimate available on the convergence and distribution of
excitatory and inhibitory inputs from different sources onto functionally distinct interneuron classes. Because inputs such as the
perforant path and nucleus reuniens thalami fibers (Wouterlood et al.,
1990) in stratum lacunosum-moleculare, the Schaffer collaterals in
strata radiatum and oriens, and the local pyramidal cell collaterals in
stratum oriens (Amaral and Witter, 1989) terminate in specific layers
in the CA1 area, the laminar distribution of the dendrites and their
synaptic densities determine the set and relative weight of possible inputs.
In this paper we first describe the geometry of immunocytochemically
visualized PV-, CB-, and CR-containing cells at the light microscopical
level. Using serial reconstructions from electron microscopic sections
immunostained for GABA we then estimated the absolute and relative
densities of excitatory and inhibitory inputs arriving onto the somata,
axon initial segments, and different dendrites of the three examined
inhibitory cell populations in the CA1 region. This way the total
number of converging excitatory and inhibitory afferents were
calculated to reveal the relative contribution of different excitatory
input pathways segregated to different layers, and the distribution of
inhibitory afferents in different domains of the inhibitory cells were mapped.
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MATERIALS AND METHODS |
Immunocytochemistry
Perfusion and pretreatment. The study was
conducted in accordance with the principles and procedures outlined in
the National Institutes of Health Guide for Care and Use of
Laboratory Animals. Male Wistar rats (Charles River Laboratories,
Budapest, Hungary; 250 gm) were perfused under deep Equithesine
anesthesia (chlornembutal 0.3 ml/100 gm), first with physiological
saline (1 min) and then with 300 ml of one of the following fixatives:
one containing 0.05% glutaraldehyde (TAAB Laboratories Equipment), 4%
paraformaldehyde (TAAB), and 0.2% picric acid dissolved in 0.1 M phosphate buffer (PB; pH 7.4) for the light
microscopical measurements (group A; n = 10 rats), and
another containing 2% glutaraldehyde, 3% paraformaldehyde, and 0.2%
picric acid dissolved in PB for the electron microscopical measurements
(group B; n = 10 rats). After fixation, the dorsal hippocampi were dissected together with the overlying neocortex and
sectioned on a vibratome at 60 µm. To allow serial reconstruction of
dendritic arbors, after immunostaining 10-11 consecutive sections were
kept in order (group A). After extensive washes in PB, two sections
from each vial were selected, marked, and the outlines and laminar
boundaries of the hippocampi were drawn using a camera lucida and a
10× objective, to record the size of sections before immunostaining
and embedding. The sections were then immersed in a mixture of 25%
sucrose and 10% glycerol in 0.01 M PB and freeze-thawed in liquid nitrogen to increase the penetration of antisera used for immunostaining. After repeated washes, the sections of animals from group B were treated with 1%
NaBH4 for 30 min to reduce free aldehyde groups
and enhance immunostaining.
Pre-embedding immunostaining for PV, CB, and CR. The
sections were incubated first in 5% bovine serum albumin (BSA; 45 min), then in rabbit anti-parvalbumin (1:5000; Baimbridge and Miller, 1982), anti-calretinin (1:3000; Rogers, 1989), or anti-calbindin D28k antiserum (R8701; 1:3000; Baimbridge and
Miller, 1982) for 2 d. This was followed by incubation in
biotinylated goat anti-rabbit IgG (1:100; Vector Laboratories,
Burlingame, CA; 4 hr). Finally, the standard ABC kit (1:100; Vector
Laboratories; 3 hr) was used. The sections were washed three times for
30 min between each serum. All the washing steps and the dilution of
the antisera were performed in 50 mM Tris-buffered saline
(TBS; pH 7.4). In group A, the peroxidase reaction was developed by
nickel-intensified 3,3'-diaminobenzidine (DAB)-4HCl (Sigma, St. Louis,
MO) as a chromogen. In group B, the peroxidase reaction was developed
using DAB-4HCl (Sigma) as a chromogen and shorter development time to
avoid the masking of the postsynaptic densities by DAB precipitate.
After the final washes in PB, the sections were treated with 1%
OsO4 for 1 hr, dehydrated in ethanol, and
embedded in Durcupan (ACM; Fluka, Buchs, Switzerland). The sections
that were drawn and marked at the beginning of the staining procedure
were drawn again, and the shrinkage in the x,y plane was calculated.
Postembedding immunostaining for GABA. The immunostaining
procedure (in material from group B) followed those described by Somogyi and Hodgson (1985), with small modifications, using a well-characterized antiserum against GABA (Hodgson et al., 1985). The
steps were performed on droplets of Millipore-filtered solutions in
humid Petri dishes, as follows: 2% periodic acid
(H5IO6) for 10 min; wash by
dipping in several changes of double-distilled water; 2% sodium
metaperiodate (NaIO4) for 10 min; wash as before; three times for 2 min in TBS, pH 7.4; 30 min in 1% ovalbumin dissolved in TBS; three times for 10 min in TBS containing 1% normal goat serum
(NGS); 1-2 hr in a rabbit anti-GABA antiserum (code number 9; diluted
1:1000 in NGS-TBS); two times for 10 min in TBS; 10 min in 0.05 M Tris buffer, pH 7.4 containing 1% BSA and 0.5% Tween 20; goat anti-rabbit IgG-coated colloidal gold (12 nm; Jackson ImmunoResearch, West Grove, PA) for 2 hr (diluted 1:20 in the same buffer); wash two times for 5 min in double-distilled water; saturated uranyl acetate for 30 min; wash in four changes of
double-distilled water; staining with lead citrate; and wash in
distilled water. Profiles showing a density of colloidal gold particles
at least 5× background level, in two or three adjacent sections were
considered GABA-immunoreactive. Axon terminals forming asymmetrical
synapses (presumed glutamatergic) were used to establish background density.
Controls. The specificity of the primary antisera has been
tested extensively by the laboratories of origin (Baimbridge and Miller, 1982; Somogyi and Hodgson, 1985; Rogers, 1989). Controls of the
methods in the present experiments included replacement of the primary
antisera with normal serum (1:200). In these sections, no staining was
visible apart from a faint background limited to the surface of the
sections. Replacement of the GABA antiserum with normal rabbit serum in
the postembedding immunogold reaction resulted in a loss of specific
staining, i.e., no signs of colloidal gold accumulation could be
detected over any profiles.
Light microscopical sampling
After immunostaining, the order of sections was
determined, and several cells were selected for reconstruction (PV,
n = 26; CB, n = 19; CR,
n = 29) from the middle sections of the series. Although PV, CR, and CB cells show unique distributions and dendritic arborization patterns, there are variations in location and shape within groups. For the reconstructions, we selected cells showing the
most characteristic properties of the given group (the selected cells
will be described in the Results section). We used only material in
which immunostaining was complete, i.e., we visualized the entire
dendritic tree evenly, and thus the dendrites could be followed to
their natural ends. Usually several neighboring cells were drawn from a
section onto tracing paper using a camera lucida and a 50× oil
immersion objective. Each section containing a portion of the dendritic
tree was drawn onto a separate sheet of paper. Capillaries cut on the
surfaces were also drawn to help matching dendritic segments when
moving onto the adjacent section. When all dendrites had been
reconstructed, dendrite portions were copied onto a single sheet. PV
and CR cells were reconstructed from three to eight sections of 60 µm
thickness. Because of the large horizontal extent of CB cells, more
sections (up to 11) were needed for their reconstruction.
