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The Journal of Neuroscience, April 15, 1999, 19(8):2876-2886
Slices Have More Synapses than Perfusion-Fixed Hippocampus from
both Young and Mature Rats
Sergei A.
Kirov1,
Karin
E.
Sorra1, 2, and
Kristen M.
Harris1, 2
1 Division of Neuroscience in the Department of
Neurology, Children's Hospital, and 2 Program in
Neuroscience, Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
Hippocampal slices have long been used to investigate properties of
synaptic transmission and plasticity. Here, for the first time,
synapses in slices have been compared quantitatively with synapses
occurring in perfusion-fixed hippocampus, which is presumed to
represent the natural in vivo state. Relative to
perfusion-fixed hippocampus, a remarkable 40-50% increase in spine
number occurs in adult hippocampal slices, and a 90% increase occurs
in slices from postnatal day 21 rats. Serial EM shows that all of the
dendritic spines have normal synapses with presynaptic and
postsynaptic elements; however, not all spine types are affected
uniformly. Stubby and mushroom spines increase in the adult slices, and
thin, mushroom, and branched spines increase in the immature slices. More axonal boutons with multiple synapses occur in the slices, suggesting that the new synapses form on preexisting axonal boutons. The increase in spine and synapse number is evident within a couple of
hours after preparing the slices. Once the initial spine induction has
occurred, no further change occurs for up to 13 hr in
vitro, the longest time investigated. Thus, the spine increase
is occurring during a period when there is little or no synaptic
activity during the first hour, and the subsequent stabilization in
spine synapse numbers is occurring after synaptic activity returns in
the slice. These findings suggest that spines form in response to the
loss of synaptic activity when slices are removed from the rest of the
brain and during the subsequent 1 hr recovery period.
Key words:
plasticity; dendritic spines; CA1 pyramidal cell; multiple-synapse boutons; serial electron microscopy
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INTRODUCTION |
Hippocampal slices are widely used
to study cellular mechanisms of synaptic transmission and plasticity
(Bliss and Collingridge, 1993 ; Bindokas et al., 1998; Diamond et al.,
1998; Fisahn et al., 1998; Luscher et al., 1998; Nayak et al., 1998).
Many approaches have been used to optimize slice health during
experimental investigations. Variables that affect slice health include
the following: (1) the age of the animal; (2) the specific composition
of the life-supporting media; (3) whether the slices are maintained at
the interface of media and oxygen or submerged; and (4) the methods of
anesthesia, killing, and cutting the slices (for review, see
Garthwaite et al., 1980; Reid et al., 1988; Hajos, 1989; Aitken et al.,
1995; Lipton et al., 1995). Although much is known about how different physiological or pharmacological parameters affect slices, relatively little is known about whether synapses are altered in slices.
Because of the slow diffusion of glutaraldehyde (~400
µm/hr), the middle of a brain slice remains hypoxic for at least 30 min during fixation by immersion in mixed aldehydes (Hopwood, 1967). To
overcome this limitation, microwave-enhanced fixation was used to speed
greatly the diffusion of glutaraldehyde to the center of the tissue
(Login and Dvorak, 1985, 1994). This procedure reveals hippocampal
slices that are optimally preserved throughout their depth within
seconds after removal from the life-support chamber (Jensen and Harris,
1989). Using this method, synapses have been quantified in well
preserved hippocampal slices after undergoing different experimental
treatments, such as long-term potentiation (Shepherd and Harris, 1998;
Sorra and Harris, 1998).
These earlier studies compared synapses among different hippocampal
slices, all of which experienced the same conditions of preparation and
maintenance in vitro. What has not been addressed quantitatively is whether synapses in slices are altered relative to
their more native state in vivo. For example, during slice preparation, glial, axonal, and dendritic processes are cut. The cut
processes release substances (neurotransmitters, growth factors, potassium, etc.) that can be inhibitory, stimulatory, or even toxic to
the intact neurons remaining in the slice. Furthermore, all spontaneous
background activity is lost when the hippocampus is removed from the
rest of the brain.
The purpose of the experiments reported here was to determine whether
slices have an altered complement of synapses relative to
perfusion-fixed hippocampus. Several parameters were tested. Slices
from both young and mature animals were tested. Slices were maintained
for varying times in vitro. The brains were perfused in situ with either fixative containing 2.5%
glutaraldehyde, which is standard, or 6% glutaraldehyde, which matches
the concentration used to obtain rapid tissue preservation in the slices.
