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The Journal of Neurophysiology Vol. 79 No. 4 April 1998, pp. 2013-2024
Copyright ©1998 by the American Physiological Society
1 Neuroscience Graduate Program, 2 Department of Physiology, 3 Department of Psychiatry, and 4 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143
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ABSTRACT |
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 Abstract
 Introduction
Methods
Results
Discussion
References
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Hsia, Albert Y., Robert C. Malenka, and Roger A. Nicoll. Development of excitatory circuitry in the hippocampus. J. Neurophysiol. 79: 2013-2024, 1998. Assessing the development of local circuitry in the hippocampus has relied primarily on anatomic studies. Here we take a physiological approach, to directly evaluate the means by which the mature state of connectivity between CA3 and CA1 hippocampal pyramidal cells is established. Using a technique of comparing miniature excitatory postsynaptic currents (mEPSCs) to EPSCs in response to spontaneously occurring action potentials in CA3 cells, we found that from neonatal to adult ages, functional synapses are created and serve to increase the degree of connectivity between CA3-CA1 cell pairs. Neither the probability of release nor mean quantal size was found to change significantly with age. However, the variability of quantal events decreases substantially as synapses mature. Thus in the hippocampus the developmental strategy for enhancing excitatory synaptic transmission does not appear to involve an increase in the efficacy at individual synapses, but rather an increase in the connectivity between cell pairs.
How immature neural circuits develop into the highly organized patterns of connections that subserve brain function has been a subject of intense study. Most neural systems appear to follow the developmental strategy of activity-dependent refinement and remodeling of initially coarse patterns of synaptic connections into highly tuned and functioning networks (Goodman and Shatz 1993 General methods
Sprague-Dawley rats ranging from 2 days to 2 yr of age were studied. Transverse hippocampal slices (500-µm thick) were prepared as described (Wyllie et al. 1994
Experimental design and data analysis for multiplicity assessments
To generate data for multiplicity assessments, we first recorded 100-400 spontaneous EPSCs, and then added tetrodotoxin (TTX; 0.5 µM) to isolate the mEPSC population (see Fig. 5A). The multiplicity was then computed in the following manner
Analysis of AMPAR and NMDAR components
To generate AMPA/NMDA ratios, the stimulation intensity was first increased until an ~100-pA EPSC was generated while holding the cell at Evidence for a developmental increase in functional synapse number
Anatomic studies have suggested that during development the total density of excitatory synapses onto CA1 cells increases, approximately doubling from neonatal to adult ages (Harris et al. 1992 Resolution of multiple synaptic contacts between cell pairs requires high probabilities of release
A developmental increase in the total number of functional synapses (ntot) could be due to an increase in the number of synapses between individual CA3-CA1 cell pairs (nij) or to an increase in the number of CA1 target cells per CA3 cell, without a change in cell-to-cell connectivity. One method of assessing the number of functional synapses between cell pairs is to compare EPSCs generated by action potentials in single CA3 cells with mEPSCs due to spontaneous release of single quanta. Such a comparison depends on maximizing Pr, to increase the likelihood that synapses made by a single CA3 cell axon onto a single CA1 cell will release transmitter simultaneously and thereby reveal the maximum EPSC that can be generated by this connection. In the ideal case, Pr would equal one for all experiments, such that multiple synaptic inputs onto a CA1 cell from an individual CA3 cell would always release together, eliciting relatively large responses when compared with CA3-CA1 cell pairs with only a single synapse between them. The opposite extreme of a very low Pr is certainly undesirable, because synapses would seldom release simultaneously, thus masking the true degree of connectivity between cell pairs.
