Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California, 94305-5345
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ABSTRACT |
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Pavlidis, Paul and Daniel V. Madison. Synaptic transmission in pair recordings from CA3 pyramidal cells in organotypic culture. We performed simultaneous whole cell recordings from pairs of monosynaptically coupled hippocampal CA3 pyramidal neurons in organotypic slices. Stimulation of an action potential in a presynaptic cell resulted in an AMPA-receptor-mediated excitatory postsynaptic current (EPSC) in the postsynaptic cell that averaged ~34 pA. The average size of EPSCs varied in amplitude over a 20-fold range across different pairs. Both paired-pulse facilitation and depression were observed in the synaptic current in response to two presynaptic action potentials delivered 50 ms apart, but the average usually was dominated by depression. In addition, the amplitude of the second EPSC depended on the amplitude of the first EPSC, indicating competition between successive events for a common resource that is not restored within the 50-ms interpulse interval. Variation in the synaptic strength among pairs could arise from a variety of sources. Our data from anatomic reconstruction, 1/CV2 analysis, paired-pulse analysis, and manipulations of calcium/magnesium ratio suggest that differences in quantal size and release probability do not appear to vary sufficiently to fully account for the observed differences in amplitude. Thus it seems most likely that the variability in EPSC amplitude between pairs arises primarily from differences in the number of functional synapses. Injections of the calcium chelator bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid into the presynaptic neuron resulted in a rapid and nearly complete block of transmission, whereas injection of the slower-acting chelator EGTA resulted in a variable and partial block. In addition to demonstrating the feasibility of manipulating the intracellular presynaptic environment by injection into the presynaptic soma, these data, and the EGTA results in particular may suggest variability in the linkage between calcium entry sites an release sites in these synapses.
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INTRODUCTION |
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The analysis of synaptic transmission in the mammalian CNS increasingly has turned to the use of techniques designed to monitor transmission between single pairs of neurons. These include minimal stimulation, where a small number (ideally only one) of presynaptic fibers are stimulated extracellularly, and paired recording, where intracellular recording from two synaptically coupled cells is performed.
Minimal stimulation, although relatively rapid and simple to perform,
suffers from a number of drawbacks. The primary concern is that one
rarely can be certain that a single cell is being reliably stimulated.
Pair recordings, where simultaneous intracellular recordings are made
from only two synaptically connected neurons, do not suffer from this
problem and also permit direct electrophysiological characterization
and pharmacological manipulation of the presynaptic cell (Miles
and Poncer 1996). The major difficulty with pair recordings is
that incidence of synaptic connection between any two cells is often
low, and thus connected pairs can be difficult to obtain (Malinow 1991
). One way around this is to use primary
dissociated culture systems where connectivity is much higher
(Bekkers and Stevens 1990
). Of course, with dissociated
cultured preparations come questions as to the identity of the recorded
cells and whether the synaptic and connective properties are similar
enough to those of mature synapses in brain for useful comparisons to
be made.
The use of organotypic brain slice cultures partially has ameliorated
such concerns because identification of cell types is much easier than
in dissociated culture and the cells maintain a morphology and
connectivity similar to that in native brain tissue (Gahwiler et
al. 1997). Previous work on roller-tube cultures of hippocampal
slices has supported the idea that organotypic cultures have properties
closer to acute slices than dissociated cultures (Gahwiler et
al. 1997
). Another useful feature of organotypic cultures is
that they express long-term potentiation (Stoppini et al.
1991
) and other forms of synaptic plasticity including paired-pulse facilitation and depression (Debanne et al.
1996a
) and thus are useful in studying these effects in pairs
of neurons (Debanne et al. 1996b
).
Here we describe some of the properties of synaptic transmission in
pair recordings performed in synaptically connected CA3 pyramidal cells
in organotypic slices maintained in interface-type culture
(Stoppini et al. 1991). Overall we find that the
properties of these recordings compare well with those performed in
acute slices (Miles and Wong 1986
) as well as roller
cultures (Debanne et al. 1995
). In addition, we
demonstrate the feasibility of pharmacologically manipulating the
presynaptic cell by including exogenous calcium buffers in the
presynaptic recording electrode. Some of these results have been
presented in abstract form (Pavlidis and Madison 1997
).
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METHODS |
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Tissue culture
Interface cultures of hippocampal slices were prepared as
described (Stoppini et al. 1991). We used 7- to
10-day-old Sprague-Dawley rats. Cultures were maintained at 37°C for
3 days and then kept at 34°C for the remaining culture period.
Cultures were used after 7-14 days in culture. Healthy cultures
selected for recording usually had a well-defined, raised border and a
relatively clearly defined stratum pyramidale. Cultures with dark
(apparently necrotic) material present in the CA3 region or a
vacuolated ("cratered") appearance or that had extensively
flattened borders were rejected. On the basis of these criteria,
approximately one-half to two-thirds of our cultures from any given
preparation typically were judged to be sufficiently healthy for recording.