Serial electron microscopy was used to perform two types of analyses.
One analysis used unbiased sampling to compare synapse densities in
volumes of hippocampal CA1 neuropil. The other analysis used
three-dimensional reconstruction of dendrites to compare synapse number
per unit length of spiny dendrite. These complementary approaches show
that neurons in hippocampal slices have more synapses than in
hippocampus fixed by intravascular perfusion at both ages. These
findings do not occur from an artifactual decrease in slice volume,
because the changes are limited to specific subtypes of synapses.
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MATERIALS AND METHODS |
Three studies were performed, as summarized in Table
1. All rats were of the Long-Evans
strain, and all of our procedures follow National Institutes of Health
guidelines and undergo yearly review by the Animal Care and Use
Committee at Children's Hospital. In total, 20 rats were used for
these studies.
Perfusion-fixed hippocampus. All perfusion-fixed hippocampus
was obtained from animals that were under deep pentobarbital anesthesia
(80 mg/kg). Two different preparations of mixed aldehyde fixatives were
used. For study 1, the brains from two male rats had been fixed
in situ via intravascular perfusion with mixed aldehydes
containing 2.5% glutaraldehyde, 2% paraformaldehyde, 1 mM
CaCl2, and 2 mM
MgCl2, pH 7.4, at 40-45°C and 4 psi pressure under deep pentobarbital anesthesia (Harris and Stevens, 1989; Harris
et al., 1992). For studies 2 and 3, littermates of the four animals
used in the slice experiments were perfused through the heart with 5 ml
of saline, followed by the same mixed aldehyde solution, except
containing 6% glutaraldehyde to match the concentration used for the
slice experiments described below.
Hippocampal slices. Slices were prepared according to
standard procedures (Harris and Teyler, 1984; Jackson et al., 1993; Sorra and Harris, 1998). In study 1, slices were obtained after rapidly
killing the animals using a guillotine. For these slices experiments,
10 male rats were used. In studies 2 and 3, the animals were first
anesthetized with the same dosage of pentobarbital that was used for
the perfusion fixation, before guillotining, to control for any
potential artifacts of anesthesia in the perfusion-fixed tissue. In
study 1, two mature, and in study 3, two immature male rats were used.
Four to six 400 µm slices from each animal were cut at 70°
transverse to the long axis from the middle third of the hippocampus
with a tissue chopper (Stoelting Co., Wood Dale, IL) and placed into
ice-cold physiological saline containing 117 mM
NaCl, 5.3 mM KCl, 26 mM
NaHCO3, 1 mM
NaH2PO4, 2.5 mM
CaCl2, 1.3 mM MgSO4,
and 10 mM glucose, equilibrated with 95%
O2-5% CO2, pH 7.4. Slices were
transferred in this saline via the blunt end of a glass pipette
directly onto nets over wells with physiological saline, at the
interface of humidified 95% O2-5% CO2 at
32°C in a recording chamber (Stoelting Co.), and maintained
for varying times in vitro.
Physiological recordings were done to ensure slice viability (Harris
and Teyler, 1984; Jackson et al., 1993; Sorra and Harris, 1998). Two
concentric bipolar stimulating electrodes were positioned 600-800 µm
apart in the middle of stratum radiatum on either side of a single
extracellular recording electrode. Slices were judged healthy if the
stimulus-response curves were sigmoidal and the half-maximal responses
remained stable for at least 1 hr before fixation. At the end of each
experiment, the slices were fixed in mixed aldehydes containing 2%
paraformaldehyde, 6% glutaraldehyde, 1 mM
CaCl2, and 2 mM
MgCl2, pH 7.4, for 8 sec in the microwave oven,
which resulted in a measured final temperature that was always <50°C
and usually <37°C to prevent microtubule destruction that occurs
above 60°C (Jensen and Harris, 1989).