TTX-sensitive spontaneous EPSCs increase in amplitude over development
Having established that elevation of the Ca2+/Mg2+ ratio substantially increases Pr, experiments were designed to look for changes in the coupling between CA3-CA1 cell pairs. We took advantage of the observation that the frequency of spontaneous events recorded from CA1 cells significantly decreases after blockade of action potentials with TTX, suggesting that, without external stimulation, CA3 cells can randomly fire action potentials in the slice preparation. This situation allowed us to characterize large numbers of single inputs from a population of CA3 cells onto single CA1 cells, by comparing spontaneous EPSCs recorded before and after TTX application. In the absence of TTX, spontaneous events consist of both mEPSCs and responses to spontaneous action potentials in CA3 cells; in the presence of TTX, only mEPSCs are present (Raastad et al. 1992
Relative contribution of AMPA receptors increases over development
One likely mechanism for the developmental increase in connectivity is the addition of new synapses. This mechanism may occur along with a different strategy, namely the insertion or activation of AMPARs at presumably "silent" synapses that contain only functional NMDARs (Durand et al. 1996
Aging causes no apparent changes in excitatory circuitry
We next asked whether the circuit changes we observed from birth to young adulthood stabilized until old age, or whether aging brought about new changes. Animals 2 yr of age were studied, an age at the limit of the animals' life span. No difference in CA3-CA1 cell coupling was found between young adult and aged animals, as the multiplicity values are quite similar (Fig. 8A1; n = 7, aged; young adult data from Fig. 6E, P > 0.7). A representative experiment from an aged animal is depicted in Fig. 8A2, in which it can be seen that TTX application causes a decrease in spontaneous EPSC amplitude comparable to that seen in young adults. PPF was also indistinguishable between young adult and aged animals, as shown in Fig. 8B. In normal Ca2+/Mg2+, aged animals displayed PPF of 1.42 ± 0.078 (n = 7), which is not significantly different from young adults (1.51 ± 0.11; n = 9, P > 0.5). The AMPA/NMDA ratios and NMDA kinetics for both age groups are also similar (Fig. 8C; n = 8 aged, young adult data from Fig. 7, P > 0.6 for both), as are mEPSC amplitude and frequency (Fig. 8D; n = 7, aged, P > 0.4 for both comparisons).
A critical question in neurobiology is how the precise patterns of neuronal connections in the mammalian brain develop from immature circuits. Although much has been learned from anatomic studies, a number of findings suggest that the morphological identification of synaptic contacts may be insufficient and perhaps misleading. In this study, physiological approaches were used, to study changes in the organization and properties of synapses during the postnatal development of excitatory circuitry between hippocampal CA3 and CA1 pyramidal cells.
Using spontaneous EPSCs to characterize cell coupling
In each hippocampal slice preparation, there are ~10,000 CA3 cells (Andersen et al. 1994 Possible mechanisms underlying synapse development
It remains an open question as to whether the formation and modification of neural circuits over development might utilize a mechanism similar to that underlying long-term potentiation (LTP) (Bear et al. 1987 Excitatory circuit properties do not change in aged animals
It is generally agreed that, to varying degrees, the capacity to learn and remember declines with advanced age, presumably due to changes in brain structures including the hippocampus (Barnes and McNaughton 1985 Conclusions
Using a variety of electrophysiological techniques, we studied both organizational and component properties of a developing excitatory circuit. We found that CA3 cells enhance coupling with target CA1 cells by increasing the number of functional synaptic contacts between them, rather than by changing the reliability or efficacy of individual synapses. It will be interesting to discover whether an equivalent developmental strategy is adopted in other circuits in the mammalian brain.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Examples include the neuromuscular junction, at which synapse elimination occurs during early postnatal life such that all but one input is eliminated from each muscle fiber (Colman and Lichtman 1993), and the developing mammalian visual system, where selective axon retraction and outgrowth lead to the formation of ocular-dominance and orientation-selective columns from an initially coarse-grained topographic map (Antonini and Stryker 1993). Insights into these processes have been derived largely from anatomic studies, using techniques such as trans-neuronal labeling and axonal reconstruction. With the reasonable assumption that most anatomically identified synapses and axonal projections correspond to functional units, these techniques have been quite useful for determining the number of inputs into particular target regions.
) as well as in the hippocampus (Durand et al. 1996) have revealed that many immature, functioning synapses lack the diagnostic morphological correlates of mature synapses and can be overlooked by both light and electron microscopic studies. Anatomic synapse identification can also overestimate the number of functional synapses: presynaptically, there may not be active transmitter release (Faber et al. 1991; Redman 1990; Tong et al. 1996; Wojtowicz et al. 1991), and postsynaptically, receptors that are active at resting membrane potential may be absent (Isaac et al. 1995; Liao et al. 1995). Together these findings underscore the importance of physiological measures of the number of functional synapses, as well as the reliability and strength of synapses.
; Collin et al. 1997; Durand et al. 1996; Liao and Malinow 1996). Of particular interest is the proposal that early in development, a significant proportion of synapses may be functionally silent because they contain only N-methyl-D-aspartate receptors (NMDARs) and no functional 
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) (Durand et al. 1996; Isaac et al. 1997; Liao and Malinow 1996). In this paper we looked for changes during development in the three quantal parameters of synaptic transmission: the probability of neurotransmitter release (Pr), quantal size (q), and the number of functional synapses (n). We also used a more specific assay for n, to study possible developmental changes in the connectivity between cell pairs. Finally, we examined the ratio of AMPAR-mediated to NMDAR-mediated synaptic transmission, to determine whether this ratio might change during development.