Electrophysiology
Individual slice cultures were transferred to a recording chamber perfused at 2-3 ml/min with artificial cerebrospinal fluid (ACSF) with the following composition (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose, pH 7.4, saturated with 95% O2-5% CO2. ACSF reagents were of molecular biology grade (Fluka). All experiments were performed at room temperature (21-23°C). 6-nitro-7-sulphamoylbenzo[ f ]quinoxaline-2,3-dione (NBQX), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), TTX, and picrotoxin were from RBI; all other reagents were from Sigma.
Whole cell recordings from CA3 pyramidal cells were made blindly
(Blanton et al. 1989) or using an infrared-DIC
microscope (Dodt and Zieglgansberger 1990
). Recordings
of excitatory postsynaptic current and potentials (EPSC and EPSPs) were
made using an Axopatch 1C or Axoclamp 2A (Axon Instruments, Foster
City, CA). Presynaptic current-clamp records were made with an Axoclamp
2A. Pre- and postsynaptic events were sampled at 10 kHz and low-pass
filtered at 1-2 kHz. Series and input resistances of voltage-clamp
recordings were monitored throughout experiments and did not vary by
>20% over the course of the recording within experiments included in the data set.
In many experiments, we used the perforated-patch technique for the
postsynaptic recording. Amphotericin (Fluka) was prepared at a
concentration of 200-300 µg/ml by dilution of a 60 mg/ml DMSO stock
(prepared at least weekly) into a solution of (in mM) 55 Cs
methansulfonate, 75 Cs2SO4, 10 HEPES, and 8 MgCl2 (pH 7.2 with CsOH). This solution was sonicated
briefly to disperse the amphotericin and was usable for 1-2 h after
preparation. The same solution without amphotericin was used to fill
the tips of electrodes (2-5 M), whereas the amphotericin solution
was used for backfilling. Series resistances stabilized in 10-60 min
between 15 and 40 M
. In experiments using broken patch whole cell
mode, series resistance varied from 10 to 25 M
.
To establish a pair recording, a second whole cell recording was obtained in an adjacent area of the CA3 cell body layer (typically ~100-300 µm separation between cells in blind recordings, 10-100 µm in visualized recordings). The presynaptic electrode solution composition was (in mM) 120 K gluconate, 40 HEPES, 5 MgCl2, 2 NaATP, and 0.3 NaGTP (pH 7.2 with KOH; in some experiments, we used MeSO4 as the major cation). When EGTA or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) was included in the pipettes, the K gluconate concentration was lowered slightly so as to maintain osmolarity at 290 mOsm. This solution also was used for postsynaptic recordings in some experiments. Presynaptic cells were held in current clamp and induced to fire single action potentials by brief injection of depolarizing current (typically 20-50 pA for 20 ms). When a successful pair was obtained (i.e., a monosynaptic EPSC was evoked by a presynaptic action potential), the presynaptic cell was stimulated by current injection at 0.03-0.1 Hz throughout the experiment.
Data analysis
On- and off-line data analysis was performed using custom
software developed in our laboratory in the Labview programming environment (National Instruments). Because the exact time of action
potential occurrence during the depolarization of the presynaptic cell
could vary slightly from trial to trial, analysis windows used for the
postsynaptic EPSC were locked to the time of occurrence of the peak of
the action potential. Sweeps in which no action potential occurred or
in which the postsynaptic recording was distorted by spontaneous
synaptic activity were excluded from analysis. In some experiments,
polysynaptic events obscured the peak of the event in many sweeps, so
in these cases the initial slope of the event was analyzed rather than
the amplitude. Spontaneous synaptic events (mEPSCs) were detected
automatically and measured as described (Ankri et al.
1994).
Histology
In some experiments, neurobiotin (Vector Laboratories) was included in the recording electrodes (0.5%) to allow anatomic reconstruction of the cells. Cells were filled for 15-60 min before the electrodes were withdrawn gently after which the culture was usually left in the recording chamber for an additional 15-60 min. A sketch of the location of the cells within the slice was made to allow later identification of the pre- and postsynaptic cells. The cultures were fixed overnight at 4°C in 1% glutaraldehyde/1% paraformaldehyde in phosphate-buffered saline (PBS). The cultures then were washed in PBS, teased away from the support membrane, then permeabilized by freeze-thaw on dry ice or liquid nitrogen and stained using the ABC Elite Kit (Vector Labs) with nickel enhancement. Stained cultures were whole-mounted in Permount. Selected well-filled pairs were traced using a Neurolucida system (Microbrightfield). Tracing was carried out with ×63 and ×100 oil-immersion objectives (Zeiss), and identification of potential contact sites was performed at ×100 and with the condenser diaphragm fully open to give the narrowest plane of focus. The photographs taken to illustrate the contacts in Fig. 4 were not taken on the same microscope used for tracing [a ×63 water-immersion objective was used (1.2 NA), and images were captured digitally using the transmitted light detector of a BioRad confocal microscope]. Images were adjusted for contrast and composed using Corel PhotoPaint.