Light and electron microscopy. For study 1, perfusion-fixed
tissue and slices were hand processed by routine procedures as described previously (Harris and Stevens, 1989; Harris et al., 1992;
Sorra and Harris, 1998). For studies 2 and 3, the fixed tissue was
stored overnight in the fixative at room temperature, and then 400 µm
slices were cut from the perfusion-fixed hippocampus embedded in agar
and processed with the slices that had been maintained in
vitro. All tissue slices were rinsed five times in buffer with repeated agitation. Each slice was manually trimmed under a dissecting microscope to a region containing only area CA1. Slices were soaked briefly in 1% osmium and 1.5% potassium ferrocyanide in 100 mM cacodylate buffer while being cooled in an ice bath
until they reached a temperature <15°C and then microwaved for 2.5 min at 37°C using the Pelco 3450 Laboratory Microwave Processor (Ted Pella Inc., Redding, CA). After several buffer rinses, the slices were
put into 1% osmium in 100 mM cacodylate buffer, cooled,
and microwaved for 2.5 min at 37°C. Slices were then rinsed four to five times in buffer and twice in water, stained en
bloc with 1% aqueous uranyl acetate while cooled on ice,
and microwaved for 2.5 min at 37°C, followed by two brief water
rinses. Samples were dehydrated in an acetone series (50, 70, 90, and
100%) for 40 sec each in the microwave oven at 37°C. Infiltration
began with acetone and 1:1 Epon/Spurr's resins for 1 hr on a rotator at 25°C, followed by 2:1 acetone Epon/Spurr's resins overnight. After replacement with fresh 100% resin for several hours, samples were embedded in coffin molds with the dendrites orthogonal to the
cutting plane. Samples were cured for 48 hr at 60°C.
The blocks were trimmed to contain a region spanning the width of the
slices and in the middle of stratum radiatum midway between area CA3
and the subiculum. Then, several 1-µm-thick and 60-nm-thin test
sections were taken spanning the full width of the slices. Thick
sections were stained with 1% toluidine blue to guide subsequent
trimming. Thin sections were mounted on Pioloform-coated (SPI Supplies,
West Chester, PA) slot grids (Synaptek; Ted Pella Inc.) and counter
stained with saturated ethanolic uranyl acetate, followed by Reynolds
lead citrate, each for 5 min. Sections were examined with a JEOL
(Peabody, MA) 1200EX electron microscope to choose an area midway
between the air and net surfaces of the hippocampal slice for
subsequent serial thin sectioning. At an optimal depth between 100 and
200 µm from the cut surfaces, excellent tissue preservation was
found, as evidenced by well preserved dendrites, with intact
mitochondria, microtubules, and synapses, and the relative absence of
dark or swollen neuronal processes (see Figs. 2-5). A square diamond
trimming tool (Electron Microscopy Sciences, Fort Washington, PA) was
used to prepare a small trapezoidal area <100 µm on a side for study
1 and <50 µm on a side for studies 2 and 3. Serial thin sections
were cut on the Leica (Malvern, PA) Ultracut S ultramicrotome, mounted,
and counter stained as above for the test thins. Individual grids were
placed in grid cassettes (Advance Microscopy Techniques, Danvers, MA),
stored in numbered gelatin capsules (Electron Microscopy
Sciences), and mounted in a rotating stage to obtain uniform
orientation of the sections on adjacent grids. All studies were
photographed at 150-200 µm from the CA1 pyramidal cell body layer.
The series of sections were photographed at 4000-6000× magnification
for study 1 perfused, early, and set 1 of the late slices, and at
10,000× magnification for the second set of late slices in study 1 and
all conditions in studies 2 and 3. Calibration grids (Ernest Fullam
Inc., Latham, NY) were photographed with each series. In total, 35 EM
series were analyzed, ranging from 25 to 134 serial sections (Table
1).
Adjusted synapse density analysis. The adjusted
synapse density (ASD) was computed for the conditions of study 1, as
outlined in Table 1, using the following equation (for review, see
Harris, 1994; Sorra and Harris, 1998):
Synapse number (nsyn) was
computed by counting the number of postsynaptic densities (PSDs)
occurring within a sample area (SA) or on two of the four lines
defining the sample area. Synapses were counted if the PSD was evident
on the sample section and if the presynaptic vesicles occurred
on the sample section or on the adjacent section for obliquely cut
synapses. Because synapse density is markedly influenced by elements
occurring nonuniformly in the SA (i.e., myelinated axons, cell bodies,
and large dendrites with section profiles >0.94
µm2), the areas of these elements were measured
and subtracted from the sample areas to obtain the homogeneous neuropil
area (HNA). Approximately half of each sample field was analyzed for
synapses by two investigators.