The dendritic tree of a cell consists of segments of distinct diameter
and appearance. Therefore, after several selected cells were drawn,
dendrite subclasses were defined on the basis of diameter and
morphology for each cell type (Table 1).
Thick (T), medium (M), and thin (t) segments were distinguished in each
layer. Most cell types possessed beaded as well as smooth dendrites,
but since the occurrence of beads depend both on the quality of
fixation and cell type, we did not use this feature for classification. To give a single average diameter value for each subclass, camera lucida drawings (100× oil immersion objective) or electron micrographs (of dendrites running parallel with the surface of the section) were made, then the diameters of the dendrites were measured at evenly
spaced intervals along the longitudinal axis of the dendrite (extremes
of the dendritic ends were excluded) and averaged. This method is
essentially equivalent to measuring the cross-section surface and
dividing it by the length of the dendrite section to calculate average
diameter. Measurements were made using the publicly available
morphometry program NIH Image
(http://rsb.info.nih.gov/nih-image/download.html) on a Macintosh Quadra
computer. The values measured at the light and electron microscopic
levels matched well (see Results).
On the drawings, dendritic segments were labeled with color codes
according to their subclass. A pseudo three-dimensional (3D)
reconstruction program called ARBOR (developed by S. Pomaházy and
modified by A. I. Gulyás; see Wolf et al., 1995) was then used to reconstruct the dendritic arbors. The two-dimensional (2D)
projections of the dendritic trees were traced on a digitizing tablet,
and the points where dendrites crossed section boundaries were also
indicated. The program interpolated the missing z values with the help of the available x, y coordinates
and the section border crossing points, carrying information on
z coordinates, assuming that dendrites cannot break at any
point. The feasibility of this approach has been verified earlier (Wolf
et al., 1995). We also tested the accuracy of the program on
intracellularly filled mossy fibers already reconstructed using
Neurolucida. The difference in the measured values was <3%. Because
we noticed almost twofold variability in the dendritic length of
individual cells, this small error is not significant. The program
supplied data in spreadsheet format on several dendritic tree
parameters. The values were further processed using Excel. We used the
pseudoreconstruction algorithm because it does not require expensive
hardware, it is much faster than other reconstruction programs, and its
accuracy is not inferior.
To calculate the surface of the cell bodies, 20 somata were selected
from each cell population and reembedded for serial sectioning. The
cross sections of the somata were drawn from 13-26 semithin sections,
1 µm each. The perimeters were measured using NIH Image (see above).
The somatic surfaces were then calculated for each cell using the
perimeter values and the section thickness assuming that two subsequent
cross sections form a truncated cone with upper and lower perimeters
equal to the perimeter of the cross sections (which is reasonable for
surfaces not containing extreme convexities or concavities).
Electron microscopical sampling
Three to five dendritic segments were reembedded from
each dendritic subclasses into Durcupan blocks from each layer and from each interneuron group. For sampling we selected only dendrite segments
that could be traced back to somata of those cell types that were
analyzed at the light microscopic level. The embedded dendrites in the
blocks were drawn using a camera lucida for length measurement. Long
series of ultrathin sections (70-150 sections of 60 nm thickness) were
cut on a Reichert Ultracut S ultramicrotome. We selected straight
dendritic segments and thus, using the Pythagorean distance (and
shrinkage correction) we could calculate the true length of the sampled
segments (between 4 and 85 µm, 26 µm on average) by measuring the
projected 2D length of the dendrites (from camera lucida drawings) and
the depth spanned by the serial sections. The series containing known
lengths of classified dendrites were immunostained against GABA and
serially reconstructed. For the reconstruction, a Hitachi-7100 electron
microscope was used. The positions of the synaptic contacts on the
selected dendritic profiles were recorded for each section. The
terminals forming synapses were numbered consecutively, and their GABA
immunoreactivity (based on the density of gold particles on the
adjacent sections) was determined (a total of 2665, 385, and 764 synapses for the three cell populations). In the case of PV and CR
dendrites, the possible PV or CR immunoreactivity (respectively) of the
presynaptic terminals was also recorded. To calculate the density
values, the number of synapses on all sampled dendritic segments of a given subclass were added and divided by the sum of lengths of the same
dendritic segments.
Ten to thirteen serial sections were cut from PV, CB, and CR-IR somata
(n = 11, 6, and 10, respectively) to estimate the
density of synapses per 100 µm2. The
synapses were serially reconstructed and numbered. The surfaces of the
somatic slices were calculated similarly to the soma surface calculation (see above).
The axon initial segments of three neurons were reembedded from each
population, serially sectioned, and reconstructed similarly to the dendrites.
In the light microscopic analysis of the dendritic arborization
patterns, the SE of the dendritic lengths has been specified. In
case of the input density measurements of this paper, SEs are not
given, because of the rather laborious nature of the sampling, the
sample size for individual dendrite subclasses from each layer was
small (three to five segments).
Correction for shrinkage
Shrinkage may arise at different steps of the histochemical
procedure. We recorded the sizes of sections before the beginning of
the immunocytochemical procedure. For the LM sampling, we had to
calculate the x,y shrinkage caused by the immunostaining (by comparing section sizes before and after immunostaining) and corrected the calibration of the 3D reconstruction program with this value. In
this phase, we did not need shrinkage correction in the z
axis because we assumed that the mechanics of the vibratome on which the 60 µm sections were cut is accurate. Even if individual sections slightly vary in thickness this variance is averaged over the consecutive sections we used for reconstruction. Thus, for the 3D
reconstruction program we gave the z value used for
sectioning. Whatever the shrinkage is along the z axis
during processing, because of the algorithm of the program, it would
not effect the results.
In the EM analysis, correction for shrinkage had to be applied for both
the x,y axes and the z axis. The x,y
correction was done similar to that at the LM level. For the
z correction, small pieces of resin-embedded sections were
removed from the glass slides, then aligned perpendicular to the slide
and embedded into a block. The width of the sections were then measured
along the x axis (previous z became x
this way) using a camera lucida and a micrometer-scaled slide. This
method is superior to the method using the focusing micrometer scale of
the microscope, because of the difference in refractory index of the
air, oil, and the embedding material, accurate measurements cannot be
done that way. The shrinkage found along the z axis was
small: 0.5%. We did not measure the actual thickness of the ultrathin
sections, because we assumed that even if the mechanics of the
ultramicrotome is not totally accurate for neighboring sections, this
error is, again, averaged over the long serial sections. Thus, for
section thickness we used the values indicated by the ultramicrotome, corrected with the small shrinkage along the z axis.