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
), and after at least a 1-h recovery period, transferred to a submersion chamber where they were continuously superfused (~2 ml/min) with a room temperature (22-25°C) artificial cerebrospinal fluid (ACSF) solution saturated with 95% O2-5% CO2. Our "normal" external solution was composed of (in mM) 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 Mg2SO4, 1.0 NaH2PO4, 26.2 NaHCO3, and 10 glucose. In cases where the Ca2+/Mg2+ ratio was elevated to increase Pr, CaCl2 was raised to 4.0 mM and Mg2SO4 was lowered to 0.5 mM. All experiments were performed in the presence of 0.1 mM picrotoxin. Recording, stimulation, and data collection techniques were similar to those described previously (Wyllie et al. 1994). Except for Figs. 1 and 3, all experiments were performed in the whole cell configuration, and unless otherwise specified, cells were voltage clamped at 
70 mV. The whole cell pipette solution was composed of (in mM) 122.5 Cs-gluconate, 11 ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 10 CsCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 8 NaCl, 10glucose, 1 CaCl2, 4 Mg-ATP, and 0.3 Na3-GTP (pH 7.2 with CsOH, 280-290 mosM).
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FIG. 1.
Field excitatory postsynaptic potentials (EPSPs) are comparatively larger in older animals. A: for equal fiber volley amplitudes (arrow), larger field synaptic responses were obtained in young adults. Example recordings from a 12-day-old and 2-mo-old animal are shown (each trace is an average of 5 responses). B: summary graph of field input-output relations for neonatal (7-12 days) vs. young adult (2-3 mo) animals.
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FIG. 3.
Resolution of multiple synapses between CA3-CA1 cell pairs requires high probabilities of release. A: simulations of the number of release sites per CA3 cell that simultaneously release transmitter onto the recorded CA1 cell in response to an action potential, a number we call the "multiplicity," show an increasingly good estimate of average CA3-CA1 cell pair connectivity (nij) with increasing pr. Thin lines delineate potential experimental error of ±10%. B1: summary graph illustrating that quadrupling the normal Ca2+/Mg2+ ratio apparently maximizes Pr. An elevated Ca2+/Mg2+ ratio, 4-aminopyridine (4-AP; 10 µM), and Sp-8-Br-cAMPS (0.8 mM) all cause large increases in synaptic efficacy (solid bars), but neither 4-AP nor Sp-8-Br-cAMPS increase synaptic efficacy when the Ca2+/Mg2+ ratio has already been elevated (open bars). Example experiments show that a concentration of Sp-8-Br-cAMPS (0.8 mM), which dramatically increases the strength of EPSPs in a naive slice (B2), has no effect on a slice that has already been potentiated with elevated Ca2+/Mg2+ (B3). In B3 the downward arrow indicates reduction of stimulus strength to match the response size of the original baseline.
).
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FIG. 2.
Neither paired-pulse facilitation (PPF) nor quantal size changes significantly over development, whereas miniature excitatory postsynaptic current (mEPSC) frequency increases 4-fold. A: PPF remains constant across ages, either in normal external solution, or in a solution with an elevated Ca2+/Mg2+ ratio. B: amplitude of mEPSCs does not change significantly over development (B1), whereas mEPSC frequency increases with age (B2). C1: coefficient of variance (CV = 
2/mean) of mEPSC amplitudes is larger in neonatal animals than in adults (6 youngest vs. 6 oldest animals, P < 0.0007). C2: cumulative amplitude histograms for the youngest neonates and oldest adults are standardized to a common mean and variance, revealing that the shape of the amplitude distribution remains constant over development.
where a is the mean amplitude of action-potential-driven events, and q denotes mean quantal size. q was defined as the mean amplitude of mEPSCs recorded in TTX, and a was inferred by subtracting the contribution of mEPSCs to the pool of events collected in the absence of TTX: if we let b equal the mean amplitude of both action-potential-driven events and mEPSCs recorded in the absence of TTX, and denote the frequency of particular events as
events, we arrive at an expression for b as a frequency-weighted average of a and q
All multiplicity experiments were performed in an external solution with a high Ca2+/Mg2+ ratio. Spontaneous CA3 cell firing was present in these conditions, but in approximately one-half of the experiments for each age group, 4-aminopyridine (4-AP; 1.5-20 µM) was added to the superfusing solution to increase CA3 cell excitability. Since in the presence of elevated Ca2+/Mg2+ 4-AP was found to have no effect on synaptic efficacy (Fig. 3B1), PPF, or mEPSC frequency (data not shown), we were confident that 4-AP did not further increase Pr in these experiments. In addition, in four adult cells, we measured the multiplicity with and without 4-AP and found it to be unaffected (P > 0.9).