PSDs have different shapes and sizes, and the probability of capturing
them on a single sample section differs in proportion to the number of
sections they occupy. Thus, the number of sections each synapse
occupied was counted, and the average number of sections (nsect) was computed for each synapse
type described in Results. The mean inverse of the
nsect per condition was used to adjust for any
differences in viewing probability, thereby removing potential size,
shape, or orientation biases.
Sampling is also affected differentially by section thickness. Every
effort was made to obtain uniform section thickness at the time of
cutting (platinum-colored sections in the diamond boat); however, the
same section colors are not necessarily the same section thickness
(Peachey, 1958). A better estimate of section thickness was obtained
for each series by measuring the diameters (d) of
longitudinally sectioned mitochondria or dendrites, counting the number
of serial sections they occupied (n), and computing section
thickness (st) as: st = d/n.
Dendrite analysis. Spine number per unit length of dendrite
was computed for lateral apical dendritic segments only (having a
diameter of <1 µm), because the primary apical dendrites were too
large and infrequent to obtain useful statistics (Table 1). Furthermore, the primary apical dendrites were far more spiny, and thus
could not be grouped with the lateral dendrites (data not shown).
Dendritic segment lengths equaled the number of sections they spanned
multiplied by section thickness for cross-sectioned dendrites. Segment
lengths were computed with the Pythagorean theorem by triangulation for
obliquely sectioned dendrites (Shepherd and Harris, 1998, their Figure 1).
Statistical analysis. Appropriate statistical tests were
used to evaluate the significance of differences between sample
populations using SigmaStat (Jandel, San Rafael, CA).
All conditions were analyzed blind as to experimental procedures and
were replicated by two or three people.
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RESULTS |
Physiological analysis
The physiological responses were monitored for a couple of hours
in slices at both ages, beginning 15-20 min after they were transferred to the recording chamber (Fig.
1). No physiological responses were
elicited in slices during the first 10-15 min, regardless of the
stimulus intensity delivered and regardless of whether there was
anesthesia before guillotining. During the next 30 min, there was a
dramatic recovery of the field EPSP (fEPSP), which was
stabilized by ~35 min in adult and by ~1 hr in young hippocampal
slices. This recovery phase is typical of all slice experiments, and
the data are presented only to emphasize the dramatic physiological
events taking place after slicing.
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Figure 1.
Recovery of the fEPSP after slice dissection.
Recording and stimulating electrodes were placed into the slice soon
after it was cut and transferred to the recording chamber (time = 0). A constant stimulus intensity of 100 µA delivered every 30 sec
was used to assess response recovery and stabilization over time. This
stimulation intensity was determined a priori to assess
slice recovery because field EPSPs were found to be half-maximal at or
approximately at a stimulus intensity of 65 µA. The responses are
plotted as the mean ± SEM of the percent recovery determined
relative to the plateau response. Data are averaged from three adults
(1 from study 1 and 2 from study 2; there was no effect of anesthesia
on response recovery) and five young animals. Response recovery is
delayed by ~10-15 min at P21 relative to adults.
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Tissue quality and synapse composition
Light microscopy revealed well preserved tissue in the CA1
pyramidal cell layer and in stratum radiatum (Fig.
2). Optimal tissue quality occurred in
the middle 200 µm of the slice (Fig. 3a), which was unchanged up to
13 hr in vitro, the longest time studied in these
experiments (Figs. 4,
5). If a particular slice had many dark,
degenerating processes, shrunken or highly vacuolated and swollen
dendrites, disrupted microtubules, and distended mitochondria, the
tissue quality was judged unsuitable for quantitative analysis (Fig.
3b). Excellent tissue preservation was a prerequisite for all samples in the perfusion and slice conditions of these experiments (Fig. 4). All synapse and spine types were detected in each
condition (Fig. 4) for up to 13 hr (Fig. 5), the latest time
evaluated. Dendritic spines were classified as stubby, mushroom,
thin, and branched as described in Harris et al. (1992) and Sorra et
al. (1998) (Figs. 3-5).
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Figure 2.
Toluidine blue-stained sections from area CA1 in
slices maintained in vitro. a, In study
1, the sections were cut parallel to the long axis of the apical
dendritic arbors of the CA1 pyramidal cells for the perfused, the
early, and the first set of late slices. b, For the 12 series photographed from the second two late slices in study 1 and for
all of the slices in studies 2 and 3, the sections were cut
perpendicularly to the apical dendritic arbor to obtain mostly
cross-sectioned dendrites, which are optimal for computing the spine
number per unit length of dendrite. The air and net surfaces of the
slices are labeled, and the double arrow indicates the
region of optimal tissue preservation in the middle 200 µm of the
slice. The trapezoids illustrate the approximate
location of subsequent serial thin sections. Scale bar:
a, b, 100 µm.