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RESULTS |
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Recording from monosynaptically coupled pairs in CA3
We established simultaneous whole cell recordings from pairs of CA3 pyramidal cells (Fig. 1A). Each pair was tested for connection by stimulating one cell, designated as presynaptic, to fire an action potential by passing a depolarizing current pulse via the recording electrode. The postsynaptic current trace then was examined for the presence of synaptic currents occurring at short (typically <3 ms), constant latencies after the peak of the action potential. Although it was not unusual for the first potential presynaptic cell tested to be coupled synaptically to the postsynaptic cell, in most experiments several potential presynaptic cells were tested before a connection was obtained. Overall, approximately one-third of potential presynaptic CA3 cells were found to be monosynaptically coupled to the postsynaptic CA3 cell. The success rate was higher when using visualized recordings from cells that were <100 µm apart, although there was a great deal of variability in the success rate even when cells were directly adjacent to each other. Data from >150 pairs are presented in this paper.
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When the presynaptic cell was a pyramidal neuron (almost all of our
recordings), synaptic responses were blocked completely by bath
application of AMPA receptor antagonists (NBQX or CNQX, 10 µM) at a
holding potential of 65 mV (Fig. 1B). Long depolarizing pulses delivered to the presynaptic cell resulted in a train of action
potentials, and postsynaptic responses during the train showed rapidly
developing depression of transmission that was often apparent after the
first action potential. This depression was characterized both by
smaller EPSCs as well as failures of transmission (Fig. 1C).
We saw no evidence of electrotonic coupling in our pairs, as
was reported in acute slices (MacVicar and Dudek 1981).
Action potentials generated by one cell never were observed to produce nonsynaptic currents or depolarizations in the other cell. We also
tested this explicitly in five pairs by delivering large hyperpolarizing pulses to the presynaptic cell. This never resulted in
any current passing to the postsynaptic cell (Fig. 1D).
Some pairs exhibited polysynaptic connections with no apparent
monosynaptic connection. These generally had longer (>5 ms) and more
variable latencies to the first synaptic potential. Such pairs were
rejected from analysis. Typically, these polysynaptic synaptic
potentials were apparently inhibitory because they had reversal
potential of around 55 mV. Such polysynaptic inhibitory currents were
also common in pair recordings where there was also a monosynaptic
excitatory component; being present in about half of experiments (Fig.
2A). As expected these
polysynaptic potentials were blocked by CNQX because blocking
glutamatergic synapses removes excitatory links to intervening
inhibitory neurons (not shown).
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Polysynaptic inhibitory events were probably mediated by GABAA receptors, but we could not pharmacologically block GABAergic inhibition in our experiments due to the disruptive hyperactivity this produced. These polysynaptic inhibitory events always occurred later than monosynaptic excitatory synaptic potentials and generally did not prevent measurement of the early excitatory event. Thus pairs having a monosynaptic response were included in the data set whether or not polysynaptic events were also observed. However, in some experiments the presence of polysynaptic inhibitory events made the analysis of paired-pulse responses difficult or impossible because polysynaptic activity induced by the first action potential affected measurement of monosynaptic responses induced by the second action potential. Such pairs were excluded from this analysis. Pairs exhibiting polysynaptic excitatory connections were much less common and also were excluded from analysis.
In a few experiments, monosynaptic inhibitory events were observed. This can be attributed to the presynaptic cell being an inhibitory interneuron rather than a pyramidal cell. Eight such recordings were obtained in the course of our studies. The identity of the presynaptic cell as an inhibitory cell always was corroborated by differences in the electrical properties of the cells as compared with pyramidal cells. Specifically, putative interneurons had shorter action potentials [64.6 ± 10.1 (SD) mV vs. 85.4 ± 4.8 mV for pyramidal cells, P < 0.0001], briefer action potentials (2.4 ± 0.47 ms vs. 3.6 ± 0.4 ms, P < 0.001), a larger fast afterhyperpolarization (10.8 ± 2.5 mV vs. 5.8 ± 1.7 mV, P < 0.001), and fired at a higher rate without accommodation in response to long depolarizations (Fig. 2B). Such pairs were not studied in detail and are not included in the analyses described in the following text. In many experiments, the postsynaptic cell was cesium-loaded in perforated-patch mode, which precluded electrophysiological confirmation of the identity of the postsynaptic cell as a pyramidal cell based on its electrical properties. However, based on the observed frequency of presynaptic interneuron recordings, from the same population of neurons, we estimate that the accidental inclusion of a postsynaptic interneuron would have occurred only a few times in the course of our many experiments. This would be minimized further by our visualized recording technique, which permits more accurate identification of cell types. In addition, analysis of neurobiotin-labeled pairs confirmed the identity of both cells as pyramidal cells in all cases tested (see following text).
Properties of pair responses
One of the striking properties of excitatory synaptic transmission between single pairs of connected neurons was the great variability in size of the synaptic potential observed from pair to pair. In some synaptically coupled pairs of pyramidal cells, average responses were extremely small, <10 pA, and failures of transmission often were observed. In others, the responses were as large as 200 pA with few if any failures. In a representative subset of 136 pairs, the average response was 34 pA; the median response was 21 pA (Fig. 3A). We also examined transmission in a few pairs while holding the postsynaptic cell in current clamp to determine size of the depolarization generated by these events. The range of average EPSP amplitudes induced by a presynaptic action potential in eight experiments varied from ~200 to 1,000 µV, and the average size was 450 µV.