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FIG. 5.
Tetrodotoxin (TTX)-sensitive spontaneous EPSCs increase in amplitude over development. A: sample sweeps illustrating the effect of TTX on spontaneous EPSCs in a CA1 cell from a neonatal (A1) and young adult (A2) animal. At both ages, TTX more than halves the frequency of spontaneous events, yet only in the young adult does TTX cause a reduction in spontaneous EPSC amplitude. B: cumulative amplitude distributions obtained from the cells illustrated in A. Each distribution is normalized to the average amplitude of mEPSCs in TTX. C: the increasing effect of TTX on spontaneous EPSC amplitude with development is illustrated by superimposing average EPSCs in the absence (thick lines) and presence (thin lines) of TTX, for cells from animals ranging from 4 days to 3 mo of age. Events are scaled such that the average mEPSCs are of equal amplitude across ages. D: the average EPSC in the absence of TTX for the 3-mo-old animal in C is scaled down to the amplitude of the average mEPSC. The exact superimposition argues against temporal overlap of individual events in the absence of TTX.
70 mV. In those very young (2-4 days) animals in which either no or very little response could be elicited at this potential, the stimulus strength was set at 2-4 times that of experiments in older animals. The cell was then depolarized to +50 mV, and after 25-50 responses had been collected, D-2-amino-5-phosphonovaleric acid (D-APV; 50 µM) was applied. The average AMPAR EPSC was taken as the average of 25-50 responses taken after the effect of D-APV had stabilized. This average AMPAR EPSC was then subtracted from the average EPSC in the absence of D-APV to obtain the average NMDAR EPSC. The peak amplitude of the AMPAR EPSC divided by the peak amplitude of the NMDAR EPSC yielded the AMPA/NMDA ratio for the cell. The average NMDAR EPSC was also used to calculate the time to half-decay of NMDAR-mediated responses.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
). If the anatomically observed increase in synapse density corresponds to the formation of functional synapses, we would expect responses to field stimulation to increase with age. We therefore compared field EPSP input-output relations in neonatal (<2 wk) versus young adult (2-3 mo) animals. In Fig. 1A, superimposed field responses are shown from a 12-day-old and 2-mo-old animal. These responses illustrate that for equal fiber volley amplitudes (arrow), the accompanying synaptic response is larger in the 2-mo-old animal. The fiber volley is an indirect measure of the number of axons activated. As shown in Fig. 1B, for given fiber volley amplitudes, young adults consistently exhibit more robust synaptic responses (n = 7, neonatal;n = 10, adult; P < 0.05).
; Manabe et al. 1993; Zucker 1989), an increase in Pr would be reflected in decreasing degrees of PPF over development. Figure 2A1 shows that no changes in PPF could be found across ages. PPF was examined in two different Ca2+/Mg2+ ratios, one that consistently yielded ~50% facilitation (n = 6-7 per group, P > 0.7), and one in which PPF was absent (n = 6-15 per group; P > 0.4). To address the possibility of a developmental change in q, we compared the average amplitudes of mEPSCs, which correspond to responses to a single quantum of transmitter released from individual synapses. Again, no significant changes were found over development (Fig. 2B1; n = 4-12 per age group, P > 0.05); if anything, there is a slight decrease in q, which may reflect a developmental increase in dendritic arborization, and hence cable filtering (see DISCUSSION).
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FIG. 4.
Single-fiber stimulation reveals a high probability of release in neonatal slices bathed in high Ca2+ and low Mg2+. A: example of a single-fiber stimulation experiment carried out in elevated Ca2+/Mg2+. To isolate a single axonal input, the stimulus intensity (solid line) was decreased in a stepwise fashion, to find a consistent plateau region just above the threshold for eliciting a synaptic response. Individual (solid square) and mean (open square) EPSC amplitudes for each stimulus intensity are plotted. B: average EPSCs (30 responses each) for each stimulus intensity are displayed, illustrating that the mean EPSC was quite constant during the plateau period. C: plot of the success rate for each stimulus intensity. During the plateau period, the average Pr was 0.82. D1: paired-pulse depression was observed during the plateau period (average of 93 responses). D2: the successful EPSCs in response to the 2nd pulse, although far more seldom, scale equally to the successes of the 1st pulse when superimposed, suggesting activation of a single release site. For this experiment (13-days old) and 5 others in which putative single synapses were isolated (7-13 days), the measured Pr was 0.74 ± 0.04.