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RESULTS |
For the sampling we had to solve two problems. First, the cell
populations visualized by the three markers are not entirely homogeneous. Two subtypes can be distinguished in the case of CB (Toth
and Freund, 1992; Gulyás and Freund, 1996) and CR (Gulyás et al., 1992) cells, and there is inhomogeneity in the case of PV cells
as well. Because the reconstructions are rather laborious, we decided
to select cells for reconstruction with the most typical morphologies.
The variations and the sampled morphologies will be described below.
The second problem arose from the fact that proximal dendrites are
thicker, whereas distal, second, or third order dendrites are gradually
thinner. Furthermore, different dendritic segments can be smooth or
beaded to various degrees. The density of synapses might be different
at these segments and in different layers where the afferents arrive
from different sources. Therefore, we had to divide dendrites in all
layers into subclasses (see Materials and Methods) for a more accurate
calculation of the number of afferent synapses. The established
dendrite subclasses and their corresponding diameters, which were used
for drawing subclass boundaries, will be described below and summarized
in Table 1. The dendrites sampled for electron microscopy were
classified and selected on the basis of the same criteria. We used
different diameter values for the subclass boundaries for different
cells and different layers, because this way the dendritic trees could be subdivided more accurately into subclasses.
Finally, we have to define the coordinate system we used. We prepared
coronal sections from the dorsal hippocampus, thus according to our
arrangement of the sections on the slides, the apical dendrite of the
pyramidal cells and thus the radial-vertical direction is parallel
with the y axis. The x axis is parallel with the
laminar boundaries (horizontal direction) and points toward the
subiculum and the CA3a area. The z axis is perpendicular to
the plane of the section and thus points to the rostral and caudal
directions. Subsequent sections follow each other along the
z axis.
The detailed geometry of the cells and several measured parameters not
detailed in this paper are available at the following worldwide
web (www) page: http://www.koki.hu/~gulyas/ca1cells.
Light microscopical properties of reconstructed interneurons
Immunostaining for the three interneuron populations revealed
characteristic distribution patterns as well as dendritic and axonal
arborizations throughout all layers and areas of the hippocampus similar to those described previously in several papers (Baimbridge and
Miller, 1982; Kosaka et al., 1987; Sloviter, 1989; Gulyás et al.,
1992; Miettinen et al., 1992; Toth and Freund, 1992; for review, see
Freund and Buzsaki, 1996). Because we studied only the inputs of CA1
interneuron populations, here we briefly describe only the dendritic
trees, without the characteristics of their axonal arborization (for
those details see the papers cited above).
Dendritic tree of PV-containing neurons
Immunostaining for PV visualized only interneurons.
Labeled neurons were predominantly found in stratum pyramidale and in the upper third of stratum oriens. The majority of neurons located in
the principal cell layers showed the characteristic features of
pyramidal basket cells. Occasionally, PV cells could be found in
stratum radiatum. In most cases they had large to medium size cell
bodies, but occasionally cells with small somata were also found in
stratum radiatum. Dendrites ran radially spanning all layers. For
reconstruction, we selected cells with large to medium cell bodies
located in stratum pyramidale and at the border of strata pyramidale
and oriens. Twenty-six cells were selected, and all dendrites were
reconstructed to their natural ends from three to eight serial sections
(60 µm). Two to six (5.50 ± 1.24) primary dendrites arose from
the soma and ran radially into stratum oriens or through stratum
radiatum into stratum lacunosum-moleculare, branching infrequently. As
seen in Figures 1,
2, and Table
2, PV cells had the most extensive
dendritic tree (4347.74 ± 1124.95 µm) among the three examined
neuron populations with a large variation among individual cells. Cells
with a large dendritic tree can have twice as long total dendritic
length than those with small trees. The distribution of dendrites among
layers (Tables 2, 3) shows that the cells
are likely to collect the majority of their inputs in strata radiatum
and oriens, and only a smaller portion in strata lacunosum-moleculare
and pyramidale.
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Figure 1.
Reconstructed dendritic trees of PV-, CB-, and
CR-containing interneurons from the CA1 region of the rat hippocampus.
Four examples are shown from each reconstructed cell population,
illustrating the characteristics of branching patterns. Different types
of dendritic segments separated on the basis of their diameter are
indicated with different colors. Note the variance in the total length
of dendrites of individual cells within a cell group and the
differential distribution of dendrites in distinct layers for the three
cell populations. Parvalbumin cells had the largest dendritic tree, and
calretinin cells had the smallest. The horizontal extent of the
dendritic tree was widest for calbindin cells and narrowest for
calretinin cells. Scale bar, 50 µm. L.M., Stratum
lacunosum-moleculare; S.R., stratum radiatum;
S.P., stratum pyramidale; S.O., stratum
oriens.
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Figure 2.
Total length of dendrites
(A), and surfaces of somata
(B) of CA1 area interneurons. A,
PV cells have the longest total dendritic length, and CR cells have the
shortest. However, as indicated by the small open
squares representing the length of individual dendrites, there
was a high variability among individual cells within a group. Only the
length of CR dendrites showed significant (p < 0.05) difference from the other two cell populations.
B, As for the total dendritic lengths, the soma surface
was largest for PV cells and smallest for CR cells with considerable
variance. The values are given in square micrometers. The differences
among the groups were significant (n = 20;
p < 0.05).
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Table 3.
Average length of different dendrite subclasses for the
three sampled cell populations in the different layers
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Although PV cells include both basket and axo-axonic cells, the smooth
transition of morphological features among the reconstructed cells does
not justify a division into two separate morphological classes. It is
highly unlikely that our sample contained only basket cells but no
axo-axonic cells, thus, the results do not support the conclusion
of Li et al. (1992) that basket and axo-axonic cells considerably
differ in the length of dendrite they have in stratum
lacunosum-moleculare.
The subclasses of dendrites classified in the case of PV cells are
shown in Table 1. The distribution of thick, medium, and thin dendrites
is shown in Figure 1. Generally, thick or medium dendrites arose from
the somata. Thick dendrites were smooth and became thinner after the
first branchpoint or after a few hundred micrometers, even if the
dendrite did not branch. Medium dendrites were smooth or irregular in
appearance. Thin dendrites bore beads in all layers. The diameter of
the dendrites varied between 0.3 and 2.7 µm among the different
subclasses. As can be seen from Table 1, PV cells had the thickest dendrites.
Dendritic tree of CB-containing neurons
The highest numbers of CB-containing nonpyramidal cell
somata were found in stratum radiatum, with a peak density near the border with stratum lacunosum-moleculare. A smaller number of cells
were seen in strata oriens and pyramidale. Cells were only occasionally
seen in stratum lacunosum-moleculare. As in earlier studies (Toth and
Freund, 1992; Gulyás and Freund, 1996), we could distinguish two
types of CB cells on the basis of their location and dendritic
morphology. Type I cells were most numerous in stratum radiatum, but
could also be found in strata pyramidale and oriens in much lower
numbers. These cells were multipolar or bitufted with two to five
primary dendrites running in all directions, often descending to
stratum oriens, but only very rarely entering stratum
lacunosum-moleculare. Type II cells could be found exclusively in
stratum oriens. They had large, fusiform cell bodies and several long,
horizontally oriented dendrites. Because type II cells were found to
project to the medial septum and thus are not typical local
interneurons, we did not include them in our samples.