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Paired-pulse characteristics of pair responses
We have examined the postsynaptic responses in pairs of pyramidal cells in response to two presynaptic action potentials, delivered 50 ms apart. In general the synaptic response to the second presynaptic action potential (EPSC2) could be larger or smaller than the response to the first (EPSC1), and this varied from trial to trial in a given pair (Fig. 3C). When the average paired-pulse ratio (EPSC2/EPSC1; PPR) was calculated for each pair, most pairs were found to exhibit paired-pulse depression (PPR < 1; the average EPSC2 was smaller than the average EPSC1, within a given pair recording). The average PPR varied greatly from pair to pair, from high levels of facilitation (2.5-fold) to depression (0.2-fold). The average PPR across 42 pairs was 0.88 ± 0.35 (Fig. 3B). However, there was no significant relationship between the average EPSC size in a pair and the average PPR (P > 0.1; Fig. 3B). As expected for a presynaptic effect, the inverse of the coefficient of variation squared (1/CV2) for the second pulse compared with the first pulse was correlated with the degree of depression or facilitation observed (correlation coefficient 0.79; P < 0.001; measured for 25 experiments; not shown). Specifically, in pairs showing strong depression, the ratio of CV2(pulse 1)/CV2(pulse 2) was relatively low.
When the trial-to-trial variability in PPR is examined within a single pair, there is a strong relationship between the amplitude of the first EPSC and the PPR for that trial. For trials with a small EPSC1, the PPR is typically larger, and PPR is smaller for trials with large first EPSCs (Fig. 3C). To analyze these data, the trials within each pair were ranked by EPSC1 amplitude and then divided into two groups, those with EPSC1s larger than average for that pair and those with EPSC1s smaller than that average. The mean EPSC2 was taken for each group and compared the mean EPSC2 for the experiment. If EPSC2 was independent of EPSC1 and could vary over the same range of amplitudes, the mean EPSC2 should be the same whether or not EPSC1 was large or small. In contrast, there was often a significant deviation from this expectation. Data from 50 pairs are plotted in Fig. 3D to represent the relationship across all of those paired recordings. The left-hand curve displays the cumulative probability of the deviation from the mean of EPSC2 where the EPSC1 was larger than average. The right-hand curve is the analogous data for trials when the EPSC1 was smaller than average. As can be seen, there is a tendency for trials with large EPSC1s to have small EPSC2s and vice versa (Fig. 3D). On average, across 50 experiments, EPSC2 was 6.8 ± 10% smaller than the mean EPSC2 when EPSC1 was larger than the mean EPSC1 and 6.8 ± 10% larger than the mean EPSC2 when EPSC1 was smaller than the mean EPSC1 (P < 0.01, Kolmogorov-Smirnov test).
Estimating the number of active synapses in a pair
We observed a great deal of variability in the size of responses from pair to pair, with a range of ~20-fold between the "weakest" pair and the "strongest" pair. Such variability could arise from a number of sources: variation in the number of active synapses between different pairs, variation in the probability of release between pairs, variation in quantal size between pairs, and variation in the dendritic location of synapses. Each of these could, in theory, account for the variation seen in the amplitude of EPSCs between pairs either alone or in combination.
To examine the possibility that there is a correlation between the size of the average EPSC in a pair and the number of synapses made between the two neurons, we have reconstructed the axonal arbor of the presynaptic cell and the dendritic arbor of the postsynaptic cell in two pairs, one with a very small response, and one with a larger response (Fig. 4). Analysis of these, and 10 other pairs not traced, reveals that the CA3 cells had mature morphologies with obvious basal and apical dendrites. Numerous synaptic spines covered the dendrites. What appeared to be thorny execrences were observed on many cells, extending from the region around the cell body. The cells had axonal projections much as would be expected in acute tissue, although perhaps more highly elaborated. The axon emerges from a single point on the basal side of the cell body or from the initial segment of a main basal dendrite and soon branches, sending projections to CA1 (Schaffer collaterals; not shown) as well as within CA3 (Fig. 4). Boutons were observed along the axons. The associative projections extended throughout both stratum oriens and s. lucidum/radiatum. Naturally, there was no extrahippocampal projection, but axons were observed extending nearly to the edge of the tissue. Thus the overall morphology is quite similar to that of CA3 cells in native tissue.
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Close examination of the two reconstructed preparations reveal sites of
close contact between the presynaptic axon and postsynaptic dendrites.
At the light level, we cannot positively confirm that these are
synaptic sites, though groups that have done analysis of similar
preparations on the EM level have found a close correspondence between
contact sites identified at the light level and synaptic sites
(Gulyas et al. 1993; Markram et al.
1997
). The number of contact points does not represent an upper
limit for the number of synapses because some contacts could contain
multiple active zones (Sorra and Harris 1993
).
By comparing the number of potential synaptic sites in different pairs, the correlation between this and size of the synapse can be examined. One of the pairs we reconstructed had a very small EPSC, <10 pA on average, with failures of transmission in many sweeps. The other gave much larger responses, 29 pA on average. We predicted that "weak" pair would have very few contacts, perhaps only one, whereas the "strong" pair would have more contacts. Surprisingly, the pair with the small response had 19 contact sites. The pair with the larger response had 14 contact sites. Thus there is no obvious relationship between number of contacts and the EPSC amplitudes observed, and the number of contacts sites for the weak pair was much larger than we predicted based on the electrophysiology. The contacts were distributed over both basal and apical dendrites. In the weak pair, 6 contacts were observed on apical dendrites, and 13 were on basal dendrites. In the strong pair, 3 contacts were on basal dendrites and 11 on apical dendrites.