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Figure 3.
Neuropil from within the middle of stratum
radiatum of hippocampal slices. Both slices are photographed at
168-176 µm from the air surface of the slice. a, In
this adult hippocampal slice, the dendrites and axons are well
preserved, with distinct microtubules (arrows), clear
cytoplasm, nonswollen mitochondria (arrowheads), and a
uniform distribution of vesicles in presynaptic axonal boutons
(sv). Some examples of longitudinally sectioned mushroom
(m) and stubby (s) spines
are also evident. b, This slice was rejected because of
poor tissue quality, as evidenced by numerous shrunken and dark
processes (d), swollen mitochondria
(arrowheads), regions of unidentifiable whorls of
membrane (large arrow), and synaptic vesicular clumping
(svc) in presynaptic axonal boutons. There are some
dendrites of good quality interspersed among the degenerating
processes; however, it was often difficult to follow the spines past
darkened processes, and many of the spines emerging from apparently
healthy dendrites synapsed with dark degenerating axons. Scale bar:
a, b, 1 µm.
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Figure 4.
Representative neuropil in the middle of stratum
radiatum of area CA1 from each experimental condition. Perfused
conditions are on the left, and slice conditions are on
the right. a, Study 1, adult hippocampus
perfused with 2.5% glutaraldehyde. b, Study 1, adult
early slice. c, Study 2, adult hippocampus perfused with
6% glutaraldehyde. d, Study 2, adult late slice.
e, Study 3, P21 hippocampus perfused with 6%
glutaraldehyde. f, Study 3, P21 late slice. Sample
lateral dendrites are indicated by asterisks. In all
cases, the tissue was judged suitable for quantitative analysis of
synapses by the overall high quality of tissue preservation. Scale bar:
a-f, 0.5 µm.
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Figure 5.
Synapses in area CA1 from perfusion-fixed
hippocampus (left) and a hippocampal slice maintained
in vitro for 13 hr before fixation
(right). These particular examples were selected to
illustrate longitudinally sectioned spines, dendrite origins, and
presynaptic boutons. a, Perfusion-fixed thin spine
(t). b, In vitro
thin (t) and stubby (s)
spines. c, Perfusion-fixed mushroom spine.
d, In vitro mushroom spine.
e, Perfusion-fixed stubby spine on an MSB.
f, Another mushroom spine from an in
vitro slice. Scale bar: a-f, 0.5 µm.
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Quantitative analysis of synapses
The ASDs in the neuropil of perfusion-fixed hippocampus
were compared with slices fixed early or late after the onset of
incubation in vitro (Table 1, Study 1). The ASD of 213 ± 17 synapses/100 µm3 in the perfusion-fixed
hippocampus was significantly less than the ASDs of 322 ± 22 synapses/100 µm3 in the early slices and 338 ± 32 synapses/100 µm3 in the late slices
(p < 0.02) (Fig.
6). The ASDs from the early and the late
slices did not differ significantly, suggesting that synapse number is
maximally elevated by ~2 hr in vitro and then remains
stable for the duration (up to 13 hr).
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Figure 6.
ASD across experimental conditions of adult study
1 (mean ± SEM). The analysis includes 15 samples from 10 animals,
as outlined in Table 1. An overall ANOVA revealed significant
differences at p < 0.02, and the post
hoc Tukey test showed that both early and late slices had
higher densities than the perfused condition (*p < 0.05) and that no significant differences occurred among the early and
late slice conditions.
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The results from the ASD analyses were confirmed in the dendrite
analysis. The number of synapses per unit length of dendrite was also
found to be greater in the slices than in perfusion-fixed hippocampus
(Fig. 7). In adult study 1, dendritic
segments in the perfused hippocampus had 3.5 ± 0.2 spines/µm,
in contrast with 4.9 ± 0.2 spines/µm in the slices
(p < 0.001) (Fig. 7a). In adult
study 2, the dendritic segments in the perfused hippocampus had
3.5 ± 0.3 spines/µm, in contrast with 4.5 ± 0.2 spines/µm in the slices (p < 0.01) (Fig.