We selected 19 cells from all parts of stratum radiatum with variable
morphologies belonging to type I. Because the horizontal extent of the
dendritic trees were the largest in the case of the CB cells, they had
to be reconstructed from 4-11 consecutive sections to reach the
natural ends of each dendrite. CB cells had three to six primary
dendrites (4.053 ± 1.08) and a rather variable total dendritic
length (3441.12 ± 937.59 µm). The great majority of dendrites
were restricted to stratum radiatum (76.2%; Table 2). The largest
variation in the length of individual dendrites were seen in the case
of CB cells.
The sampled cells showed different morphological features. In one end
of the spectrum were cells with multipolar morphology (Fig. 1, first
cell). Here five or six dendrites arose from the cell bodies and ran in
all directions, often spanning a considerable distance horizontally.
These cells were mainly located in the middle or bottom half of stratum
radiatum. Cells on the other extreme (Fig. 1, second cell) had one or
two dendrites descending through stratum pyramidale into stratum oriens
and several dendrites extending radially toward the stratum
radiatum/lacunosum-moleculare border. These cells were more often
located in the top half of stratum radiatum. The majority of the CB
cells had morphological features somewhere between the two extremes,
with a continuous distribution. Only occasional short dendritic
segments entered stratum lacunosum-moleculare, independent from other
morphological features.
Generally, the diameter of the dendrites was small in the case of CB
cells (Table 1), and varied between 0.2 and 1.8 µm. Dendrites
arising from the soma were thick and branched within 50-100 µm
of origin. The dendrites did not necessarily change their diameter
to medium after the first bifurcation, but might taper after a few
hundred micrometers without branching. The CB cells were much less
beaded than PV cells. Thick and medium dendrites were always smooth,
whereas thin dendrites could be irregular or rarely beaded.
Dendritic tree of CR-containing neurons
Two types of CR cells, a spiny and a spine-free type, have
been described in the hippocampus (Gulyás et al., 1992). In the CA1 area, only spine-free CR cells are present (Gulyás et al., 1996), although they can be found in all layers, even in the
hippocampal fissure, showing a rather even laminar distribution. The
dendrites arise from multipolar, bipolar, or fusiform cell bodies and
run primarily radially, traversing several layers. Dendrites of
neighboring cells often form dendrodendritic contacts.
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Figure 3.
Distribution of excitatory and inhibitory inputs
on medium-diameter dendrites of PV-, CB-, and CR-immunoreactive
interneurons in CA1 stratum radiatum. The dendrites and the synapses
were reconstructed from serial ultrathin sections immunostained for
GABA. A large difference can be seen both in the absolute and relative
number of excitatory and inhibitory synapses terminating on the three
types of dendrites. The surface of the PV-positive dendrite is densely
covered by synapses in contrast to the sparse innervation of CB- and
CR-positive dendrites. On the other hand, the proportion of inhibitory
terminals compared to all synaptic inputs is lowest on the PV and
highest on the CB dendrites. Excitatory terminals are colored
light gray, GABAergic inhibitory boutons are
dark. Note the large variance in the size of axon
terminals. GABA-negative and GABA-positive axon terminals labeled
with e1-e9 and i1-i4, respectively, are
shown in electron micrographs in Figure 4. Scale bar, 1 µm.
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We selected 29 cells with cell bodies located in stratum pyramidale and
lower stratum radiatum. CR cells had the smallest total dendritic
length (2499.41 ± 946.34 µm, significantly different from both
PV and CB cells). Their dendrites ascended or descended radially
invading all layers, similar to the PV cells. However, CR cells had
fewer primary dendrites (2.97 ± 0.94), and the laminar distribution of the dendritic tree was even more balanced than in the
case of PV cells (Table 2). The sampled cells showed rather homogenous
morphological features. The diameter of the dendrites and thus the
diameter of the subclasses were similar to CB cells (varied between 0.2 and 1.6 µm; Table 1). The dendritic surface, particularly of the
thickest dendrites, was more irregular or beaded than the dendrites of
PV cells. Thick dendrites almost always had an irregular appearance.
Medium dendrites were either irregular or beaded, whereas thin
dendrites were always beaded.
Calculation of the somatic surfaces
To estimate the number of synapses converging onto the
somata, first we had to measure the surfaces of the somata for all the
three examined cell types (Table 2, Fig. 2B). The
soma diameters and surfaces correlated with the size of the dendritic
trees, thus PV cells had the largest (1006 ± 183 µm2), and CR cells had the smallest
(520 ± 154 µm2) surface. CB cells
were in between (798.56 ± 139.61). The difference between PV and
CR cells was almost twofold and statistically significant (p < 0.05).
Electron microscopic investigation of synaptic input densities
At the electron microscopic level, the examined dendrites and
somata shared all characteristic features of interneurons. The dendrites had no spines, they received excitatory and inhibitory inputs
on their shaft. The cytoplasm of both the dendrites and the somata were
electrondense and possessed numerous mitochondria. The somata contained
large amounts of rough endoplasmic reticulum and ribosomes. The nuclei
were invaginated and often had intranuclear rods. The irregularity of
the dendrite diameters apparent at the light microscopic level were
also observed in the electron microscope.
PV-immunoreactive dendrites
From the three examined populations, PV dendrites received
the most abundant innervation. As demonstrated in Figures 3, 5, and
7A and Table 4, the density of
synapses was two or three times higher than on the other two cell
groups for all layers and thickness subclass. The diameter of the PV
dendrites, as already seen at the LM level, was also larger than the
similar dendrite subclasses of the other cell populations. They
possessed the largest number of mitochondria, which were often
clustered within the thick varicosities of the medium or thin
dendrites. Thick dendrites were usually fairly even in diameter.
We distinguished three types of terminals forming synapses on the
PV dendrites (Fig.
4A,B):
(1) GABA-negative terminals forming asymmetrical synapses, (2)
GABA-positive terminals forming symmetrical synapses, and (3)
GABA-positive terminals also showing PV immunoreactivity. In the latter
case, the density of gold particles was lower than in other
GABA-positive terminals, most probably because of the DAB end product
masking the immunoreactive GABA epitopes. Although the PV
immunoreaction end product often precipitated on the postsynaptic sites, the symmetrical or asymmetrical nature of the synapses could be
established in most cases, especially in dendrites with weaker
immunostaining for the calcium-binding protein. In all cases the
GABA-positive or GABA-negative character of the terminals correlated
with the type of the synaptic active zones. We saw a large variation in
the size of presynaptic terminals (Fig. 4A, e1-e5,
i1) and in the size of synaptic active zones.