Despite the relatively large number of potential contacts identified in the reconstructions, our electrophysiological data suggest that there are relatively few functional synaptic contacts in most pairs. Spontaneous miniature EPSCs (mEPSCs), recorded in the presence of 1 µM TTX and 100 µM picrotoxin had an average amplitude of 6.3 ± 3 pA, and ranged in amplitude from <3 up to 80 pA (n = 3 cells; Fig. 5). We note that the mEPSCs we recorded would include those originating from mossy fiber terminals because the granule cells are present in these cultures. It may be that some larger events originate from these synapses, which are close to the cell body. Still, even if the smaller mEPSCs (~5-10 pA) are taken to represent a typical associational synapse, the majority of our pairs, with mean EPSCs <30 pA, would consist of fewer than five active synapses.
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Because the estimate of contact sites does not appear to correlate with
response size it is possible that the wide range in amplitudes
represents connections with roughly constant numbers of synapses, but
with widely ranging release probabilities or differences in quantal
size between pairs. One measure of the relationship between EPSC
amplitude and the underlying properties of the quantal synaptic
responses is the coefficient of variation (CV) of the EPSC amplitude
(Faber and Korn 1991). We found a significant positive
correlation between 1/CV2 and the mean EPSC amplitude (Fig.
6; correlation coefficient 0.64;
P < 0.01; n = 27). Pairs with small
mean EPSCs tended to have small 1/CV2 values while pairs
with large EPSCs had larger values. Although interpretation of such
data is not straightforward (Faber and Korn 1991
), it is
consistent with the idea that the release probability and/or the number
of release sites is higher for pairs with larger responses.
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If it is true that differences in probability of release are a
major component of the differences in 1/CV2, then it should
be possible to increase the amplitude of the response of a weak pair to
the vicinity of a strong pair by increasing release probability. We
tested this possibility by raising calcium and lowering magnesium in
the ACSF to increase the probability of release (Fig.
7). In seven of nine pairs, changing the
calcium/magnesium ratio in the ACSF from 2.5 mM Ca/1.3 mM Mg to 3.5 mM
Ca/0.7 mM Mg or 5 mM Ca/1 mM Mg did not result in an increase in the
maximal EPSC amplitude observed. In other words, there was a
"ceiling" that could not be passed simply by increasing calcium
(Fig. 7C). In two pairs there was a small increase in the
maximal response size after calcium elevation but only to a maximum of
120% of control levels. On average, the maximal EPSC in high calcium
was 105 ± 10% of that in control conditions. This lack of change
was not due to a lack of effect of raising calcium, as there were clear
effects of increasing calcium on transmission. In high calcium, the
average EPSC was 146 ± 45% of that in control conditions, and
the PPR decreased by 25 ± 13% of the control values. Furthermore there was a large increase in 1/CV2 (375 ± 349%;
range, 114-1,200%), when calcium was raised, as expected if the
probability of release had been increased. We also noted that in high
calcium, the positive relationship between EPSC amplitude and
1/CV2 was maintained (correlation coefficient 0.76;
P < 0.05; Fig. 6B). The corresponding data
for these experiments under control calcium conditions were included in
Fig. 6A and also were correlated significantly with mean
EPSC amplitude when considered independently (0.87; P < 0.05). In two pairs, we tested the effects of lowering calcium/magnesium to 1.3 mM/2.5 mM (Fig. 7A). In both
experiments, there was a large decrease in the mean EPSC (67% average)
and an increase in the PPR of 21% on average.
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Pharmacological manipulation of the presynaptic cell
One of the advantages of using pair recordings is that the cytoplasm of the presynaptic cell is directly accessible to experimental manipulation by including exogenous compounds in the presynaptic recording electrode. We observed transmission between pairs of connected cells was quite stable over long periods of time (upward of 3-4 h) when using a standard whole cell recording. Thus synaptic transmission apparently is unaffected by prolonged dialysis of the presynaptic cytoplasm under our experimental conditions (Fig. 8A; n = 34). Nonetheless we have found it possible to introduce substances into the presynaptic cell and have those substances reach the axon terminals at effective concentration. As an initial demonstration of our ability to manipulate the presynaptic cell, we included high concentrations of calcium chelators in the presynaptic electrode. We selected BAPTA and EGTA, two calcium chelators whose effects on transmission have been characterized in other studies, in hippocampal synapses (by bath application of membrane-permeant analogues of these compounds) as well as at other synapses.