7b). The difference was even greater at postnatal day 21 (P21), wherein the dendrites in the perfused hippocampus had
only 2.2 ± 0.2 spines/µm, in contrast with 4.2 ± 0.5 spines/µm in the slices (p < 0.001) (Fig.
7c).
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Figure 7.
The number of spines per unit micrometer
length of dendrite was higher in slices than in perfused hippocampus
for all three studies. Data are presented as mean ± SEM;
*p < 0.01, study 1; *p < 0.001, studies 2 and 3. See Table 1 for the number of dendrites
analyzed in each study.
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Spine and synapse specificity
The increase in synapse number in slices was not uniform across
different types of dendritic spines. At all ages, thin spines predominated in both the perfused and slice conditions (Fig.
8), and in the adults, there was no
significant difference in thin spines between perfused and slice
conditions. There were more mushroom and stubby spines (Fig.
8a,b) and fewer asymmetric shaft synapses in the
adult slices (Fig. 8b). At P21, thin, mushroom, and stubby
spines occurred in approximately equal proportions in the perfused
brain; however, in the slices, there was a dramatic increase in both
the thin and mushroom spine categories (Fig. 8c). Branched
spines were rare at all ages, and there was a small significant
increase in the slices at P21 only (p < 0.05)
(Fig. 8c). Additionally, five filopodia were observed at P21
(data not shown).
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Figure 8.
The increase in synapse density affected only some
synapse populations. For the adult study 1, only thin, mushroom, and
stubby classes were distinguished. In studies 2 and 3, the rare
branched spines and two classes of shaft synapse (asymmetric,
presumably excitatory; and symmetric, presumably inhibitory) were
identified, as well. In all studies, spine synapses that were
incomplete within the series accounted for <10% of the total synapse
number. a, b, Thin spines predominated
and were comparable across the perfusion and slice conditions
(p = 0.43), whereas more mushroom
(*p < 0.005) and more stubby (*p < 0.005) spines occurred in slices from adult studies 1 and 2. In
addition, there was a small significant decrease in shaft synapses
(*p < 0.05). c, There were more thin
(*p < 0.001), mushroom (*p < 0.02), and
branched (*p < 0.03) spines in slices at P21. Data are
presented as mean ± SEM.
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In studies 2 and 3, synapses were characterized as having macular or
perforated PSDs. In the adults, only the macular PSDs increased
significantly in slices (Fig.
9a). In contrast, the P21
slices showed a large increase in macular and perforated PSDs (Fig. 9b).
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Figure 9.
a, Macular synapses were greater in
adult slices (*p = 0.05). b, P21
slices had more macular (*p < 0.001) and more
perforated (*p < 0.05) synapses. Data are
presented as mean ± SEM.
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In study 1, the presynaptic axons associated with every synapse were
followed through serial sections to identify whether there was only one
PSD [single-synapse bouton (SSB)] or more than one PSD
[multiple-synapse bouton (MSB)]. There were more MSBs in the slices
(p < 0.03) (Fig.
10).
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Figure 10.
Comparison of the relative incidence of SSBs and
MSBs. The incidence of SSBs was not statistically different between the
perfused and slice conditions (p = 0.18).
More MSBs occurred in slices (*p < 0.03). Data are
presented as mean ± SEM.
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DISCUSSION |
Hippocampal slices have 40-90% more dendritic spines, depending
on age, than comparable regions of hippocampus fixed by perfusion in vivo. The relative increase in dendrite spininess was
detected within 2 hr after slicing. Serial EM shows that all of the
dendritic spines have normal synapses with presynaptic and postsynaptic elements; however, not all spine types are affected uniformly. Stubby
and mushroom spines increase in the adult slices, and thin, mushroom,
and branched spines increase in the immature slices. MSBs increase
preferentially in the slices, suggesting that the new synapses formed
on preexisting axonal boutons.
Several potential artifacts can be ruled out as explanations for
these findings. Less than 5 min were needed to prepare the hippocampal slices, and perfusion fixation required 4-5 min of previous anesthesia and ~30 sec before the fixative cleared the brain. Previous studies have shown there are no changes in synapse number with brief periods of hypoxia-ischemia such as these (Hu et
al., 1998). Thus, the relative increase in spines is unlikely to result
from subtle differences in anoxia during slice preparation versus
perfusion fixation. In studies 2 and 3, anesthesia was introduced
before slice preparation to control for this potential variable
relative to perfusion-fixed brain, and still there were more spines in
the slices. Thus, the relative increase in spine number in slices was
not, instead, an artifactual loss of spines during anesthesia in the
perfusion-fixed brains. All of the slices had a central core of well
preserved neuropil (Reid et al., 1988; Jensen and Harris, 1989). This
excellent tissue quality, as well as the elevated spine densities, were
maintained for at least 13 hr in vitro, the longest time studied.