Since in this study we did not intend to measure these characteristics, only the number of sections containing a terminal or a synapse was
recorded. We found that qualitatively the terminal size correlated with
the size of the synaptic specialization and the number of vesicles
(Pierce and Lewin, 1994). In general, GABA-positive and PV-positive
axon terminals were larger (six to eight sections) than GABA-negative
terminals (three to six sections; Fig. 4, e vs i,
PV). The same variability and size differences among
terminal types were seen for synaptic terminals in contact with CB and CR dendrites as well.
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Figure 4.
Types of afferent boutons on the dendrites of the
examined interneuron populations. A, B,
PV-positive dendrites received synapses from GABA-negative
(e1-e5), GABA-positive (i1), as well as
PV/GABA-positive terminals. From the five excitatory terminals
(e1-e5) contacting a short section of the reconstructed
dendrite shown in Figure 3, four (e1-e4) formed
asymmetrical synapses (curved white arrows) in this
plane of section. The large GABA-positive terminal (i1)
also formed a synapse (white arrow) with the dendrite,
however, as often happens in immunostained material, the symmetrical
nature of the synapse is not evident because of the DAB precipitate in
the postsynaptic profile. B demonstrates a
GABA-negative, likely excitatory (e), a
GABA-positive inhibitory (i), and a PV-positive
(PV) terminal forming synapses on a PV-positive
(nonreconstructed) dendrite. C, D,
Electron micrographs of two parts of the reconstructed CB dendrite in
Figure 3 demonstrate the large variation in the size of GABA-negative
excitatory (e6 vs e7) and
GABA-positive inhibitory (i2 vs i3)
terminals forming asymmetrical (curved arrows) and
symmetrical (arrow) synapses. E, F,
CR-positive dendrites, besides the GABA-positive and -negative
terminals, might receive inputs from CR-immunoreactive axons as well.
Excitatory terminals e8 and e9 and the
inhibitory synapse i4 contact the reconstructed dendrite
shown in Figure 3. In F a dendrite (not shown in Fig. 3)
is innervated by a GABA-positive CR-positive axon terminal as well as
by a GABA-negative (e) terminal. Scale bars, 0.5 µm.
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In spite of the high density of total synaptic input, the density of
GABA-positive terminals was nearly equal to or lower than the density
of GABAergic terminals on the CR and CB dendrites, respectively (Fig.
5B). It follows that the
proportion of GABA-positive terminals was two to five times lower on PV
dendrites than on CR and CB dendrites in stratum oriens and stratum
radiatum (Fig. 5C). The ratio of GABA-positive terminals was
higher in stratum lacunosum-moleculare, but still below the value for
CB dendrites in other layers.
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Figure 5.
Density of total synaptic input
(A) and GABA-positive (B)
synapses, as well as the ratio of terminals positive for GABA
(C) terminating on different dendrite subclasses
of the examined interneuron populations. Densities are expressed in
number of synapses per 100 µm. The density of synapses is largest on
PV dendrites (A), regardless of the layer or
dendrite subclass. Conversely, the density of GABA-positive terminals
is largest on CB dendrites (B), which means that
the ratio of GABA-positive inputs is much higher on CB than on PV
dendrites (C). For abbreviations, see Table
1.
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Terminals immunoreactive for both PV and GABA were responsible on
average, for a quarter of the GABA-positive inputs (27.6%). They were
concentrated in the perisomatic region, where they formed 45 and 61%
of the local inhibitory terminals on thick dendrites in strata radiatum
and oriens, respectively (for somata see below). It is important to
note here that immunostaining for PV consistently visualizes the
PV-containing dendrites, but the staining of PV-positive main axons,
and especially axon terminals may vary a lot depending on the quality
of fixation. This suggests that the PV level in axon terminals is
around the detection threshold of the antibody we used. Therefore, the
above values are likely to underestimate the real ratio of PV-positive
GABAergic terminals.
CB-immunoreactive dendrites
The density of synaptic inputs on CB dendrites is lower than that
on PV cells, and is close to the values found on CR cells. The
diameters of the dendrites were generally smaller, but more uniform,
and they possessed less mitochondria. Varicose CB dendrites were rare
compared to those of PV neurons. The majority of CB dendrites are
located in stratum radiatum. Considerable amounts of dendrites can be
found in stratum oriens as well, and only a minimal number in stratum
lacunosum-moleculare. For electron microscopy, we sampled dendrites in
stratum radiatum and stratum oriens and found that the distribution of
afferent synapses was similar in these two layers. Two types of
terminals could be distinguished: GABA-negative forming asymmetrical
synapses and GABA-positive making symmetrical synapses (Fig.
4C,D). The variability and size differences of GABA-negative
and -positive terminals were similar to those seen in the case of PV
dendrites. The density of excitatory terminals was rather low compared
to PV cells and showed little variation among dendrites of different
thickness, with the highest density of inputs on stratum radiatum thick
dendrites. The density of GABA-positive terminals was slightly higher
on CB dendrites compared to PV dendrites (with some variability),
resulting in the highest ratio of GABA-positive terminals among
interneuron types examined (Table 4, Figs. 4, 5C).
CR-immunoreactive dendrites
The density of synaptic inputs was lowest on the CR
dendrites, but only slightly below the values for CB dendrites. The
diameters of the CR dendrites were similar to those of CB dendrites,
but these, being the most beaded from the three examined cell types at
the light microscopic level, showed rather large variability in diameter.
We distinguished three types of input onto CR dendrites: (1)
GABA-negative terminals forming asymmetrical synapses, (2)
GABA-positive terminals forming symmetrical synapses, and (3)
GABA-positive terminals also immunoreactive for CR, because CR
cells are known to form frequent axo-dendritic contacts with each other
(Gulyás et al., 1992, 1996). The density of excitatory synaptic
inputs onto CR dendrites, with some variability, was similar to the
values obtained for CB dendrites. However, the density of GABA-positive inputs was close to that of PV cells. This way the density of total
afferent synapses was the lowest for CR cells, and the percentage of
GABA-positive terminals higher than for PV dendrites, but lower than
for CB dendrites. The ratio of inhibitory terminals increased slightly
toward the perisomatic region (Table 4; see Fig. 10).
Similar to the PV-PV contacts, approximately a quarter of the
GABA-positive terminals on CR neurons contained CR (26.9%). However,
in contrast to the PV terminals, which were predominantly found in the
perisomatic region, CR terminals were distributed rather evenly
throughout the dendritic tree.
Density of inputs onto the somata
The reconstructions of the soma surfaces (Table 2, Figs.