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When the presynaptic electrode contained BAPTA (10 mM; potassium salt), transmission declined rapidly (Fig. 8B). Typically there was a period of several minutes during which transmission was stable, followed by a period of decline, which proceeded until transmission nearly was blocked. During this time, there was no significant differences in presynaptic action potential height and width between control and injected pairs over the course of the experiments [height ratio (mVt=20 min/mVt=0 min) for control: 0.95 ± 0.17 (mean ± SD; n = 15); BAPTA: 0.94 ± 0.18 (n = 10); difference not significant. Width ratio (mst20/mst0; measured at the base) control: 0.96 ± 0.23; BAPTA: 1.09 ± 0.12; difference not significant].
Maximal block was characterized predominantly by failure of
transmission in most trials although occasional small EPSCs continued to appear (Fig. 8B). The time course of BAPTA block was
variable with half-maximal block being observed within minutes in some cases and taking as long as 30 min in other experiments. On average, the block reached a half-maximal level after 15 min. A similar extent
of block was observed in all experiments (n = 16).
There was some indication that block was more rapid when the recording locations were close together (<100 µm, using visualized recording), but we have not systematically investigated this. In a previous study
on the effects of presynaptic BAPTA injection on transmission at the
squid giant synapse, it was calculated that 1 mM BAPTA resulted in 50%
block of transmission (Adler et al. 1991). To get a
better idea of the sensitivity of the release mechanisms in our
preparation, we tested the effects of a lower BAPTA concentration, 1 mM
(n = 5). We found that the effects of 1 mM BAPTA were
quite similar to 10 mM with a progressive block of transmission
observed soon after break-in. Although these recordings were maintained only for 20 min, in some experiments, the effects of 1 mM BAPTA appeared to stabilized within the recording period at a level of
20-70% of control levels (Fig. 8C). The BAPTA data are
summarized in Fig. 8D.
We also tested the effects of including EGTA in the presynaptic
electrode (Fig. 9). With 1 mM EGTA in the
pipette, no effect was seen on transmission (Fig. 9C;
n = 4). Similarly, in many experiments with 10 mM EGTA,
there was no significant effect on transmission (Fig. 9C).
However, in some cases (7/14), there was an appreciable block of
transmission, ranging 75%. An example of such an experiment is shown
in Fig. 9B. On average, 10 mM EGTA blocked transmission
~20% (Fig. 9D).
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DISCUSSION |
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These data represent an initial characterization of pair
recordings of CA3 pyramidal cells in the interface cultures, and can be
compared with work done with roller cultures (Debanne et al.
1995) and acute slices (Miles and Wong 1986
).
Besides demonstrating that these cultures are a good model system and
outlining the properties of synaptic transmission between single
connected neurons, we have shown that pharmacological manipulation of
the presynaptic cell is feasible for small molecules. In doing so, we
have tested hypotheses concerning the sensitivity of the release
machinery to exogenous calcium buffers.
Comparison to acute slices
The synaptic responses we obtained are quite similar to those
obtained in acute hippocampal slices by (Miles and Wong
1986). In this study, it was reported that the quantal content
of pairs was low, consistent with our findings, at least for most
pairs. Second, although most of our experiments were conducted in
voltage clamp, the responses we have examined in current clamp showed depolarizations of similar magnitude to those seen in the acute slice.
Miles and Wong (1986)
also observed polysynaptic
inhibition that could be elicited by a single action potential in the
presynaptic pyramidal cell, as we have (Fig. 2).
On average, our recordings were dominated by paired-pulse depression.
This is in contrast to the ~25% facilitation seen by Miles and Wong
(1986) in CA3-CA3 pair recordings in acute slices (in 2 mM Ca and 1.6 mM Mg). Debanne et al. (1996)
found a slight facilitation (8%) in CA3-CA3 pairs in roller tube cultures with an
Ca/Mg composition of 2.8/2.0 mM. The difference in our results might be
accounted for by the fact that we routinely used 2.5/1.3 Ca/Mg, which
may yield a slightly higher probability of release than the Ca/Mg
compositions used by Miles (1986)
and Debanne et al. (1996)
. On the other hand, using field stimulation in
2.5/1.3 Ca/Mg, Zalutsky and Nicoll (1990)
observed
paired-pulse facilitation in acute slices. Thus the predominance of
depression could indicate that the probability of release in cultures
is higher on average than in acute slices. The finding that raising
calcium did not result in the uncovering of low-probability release
sites is consistent with this idea (Fig. 6). The probability of release
in acute slices has been reported to be high in young rats (<2 wk) and
to decrease with age (Bolshakov and Siegelbaum 1995
).
Because our cultures were prepared from 1-wk-old animals and cultured
for 1-2 wk, our results may reflect this developmental difference. An
alternative explanation is that the synapses in our cultures do not
fully express the facilitation mechanism.
Variability between pairs
We observed a great deal of variability in the response amplitudes between different pairs. This could arise from a number of sources: differences in the number of active synapses between pairs, differences in the probability of release, or in quantal size. Additional variability could arise from differences in voltage-clamp errors in measuring EPSCs arising from distant synaptic sites or from different distributions of synapses on apical and basal dendritic trees. Each factor in theory could account for the observed variability alone or in combination with the others. For purposes of discussion, we first will consider the possibility that one of these factors alone might account for most or all of the observed variability.