Another question is whether the slices experienced more shrinkage than
in perfusion-fixed brain. If so, this would artificially produce a
higher spine density in the slices. Several observations rule against
this potential artifact. First, there is a spine and synapse
specificity to the effect. If there were a generalized shrinkage in the
slices, with no other change, then all types of synapses should be
affected uniformly. Wenzel et al. (1994) also found a specific
effect of slicing in adult hippocampus, wherein somatic spines were
found on dentate granule cells from adult slices, which do not normally
occur in hippocampus perfusion-fixed in vivo. Second, the
dendrite diameters were uniform across conditions, showing that no
generalized shrinkage of individual processes occurred (data not
shown). Jensen and Harris (1989) also found a constancy in
mitochondrial diameters across the slice and perfused conditions. Other
studies have found that the extracellular space (ECS) should occupy
~20% of the brain volume (Nicholson and Sykova, 1998); however, some
of the ECS is lost during processing of perfusion-fixed tissue, thereby
accounting for a net 5-15% overall tissue shrinkage (Hillman and
Deutsch, 1978; Cragg, 1980; Schuz and Palm, 1989). Similarly, EMs from
the hippocampal slices show very little extracellular space. The living
slices were originally cut at ~400 µm thickness, as determined by
the Vernier scale on the tissue chopper, but were ~350 µm thick
after processing (Fig. 2b), accounting for a 12.5%
shrinkage that is comparable to perfusion-fixed tissue.
The ASD analysis was used to obtain an unbiased estimate of synapse
densities in the slices and perfused hippocampus. This analysis could
be influenced by other factors, such as subtle changes in the density
of small glial processes or dendrite diameter, which could alter the
relative density of synapses. This potential artifact was minimized by
computing the HNA. In addition, the dendrite analysis of spine number
per unit length is not sensitive to these potential volume artifacts.
Because both approaches revealed the same results, it strengthens the
findings from all of the ASD analyses.
The increase in MSBs in slices is consistent with other studies showing
that when spines form quickly they make new synapses with preexisting
axonal boutons (Woolley et al., 1996). It will also be
interesting to learn whether there is a significant portion of
nonsynaptic boutons in perfusion-fixed brain that could be the
source of the nonsignificant trend toward an increase also in
the SSBs.
The increase in stubby spines in the adult slice preparations
recapitulates processes that occur during hippocampal development (Harris and Stevens, 1989; Harris et al., 1992; Fiala et al., 1998).
During the first postnatal week, hippocampal synapses are recruited to
dendritic shafts via filopodia. Then, shaft synapses give rise to
stubby spines, which predominate for the next few weeks. As the animals
mature, the shaft and stubby spine synapses are markedly reduced while
thin and mushroom spines are formed, and eventually the thin spines
become the dominant spine shape. Curiously, in slices from P21, the
thin and mushroom spines reach numbers comparable with those found in
the mature hippocampus in vivo and in vitro.
These findings emphasize that spine induction is more profound in the
developing slices than in mature slices, possibly accelerating an
ongoing developmental process.
Together, these arguments support the conclusion that new spines are
formed in hippocampal slices. One possible mechanism is that spines are
induced by excessive synaptic activation when glutamate and potassium
ions are released from the cut processes. This possibility seems
unlikely because recent findings show that many more spines are induced
when synaptic transmission is completely blocked in slices (Kirov and
Harris, 1998). These findings are consistent with observations from
developing cortex, which show that blocking synaptic transmission
results in spinier developing dendrites (Dalva et al., 1994; Rocha and
Sur, 1995). Conversely, excessive activation caused by elevated NMDA
results in spine loss (Segal, 1995; Halpain et al., 1998). The spine
induction observed in our experiments occurs within the first 2 hr,
pointing to the loss of spontaneous synaptic activity when slices are
removed from the rest of the brain and the "synaptically silent"
recovery period as the candidate mechanisms for induction of new spines in slices.