6A,
7) and the calculation of the
synaptic densities (Table 4; Fig. 8)
showed that, similar to the dendritic trees, PV-positive somata
received the highest density and CR cell bodies the lowest density of
synaptic input. Besides the GABA-positive terminals, the somata of the
examined interneuron populations also received abundant GABA-negative
asymmetrical synapses as well (Fig. 6B-E), which is
a characteristic feature distinguishing interneurons from pyramidal
cells. The proportion of GABAergic terminals was higher on the somata
than on the dendrites for all three cell types (Table 4). Similar to
the dendrites, CB somata received the strongest GABAergic innervation,
and PV cell bodies the weakest. In the case of PV cells, a large
portion (70%) of the GABA-positive terminals also contained PV
(Fig. 4B). In the case of CR somata, the ratio of CR-
and GABA-positive terminals represented only 11.5% of all
GABA-positive terminals, supporting the conclusion drawn from the
distribution of CR-positive terminals that inputs from other CR cells
are not concentrated in the perisomatic region, unlike in the case of
PV-PV connections.
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Figure 6.
Afferents on the somata and axon initial segments
of interneurons. A, Partial reconstructions of the
somatic inputs of a PV, a CB, and a CR neuron, as well as the
distribution of axo-axonic synapses on the axon initial segment of a PV
neuron. Inhibitory cells receive both excitatory (light
gray) and inhibitory (dark gray and
black) inputs onto their somata. A large proportion of
the somatic inhibitory terminals on the PV somata came from PV-positive
axon terminals (black). Eight to twelve axo-axonic
synapses terminate on the axon initial segment of an interneuron.
Innervation of the axon initial segment of a PV neuron is shown at the
bottom of the panel. The axo-axonic synapses clustered
close to the soma at the very beginning of the axon initial segment.
Terminals labeled i1-i6 and e1-e3 are
shown in electron micrographs in B-G. B,
The three types of somatic input arriving onto PV cells are shown in
the electron micrograph. Bouton e1 is a GABA-negative
(presumed excitatory) terminal. Similar to the dendritic contacts,
GABA-positive terminals could be PV-positive (PV)
or negative (i1). C-E, CB- and
CR-positive somata also received GABA-negative (excitatory, e2,
e3) and GABA-positive (inhibitory, i2,
i3-i4) synapses, but in a lower density than
PV-containing somata. F, G, Low- and
high-power electron micrographs of the axon initial segment
(A) receiving symmetrical synapses. Note in
A that the proportion of inhibitory terminals is higher
in the somatic region than in the dendritic tree for all examined
populations. In B-E and G, curved
arrows indicate asymmetrical synapses, arrows
indicate symmetrical synapses. Scale bars: A, 2 µm for
somata, 1 µm for AIS; B, D,
E, G, 0.5 µm; C, 0.25 µm; F, 5 µm.
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Figure 7.
Absolute number (A) and
proportion (B) of excitatory and inhibitory
synapses converging onto the three examined cell populations.
PV-positive cells received several times more excitatory input than CB
or CR cells. The ratio of inhibition was highest on CB cells and lowest
on PV cells. The SEM seen in the Tables derives from the SEM of
dendritic length measurements and is not indicated here.
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Figure 8.
Characteristics of the somatic input.
A, Density of somatic synapses (number of synapses per
100 µm2) and the ratio of GABA-positive terminals
(expressed as percentage). The density of synapses, similarly to the
values on the dendrites, were highest for PV cells and similar for the
other two cell populations. The ratio of GABAergic terminals was higher
than on the dendrites. B, Calculated total number of
excitatory and inhibitory synapses on the somata of PV, CB, and CR
cells, as well as the number of inhibitory synapses converging onto the
axon initial segments. Because of their larger size, PV cells received
many more inputs than the CB and especially the CR cells.
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Innervation of the axon initial segments
Serial reconstructions (Fig. 6A;
n = 3-3) showed that, in contrast to earlier data,
interneurons do receive innervation onto their axon initial segments
(AISs). Eight to twelve axon terminals formed symmetrical synapses on
the first 10-15 µm of the AISs (Fig. 6F,G).
Further synapses were not found on the examined first 30 µm of the
AIS. The average number of axo-axonic terminals per cell did not differ
significantly for the examined cell populations (Table
5). In the case of PV-immunostained
material, none of the terminals forming symmetrical synapses on AISs
contained PV (Fig. 6A).
Calculation of the total number and distribution of
afferent synapses
Using the data set on the total length of different dendrite
subclasses in different layers and the respective electron microscopic data sets for the three examined cell populations, we calculated the
total number and distribution of excitatory and inhibitory inputs onto
PV, CB, and CR cells. For the calculations, the average length of a
given dendrite subclass was multiplied with the density of synapses on
the same subclass. There were however some dendrite subclasses in the
light microscopic data set that were not sampled for electron
microscopy because they contributed only minimally to the total length,
and therefore the time-consuming process of sampling was not feasible.
For those subclasses where the synaptic densities were not sampled, we
used density values of dendrites from a neighboring layer with the same
thickness, because these dendrites were the most similar in appearance
to the nonsampled segments (for the replacements, see Table 4
footnote). To calculate the convergence in the somatic region, we
multiplied the average soma surface with the density of input synapses.
Total number of inputs on PV, CB, and CR cells
First we calculated the total number of excitatory and
inhibitory inputs converging onto all dendritic classes of each
neurons. The values for dendritic convergence are shown in the first
row of Table 5. Values for the somatic convergence and the input of
axon initial segments are shown in Figure 8B and
Table 5. Averages and SEs in Table 5 derive from the calculated
convergence onto each light microscopically reconstructed cells. For
detailed values of convergence onto individual cells, see the
above-mentioned www page. Concerning both the total convergence, as
well as the inputs onto different domains (dendritic, somatic, AIS), PV
cells received the largest number of synapses (Fig. 7, Table 5). The total number of synapses converging onto an average PV cell was six to
eight times larger than that onto CB and CR cells. This large
difference is because of the fact that PV cells have the largest
dendritic tree, and the density of synaptic inputs is the highest on
their dendrites. As expected from the density data, and as demonstrated
in Figure 7B, the ratio of excitatory versus inhibitory
terminals is strikingly different on the three cell populations. PV
cells receive the smallest proportion of inhibitory synapses (6.4%),
whereas CR (20.7%) and especially CB cells (29.4%) receive much more
(Fig. 7B). Differences in the somatic input followed the
pattern seen for the dendrites: PV cells received the most numerous
synaptic inputs and CR cells the least (Table 5). The largest
proportion of inhibitory terminals arrived onto CB and the smallest
onto PV cell bodies. The percentage of inhibitory synapses was higher
for the somata than the dendritic region for all cells.
Layer- and cell compartment-specific distribution of
excitatory and inhibitory inputs
The distribution of total synaptic input (A, C)
and inhibitory (B,D) terminals among layers is demonstrated
in Figure 9. In C and
D, which show the relative distribution of inputs, CB cells are seen to receive the vast majority of their excitatory and inhibitory input in stratum radiatum. Thus, they receive excitatory input predominantly from Schaffer collaterals. The inputs are more
evenly distributed among the layers for CR and PV cells, although the
excitatory input onto PV cells relative to the inhibitory input is
smaller in stratum lacunosum-moleculare than in other layers. The
relative contribution of perforant path and/or reuniens thalami fibers
is highest on CR cells, but it is still only about a third of the
Schaffer collateral input.