The first possibility that we considered is that large variability in
the average EPSC amplitude between pairs arises simply because the
number of active synapses simply varies from pair to pair. However, the
number of potential synaptic contacts we found anatomically did not
correlate with the EPSC sizes observed in a pair, and furthermore the
pair that had a very small response had many more potential contacts
than would be predicted from the physiology. There is no obvious
difference in the distance of the contacts from the cell body in the
two pairs we reconstructed that could account for the difference in the
observed current amplitude, though the weak pair had a larger
proportion of contacts on basal dendrites (Fig. 4). We note that other
investigators who have reconstructed pairs also observed little
correlation between the number of contacts and response size
(Deuchars et al. 1994; Markram et al.
1997
), although the numbers of contacts observed in these cases
were smaller than in our pairs, and the synapses were in acute slices
of neocortex rather than cultured hippocampal slices.
A second possibility is that there is large variability in the average
probability of release between pairs. There was a positive correlation
between the coefficient of variation and the average size of the EPSC
in a pair, suggesting that stronger pairs might possess a higher
release probability or have more release sites than weaker pairs.
Because paired-pulse facilitation is thought to be due to a temporary
increase in the probability of release (Zucker 1989),
the PPR should decrease with increasing initial probability of release
as demonstrated by Dobrunz and Stevens (1997)
. Thus if
all other parameters are equal, pairs with a relatively low PPR would
be expected to have a higher probability of release, and thus exhibit
relatively large average EPSCs to a single action potential. The lack
of any such relationship between PPR and average EPSC size suggests
that any differences in the probability of release cannot entirely
explain the range of response sizes we observed (Fig. 3B).
Furthermore if smaller responses occurred because release probability
in weaker pairs was consistently lower than that in stronger pairs, it
would be expected that the amplitude of average small EPSCs could be
increased to a size approaching that in strong pairs by increasing
release probability. Raising the probability of release with calcium
did not readily convert small responses to large responses (Fig. 6).
This suggests that there is no subpopulation of very-low-probability
synapses between pairs that can be revealed by raising probability of
release. In addition, taken with the fact that most pairs were
dominated by paired-pulse depression, these data suggest that the
probability of release in most pairs is fairly high.
A third explanation for the differences in mean EPSC amplitude among
pairs is that the quantal size is larger in pairs with larger
responses. Thus pairs with large responses might consist of the same
number of active synapses as those with small EPSCs, but with a larger
quantal size. This possibility is supported by the broad distribution
of the mEPSCs, though mossy fiber synapses may account for some of the
larger events. There is some evidence against differences in quantal
size as the sole explanation for interpair variability. First, there
was a positive correlation between 1/CV2 and EPSC
amplitude. Although this type of analysis can be problematic (Faber and Korn 1991), in the simplest case, the inverse
square of the CV is predicted to increase with increasing probability of release or increasing number of release sites but should not change
when only the quantal size is increased. Second, under conditions where
the probability of release is lowered, strong pairs readily become
weak. For example, when stimulating pairs with trains of action
potentials, which caused depression, much smaller events and failures
appeared in the same pair that gave rise to large events (Fig.
1C). We also found that loading of the presynaptic cell with
BAPTA or EGTA causes a graded decline of transmission, and we observed
possible quantal events after BAPTA block was maximal (Fig.
7B). A similar effect was seen after lowering extracellular
calcium (Fig. 6A). These small events are comparable in size
with the smallest events observed in any pairs (<10 pA). Thus the
large EPSCs in these pairs are apparently made up of the sum of smaller
events with a quantal size similar to those making up the events in
weak pairs.
An alternative hypothesis is that the synapses in weak pairs are located further from the cell body than those in strong pairs, resulting a larger voltage-clamp errors and thus underestimation of EPSC amplitudes for distal synapses. If this is the case, it is not evident from our reconstructions because the putative synapses in both pairs appear to be distributed widely over the dendritic tree. The same is likely to be true for the other stained pairs that were not reconstructed because the presynaptic axons always projected widely among the dendritic layers. Thus there is no obvious segregation of synapses to proximal or distal dendrites that could readily account for the difference in response amplitudes, although this easily could be a contributing factor.
On the basis of the whole of our evidence, including paired-pulse
analysis, manipulations of calcium/magnesium ratio, and 1/CV2 analysis, we conclude that differences in quantal
size and release probability do not appear to vary sufficiently to
fully account for the observed differences in amplitude. Thus it seems
most likely that the variability in EPSC amplitude arises primarily from variation in the number of synapses formed between different pairs
despite the results of the reconstruction of two pairs. It is likely
that the limited anatomic analysis of potential contacts we performed
does not provide an accurate representation of the number of active
synapses. This may be because some synaptic contacts are effectively
nonfunctional either presynaptically (due to a very low or a zero
probability of release) or postsynaptically (due to the lack of
functional AMPA receptors but not necessarily a lack of NMDA receptors)
(Malenka and Nicoll 1997).