There are several ways in which the new spines could form. One
possibility that has been widely suggested, without convincing morphological evidence, is that new spines form from the splitting of
previously existing spine synapses. This process is unlikely because
branched or "splitting" spine heads never share the same presynaptic axon in hippocampal area CA1 in vivo or in
slices (Sorra et al., 1998). Another possibility is that spines form in
response to sprouting axons that are replacing the cut and degenerating
processes. This possibility also seems unlikely because degeneration,
removal, and sprouting of new axons requires several days, and even
then synapse number never reaches the normal predeafferentation level
(Wheal et al., 1998). Rapid spine formation in the slices might
recapitulate development, namely that after quieting of the neuron,
filopodia are induced that guide axons to the dendritic shafts, and
spines arise from those shaft synapses. If this is the process, then we
would expect to see an increase in shaft synapses and filopodia at an
earlier time point (such as within 30 min after preparing the slices).
Some evidence for this process was detected in the P21 slices in which
a few filopodia were encountered, even at 9-10 hr after preparing the
slices, whereas none were detected in the perfused hippocampus at this
age. Alternatively, spine formation in slices may start to recapitulate
development in that protrusions are extended, but because the compact
neuropil is full of axonal boutons at both P21 and in adult slices, the protrusions immediately encounter good candidates with which to form
synapses. This contrasts with early development (P1-P6), wherein
filopodia traverse a much looser neuropil that has very few axonal
varicosities containing vesicles (Fiala et al., 1998). Because many of
the more mature axons already have other synapses on them (i.e., the
MSBs) demonstrating their suitability for making synapses, the
protrusions develop immediately into dendritic spines without the
intermediate stage involving retraction to the dendritic shaft. Further
experiments will be needed to assess whether there are intermediate
stages in spine formation during the 1 hr recovery period.
In summary, dendritic spines are remarkably plastic in both adult and
immature hippocampus as demonstrated here by their profound induction
in hippocampal slices. The most likely explanation for this process is
that more spines form because the slice is relatively quiet in terms of
overall synaptic activity. Once the initial spine induction has
occurred during the recovery period, spine and synapse numbers
stabilize at the elevated level when synaptic activity resumes in
slices maintained for longer periods in vitro.
| |
FOOTNOTES |
Received Nov. 24, 1998; revised Jan. 26, 1999; accepted Jan. 28, 1999.
This work was supported by National Institutes of Health Grants NS21184
and NS33574, Mental Retardation Research Center Grant P30-HD18655 (K.M.H.), Program in Neuroscience and Division of Medical
Sciences, Harvard University, and the Natural Sciences and Engineering
Research Council of Canada (K.E.S.). We thank Dr. John Fiala for
thoughtful discussions and Drs. John Davis and John Fiala for
assistance with the reconstruction system and image analysis in the
Image Graphics Laboratory at Children's Hospital. We thank Marcia
Feinberg for assistance on the serial electron microscopy and Karen
Szumowski for assistance with the figures.
All three authors contributed equally to this work.
Correspondence should be addressed to Dr. Kristen M. Harris, Division
of Neuroscience, Enders 260, Children's Hospital, 300 Longwood Avenue,
Boston, MA 02115.
| |
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The Establishment of GABAergic and Glutamatergic Synapses on CA1 Pyramidal Neurons is Sequential and Correlates with the Development of the Apical Dendrite
J. Neurosci.,
December 1, 1999;
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[Abstract]
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A. Dunaevsky, A. Tashiro, A. Majewska, C. Mason, and R. Yuste
Developmental regulation of spine motility in the mammalian central nervous system
PNAS,
November 9, 1999;
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[Abstract]
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H. L. Atwood and J. M. Wojtowicz
Silent Synapses in Neural Plasticity: Current Evidence
Learn. Mem.,
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542 - 571.
[Abstract]
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K. M. Harris
Calcium from internal stores modifies dendritic spine shape
PNAS,
October 26, 1999;
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[Full Text]
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S. Oleskevich
Cholinergic Synaptic Transmission in Insect Mushroom Bodies In Vitro
J Neurophysiol,
August 1, 1999;
82(2):
1091 - 1096.
[Abstract]
[Full Text]
[PDF]
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G. M. G. Shepherd, M. Raastad, and P. Andersen
General and variable features of varicosity spacing along unmyelinated axons in the hippocampus and cerebellum
PNAS,
April 30, 2002;
99(9):
6340 - 6345.
[Abstract]
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[PDF]
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TOP
ABSTRACT
INTRODUCTION