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Figure 9.
Distribution of all (A, C) and
inhibitory (C, D) terminals on dendrites in different
layers. A and B show the absolute,
whereas C and D show the relative weight
of inputs within different layers. CB cells receive most of their
excitatory and inhibitory inputs in stratum radiatum. The input of the
PV and especially the CR cells is more balanced.
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We examined the proportion of inhibitory terminals on the soma, as well
as on dendrites of different thickness, at different distances form the
soma and in different layers (Fig. 10).
Our first finding was that the ratio of inhibitory terminals is always higher on the somata than on the dendrites, irrespective of the laminar
location (Fig. 10A). The second observation along the
same lines was that, for CB and CR cells, inhibitory synapses on thick dendrites were always larger in number than on the medium diameter and
thin dendrites (Fig. 10B, Table 4). If data were
summed for all layers, the ratio of inhibitory terminals correlated
with the dendritic diameter. For PV cells, inhibitory contacts were of
similar density on thin dendrites than on thick dendrites. Dendrite
thickness does not necessarily correlate with the distance from the
soma, because for a PV or CR cell, a stratum lacunosum-moleculare medium dendrite is further away from the soma than a stratum radiatum thin dendrite (the diameter of dendrite subclasses was defined separately within each layer). Therefore, we calculated the proportion of inhibitory synapses on proximal, medium distal, and distal dendrites
as well. The distribution matched the findings for the simple dendrite
thickness calculations. Namely, proximal dendrites of CB and CR cells
received relatively larger amounts of inhibitory terminals than the
distal segments. For PV cells, the proportion of inhibitory synapses on
dendrites did not correlate with the distance either.
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Figure 10.
Ratio of inhibitory inputs on different
compartments calculated from total convergence values.
A, The ratio of inhibitory synapses on somata was higher
than on dendrites for all cell populations. B, Ratio of
inhibition versus dendrite thickness. Thinner and thus more remote
dendritic regions had a lower percentage of inhibitory terminals than
the proximal dendrites in the case of CB and CR cells. A similar trend
was seen on PV cells in the case of thick and medium dendrites. The
trend broke in the case of thin dendrites, most probably because a
large amount of thin dendrites were found in stratum
lacunosum-moleculare, where the ratio of inhibition is generally
higher. C, The ratio of inhibition in different layers
for the three cell types.
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The analysis of proportion of inhibition in different layers revealed
that the ratio of inhibitory terminals differs among layers for each
cell population (Table 4, Fig. 6C). In the case of CB and CR cells the
variation is relatively small, but PV cells receive considerably more
inhibition onto their dendrites in stratum lacunosum-moleculare
(~18%) than in other layers (~5%). This suggests that, for PV
cells, the inputs from the perforant pathway and/or the nucleus
reuniens thalami are under a stronger inhibitory control than the
synapses formed by Schaffer collaterals in strata radiatum and oriens.
Analysis of the relative distribution of inhibitory synapses among
different domains (Fig. 11) revealed
that relative to its size, the soma receives a disproportionately high
ratio of the total inhibitory inputs. PV cell somata possess the
largest share of inhibitory terminals (16.9% of all GABAergic
boutons), and those of CB cells possess the lowest (11.7%). The
contribution of terminals on the axon initial segments is negligible,
at least regarding their number.
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Figure 11.
Relative distribution of GABA+
inhibitory terminals on different domains of the examined cell types.
Note that although the relative surface of the somata is small compared
to the total dendritic surface, a large portion of the inhibitory
inputs (15-20%) converges onto the perisomatic region (darker
shades, soma, and AIS) in the case of PV cells. The
contribution of dendritic inhibition (lighter shades,
thin, medium, thick dendrites) is highest for CB cells and lowest for
PV cells.
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DISCUSSION |
The main findings of the present study are: (1) the total
dendritic length and diameter is largest for PV cells and smallest for
CR cells; (2) the density of inputs and thus the total number of
afferent synapses is several times higher on PV cells than on CB or CR
cells, whereas the ratio of inhibitory inputs is significantly higher
on CB and CR cells, suggesting that there is no obvious correlation
between the number of excitatory and inhibitory inputs converging onto
neurons; (3) the relative distribution of excitatory and inhibitory
inputs among different layers show interneuron-specific differences;
and (4) inhibitory terminals are concentrated in the perisomatic and
proximal dendritic regions, irrespective of the laminar location of the soma.
Technical considerations
The question of shrinkage and the accuracy of the pseudo-3D
reconstruction algorithm has been discussed in Materials and Methods. Error may be introduced by incomplete visualization of cells using immunocytochemistry. We minimized this problem by applying a
combination of sensitivity-increasing steps (see Materials and
Methods), achieving that dendrites could clearly be followed until
their natural ends. Throughout the study we considered GABA-negative
terminals to be mostly excitatory. This obvious oversimplification does
not introduce significant error for two reasons: first, GABA-negative terminals encountered bore the characteristic features of glutamatergic synapses, i.e., they formed asymmetrical synapses and possessed medium-size round vesicles; second, the majority of hippocampal synapses are formed either by GABAergic or glutamatergic terminals because fibers of diverse neurochemical nature arriving from
subcortical areas are relatively sparse, and most of them carry
varicosities that are largely nonsynaptic (Oleskevich et al., 1991;
Umbriaco et al., 1995)
Large differences in synaptic convergence among the examined
cell types
PV cells possessed the largest dendritic tree, soma surface, as
well as the highest density of afferent synapses, thus the total number
of synapses converging onto the cells is several times (~4-8) higher
than on CB or CR cells. Furthermore, the majority of afferents on PV
cells is excitatory (93.6%) with a sparse inhibitory input. Pyramidal
cells form mostly single synaptic contacts on their target elements
(Gulyás et al., 1993b; Sik et al., 1993), thus the number of
converging principal cells probably closely matches the number of
excitatory inputs. In contrast, interneurons form multiple contacts
with their targets (Gulyás et al., 1993a; Buhl et al., 1994;
Miles et al., 1996), thus the number of converging afferent inhibitory
cells must be severalfold lower. The large difference in the
convergence of excitatory and inhibitory inputs among the cells matches
the findings (Soltesz et al., 1995; Miles et al., 1996) that the
activity level of hippocampal basket cells (the majority of them
PV-immunoreactive) is higher than the activity level of other
interneuron populations. This however is not necessarily the
consequence of the stronger excitatory drive, because several other
factors influence the activity of a given neuron. Passive and active
membrane properties (and their distribution) might be different on the
examined cell populations, and that would significantly effect the
threshold and pattern of firing.