Variation of responses within pairs
In addition to the variability among pairs, EPSC amplitudes within
a given pair recording fluctuated considerably (i.e., Fig. 3C). Although we have not conducted a formal quantal
analysis of our EPSCs, it is likely that the major source of this
variation is fluctuation in the number of quanta released from trial to trial as at other synapses. In addition, the average quantal content in
most pairs appears to be fairly low, because failures of transmission were observed (i.e., Fig. 7) and most EPSCs in pairs were not more than
four to six times as large as typical mEPSCs (Fig. 5). One consequence
of fluctuations in transmission seems to be that when paired pulses are
delivered, there is what can be termed "competition" for synaptic
resources between the first and second EPSCs (Fig. 3D) as
has been observed previously in organotypic slices (Debanne et
al. 1996) and in motor cortex (Thomson et al. 1993
). The magnitude of the effect was quite variable although we detected competition in most but not all pairs. The limiting resource may be presynaptic, as shown by Debanne et al.
(1996)
and might reflect readily releasable synaptic vesicles.
In addition, the fact that 1/CV2 for the second pulse
compared with the first pulse is correlated with PPR in our pairs is
also suggestive of a presynaptic change. However, from our data we
cannot rule out other explanations such as postsynaptic receptor
desensitization (Arai and Lynch 1998
) or rapid feedback
presynaptic inhibition mediated by glutamate (Chittajallu et al.
1996
).
Effects of presynaptic calcium chelator injection
In most studies, in a variety of preparations, BAPTA has been
demonstrated to block transmission (Adler et al. 1991;
Borst and Sakmann 1996
; Niesen et al.
1991
), whereas EGTA variously has been reported to have no
effect (Adams et al. 1985
; Adler et al.
1991
; Atluri and Regehr 1996
; Delaney et
al. 1991
; Spigelman et al. 1996
;
Swandulla et al. 1991
) or to partially block
transmission (Borst and Sakmann 1996
; Kretz et
al. 1982
; Salin et al. 1996
). To our knowledge,
there have been no tests of the effects of calcium chelators in
hippocampus using direct intracellular injection into presynaptic cells.
Our experiments with BAPTA demonstrated a high degree of sensitivity to
this chelator at concentrations no higher than 1 mM. This is a
comparable sensitivity to that observed in the squid (Adler et
al. 1991), although it is likely that in our experiments the
concentration of BAPTA in the terminal after 20 min is still substantially lower than that in the pipette.
In our hands, EGTA was capable of blocking transmission in at least
some experiments, despite its slower binding of calcium than BAPTA, in
agreement with the data of others who used presynaptic injection to
administer the chelator to mammalian neurons (Borst and Sakmann
1996). While the effects of EGTA, when observed, may be
analogous to those of BAPTA, that is, via buffering of calcium before
release can be triggered, we cannot rule out alternatives to this
explanation. First, EGTA could be having a toxic effect perhaps related
to its release of protons when binding calcium. Another possibility is
that other events mediated by calcium, besides the rapid triggering of
release, might be perturbed. For example, if a slow calcium signal is
required for the refilling of synaptic vesicle docking sites and EGTA
blocked this signal, then eventually transmission would be blocked.
At CA3 associational synapses, effects of EGTA (using bath application
of EGTA-AM) on baseline transmission also have been observed by
(Salin et al. 1996) (40% block) but not by
Spigelman et al. (1996)
. The variability of EGTA effects
on our study likely reflects the fact that only a few synapses are
recorded in each experiment. One reason EGTA had no apparent effect in
some experiments might be differences in the concentration of EGTA
attained at the terminal. Arguing against this, if we assume that EGTA
diffuses into the cell as readily as BAPTA, it is likely that EGTA
reached a high concentration in the terminal within 20 min in most
cases, even when block was not observed. However, without an
independent measure of EGTA concentration, the possibility remains that
in some experiments, an insufficient concentration was reached in the
terminal. The more interesting possible reason for the variability of
the EGTA effect is a underlying difference in the properties of
synapses. For example, in some terminals, release sites may not be as
closely linked to calcium entry sites as in others (Smith and
Augustine 1988
).
These data demonstrate the feasibility of performing pharmacological
manipulations of presynaptic neurons in hippocampal slice cultures. The
effects of BAPTA and EGTA were very rapid with noticeable effects
within 10 min being typical. This speed suggests that such
manipulations need not need to be limited to small molecules such as
BAPTA. Indeed, we have indications from experiments with fluorescently
labeled dextrans that reasonably rapid (<1 h) access to terminals
200 µms away from the recording site might be obtained for
substances with molecular weights <3,000 (unpublished data). This
opens the possibility that types of analysis of the synaptic vesicle
release machinery that has been performed at the squid giant synapse
(DeBello et al. 1995
) might be extensible to the hippocampal slice.
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ACKNOWLEDGMENTS |
---|
We thank E. Schaible for technical assistance, I. Parada-Riquelme, J. Hirsch, and D. Prince for use of and training with the Neurolucida systems, D. Faber for the mEPSC analysis software, and R. McQuiston for discussion.
This work was supported by National Institutes of Health Grants NS-10330 to P. Pavlidis and MH-48108 to D. V. Madison.
Present address of P. Pavlidis: Center for Neurobiology and Behavior, Columbia University, New York, NY 10032.
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FOOTNOTES |
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Address for reprint requests: D. V. Madison, B115 Beckman Center, Stanford University School of Medicine, Stanford, CA 94305-5345.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 November 1998; accepted in final form 25 February 1999.
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REFERENCES |
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