Inhibition is concentrated in the perisomatic region and on
proximal dendrites
Regardless of the total contribution of inhibitory terminals to
the synapses converging onto a cell type, the ratio of inhibitory terminals was highest on the somata, still high on proximal dendrites, and lowest on thinner distal dendrites (except for PV dendrites in
stratum lacunosum-moleculare). In a modeling study (M. Megias, Z. Emri,
K. Antal, T. F. Freund, A. I. Gulyás, unpublished
observations) using realistic neurons based on the geometry of our
reconstructed cells, we demonstrated that in the model neurons the
diameter (and thus the surface/volume ratio) of a compartment affects
the interaction of EPSPs and IPSPs. In the perisomatic region, more inhibitory terminals are required to ensure the same efficacy of
inhibition (same reduction in EPSP amplitude) than in the distal dendritic region, which may explain why the relative abundance of
inhibitory terminals is higher in the perisomatic region.
The sources of inputs of different interneuron populations
The relative contribution of afferent pathways to the inputs of
the examined interneurons was different (Fig. 9). CB cells showed the
strongest input selectivity. They received most of their inputs in
stratum radiatum and almost none in stratum lacunosum-moleculare. PV,
and especially CR cells, showed a more balanced dendritic distribution,
receiving synapses in all layers of the hippocampus. Thus, CB cells
seem to receive input primarily from the Schaffer collaterals and
hardly any from the entorhinal or reuniens thalami afferents. The other
two cell populations receive input from all pathways present in CA1.
The high input selectivity of CB cells suggests that they are activated
largely in a feedforward manner by CA3 pyramidal cells. In contrast, PV
and CR cells can be activated in a feedforward manner by the Schaffer
collaterals and entorhinal/reuniens thalami afferents, as well as in a
feedback manner by the local CA1 pyramidal cell collaterals. Local
collaterals of CA1 pyramidal cells were shown earlier to terminate
primarily on horizontal interneurons in stratum oriens (Blasco-Ibanez
and Freund, 1995), as suggested also by the physiological evidence for
feedback activation of these interneuron types (McBain et al., 1994;
Ali and Thomson, 1998).
Because CB cells mediate dendritic inhibition of principal cells
(Gulyás and Freund, 1996), the laminar distribution of their dendrites suggests that dendritic inhibition of pyramidal cells in the
CA1 area is primarily driven by Schaffer collaterals in a feedforward
manner. In contrast, perisomatic inhibition, which is mediated, at
least in part, by the PV-containing basket and axo-axonic cells (Kosaka
et al., 1987) can be activated both in a feedforward and feedback
manner. The CR cells, which selectively innervate other interneurons
(Gulyás et al., 1996) and may play an important role in the
generation of hippocampal high frequency oscillations, receive both
feedforward and feedback excitation.
The proportion of inhibitory synapses in specific layers was different,
suggesting that excitatory inputs from various sources are under
different degrees of inhibition. The inhibitory control seems to be
relatively strong (18-40%) for the perforant path/reuniens thalami
input of PV cells and for all input pathways converging onto CB and CR
cells. In contrast, the inhibitory control of the Schaffer collateral
and CA1 pyramidal cell recurrent collateral excitation of PV cells
appears to be relatively weak (3-5%).
Two parallel systems of mutually connected interneurons
In the case of CR and PV cells, a considerable proportion
(~27%) of their inhibitory input arrives from axon terminals stained for the same neurochemical marker. PV cells innervate each other primarily in the perisomatic region, whereas CR cells innervate each
other throughout all somatodendritic compartments. Earlier data
indicated that CR cells do not innervate PV neurons (Gulyás et
al., 1996). On the other hand, PV terminals are confined to strata
pyramidale and proximal oriens, therefore even if they contacted CR
cells, the number of these contacts is likely to be negligible. Thus,
two groups of interneurons appear to exist where connections between
the groups are sparse or nonexistent, whereas individual members of
each group are extensively connected to each other. These two
inhibitory cell ensembles are therefore largely independent and have
profoundly different efferent connectivity. PV cells have a direct
effect on pyramidal cells, whereas CR cells influence pyramidal cells
only indirectly, via the innervation of CB cells, VIP/CCK-containing
basket cells (Gulyás et al., 1996) and somatostatin-containing
cells (Acsady et al., 1996). Mutual inhibitory connections were
suggested to be responsible for the generation of rhythmic, synchronous
activity patterns (Perkel and Mulloney, 1974; Mulloney et al., 1981;
Wang and Rinzel, 1993; Whittington et al., 1995). Modeling studies
revealed that the most important factor influencing the frequency of
the oscillation is the decay time constant of the IPSCs (Wang and
Rinzel, 1993; Traub et al., 1996). Earlier studies suggested that
different interneuron populations exert their effect via
GABAA receptors with different kinetics (Pearce,
1993; Buhl et al., 1994; Banks et al., 1998). Perisomatic, most
probably PV-containing, inhibitory cells were shown to generate faster
IPSCs than dendritic inhibitory cell populations. Thus, the two systems
might generate oscillations with different parameters. It is noteworthy
that only those cells form mutually interconnected networks (PV and CR)
that are also driven by entorhinal and/or recurrent input rather than
by the Schaffer collaterals alone.
In summary, the present paper provides the first comprehensive set of
data about the absolute and relative numbers of inhibitory and
excitatory synaptic inputs of three functionally distinct types of
GABAergic interneurons. These data are of crucial importance for
modeling studies, as well as for interpreting physiological and
pharmacological results. In addition, they predict the behavior of
these neurons during different network activity patterns in the
hippocampus. The PV cells were shown to receive six to eight times more
excitatory input than CB or CR cells, and the ratio of inhibitory
terminals among their afferent boutons is rather low. Consequently, in
PV cells the contribution of individual EPSPs to somatic depolarization
is small, but because of their large number they more effectively
depolarize the cell than afferents of CB or CR cells. Therefore, the
firing of PV cells will follow the average background activity level
more faithfully than the output of CB or CR cells, which are more
effectively influenced by individual inputs. CB cells are largely
activated in a feedforward manner by Schaffer collaterals, whereas PV
and CR cells receive additional feedforward input from the entorhinal
cortex and local feedback inputs from recurrent collaterals. However,
as revealed by the relative density of synaptic inputs, the feedforward
drive of PV cells from the CA3 area is probably under a much weaker inhibitory control then their entorhinal inputs or the inputs of CB or
CR cells in all layers.
| |
FOOTNOTES |
Received July 26, 1999; revised Sept. 7, 1999; accepted Sept. 9, 1999.
This work was supported by the Howard Hughes Medical Institute, the
McDonnell Foundation, National Institute of Neurological Disorders and
Stroke (30549), FPI grants, Ministerio de Educación y
Ciencia, Spain, and OTKA (National Science Foundation of Hungary) (T23261). We are grateful to Drs. G. Buzsáki, K. Kaila, R. Miles, and I. Mody for helpful discussions and comments on this manuscript and
to E. Borók, G. Góda, and E. Oswald for excellent technical assistance.
Correspondence should be addressed to Attila I. Gulyás, Institute
of Experimental Medicine, Hungarian Academy of Sciences, P. O. Box
67, H-1450 Budapest, Hungary. E-mail: gulyas{at}koki.hu.
| |
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[Full Text]
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TOP
ABSTRACT
INTRODUCTION