). As shown in the leftmost bar of Fig. 3B1, this change greatly increases the size of field EPSPs(n = 8). In fact, increasing the Ca2+/Mg2+ ratio appears to maximize Pr, for other manipulations that increase Pr, 4-AP (10 µM, n = 4) and Sp-8-Br-cAMPS (0.8 mM, n = 6; solid bars), no longer do so in conditions of elevated Ca2+/Mg2+ (open bars, n = 6 each). For example, a concentration of Sp-8-Br-cAMPS (0.8 mM), which dramatically increases synaptic strength in a naive slice (Fig. 3B2) (see also Chavez-Noriega and Stevens 1994), has no effect on a slice to which elevated Ca2+/Mg2+ has already been applied(Fig. 3B3).
). To maximize the likelihood that this single axonal input made only one functional synapse onto the recorded cell, we performed an additional paired-pulse test (Stevens and Wang 1995). Paired-pulse depression was observed during the plateau period (Fig. 4D1), presumably due to a lower probability of release in response to the second stimulus. If two or more synapses were being activated during the plateau period, the lower Pr on the second stimulus would make it less likely that synapses would release simultaneously. Thus in this case, the amplitude of successful responses to the second stimulus would be expected to be smaller than successful responses to the first. On the contrary, the average amplitude of successful responses to the first and second stimulus were very similar (Fig. 4D2), consistent with the activation of a single synapse. In this experiment, and in five others that also passed the above tests, the average Pr was0.74 ± 0.04 (mean ± SE). These experiments were performed in animals ranging from 7 to 13 days of age. In a previous set of experiments from our laboratory in which the Ca2+/Mg2+ ratio was also elevated (Isaac et al. 1996), animals 15-18 days of age were studied, and Pr was found to be similarly high (0.84 ± 0.05, n = 5).
). We reasoned that if individual CA3 cells contact individual CA1 cells through multiple synapses, action potentials in CA3 cells would result in responses larger than the average mEPSC, and these larger responses would be abolished by TTX. The extent to which TTX reduces spontaneous event amplitude would then correlate with the degree of coupling between CA3-CA1 cell pairs.
). In the seven cells with the largest spontaneous EPSCs in the absence of TTX, random firing was always observed (R ranged from 0.94 to 0.99). Finally, neonatal and adult animals might have similarly large responses to action potentials in CA3 cells, but such responses in the neonates could be masked if in the neonates a larger proportion of events in the absence of TTX were mEPSCs. Yet in Fig. 6B it can be seen that the frequency of spontaneous events in the absence of TTX increases in step with the developmental increase in mEPSC frequency, such that the percentage of mEPSCs in the absence of TTX remained constant at ~40% for all ages studied (Fig. 6C, n = 4-12 per group).
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FIG. 6.
Comparison of the size of TTX-sensitive spontaneous EPSCs with mEPSCs yields a measure of CA3-CA1 cell coupling that increases dramatically over development. A: histogram of interevent intervals for spontaneous EPSCs recorded in the absence of TTX, plotted on a semi-log scale. The exponential distribution of intervals indicates that events occurred randomly and independently in this cell from the adult animal in Fig. 5A. Exponentially distributed intervals were observed in all 7 cells with the largest spontaneous EPSCs in the absence of TTX (R ranged from 0.94 to 0.99). B and C: the frequency of spontaneous EPSCs in the absence of TTX ( 
) increases in step with the frequency of mEPSCs (
) over development (B), such that at all ages, ~40% of spontaneous EPSCs in the absence of TTX were mEPSCs (C). D: mEPSCs were stable in both amplitude and frequency. Individual experiments are depicted, with each experiment having an amplitude () and frequency () pair that was tracked for 5-20 min. Each value is determined from a 2- to 4-min time window. E: because mEPSCs were found to be stationary over time, their contribution to the population of spontaneous events recorded in the absence of TTX could be subtracted, to reveal the mean TTX-sensitive spontaneous EPSC (see Experimental procedures). Because TTX-sensitive events correspond to responses to spontaneous action potentials in CA3 cells, dividing their mean amplitude by the mean amplitude of mEPSCs yields a measure of the number of release sites per CA3 cell that simultaneously release transmitter onto the recorded CA1 cell, or the multiplicity. The multiplicity was found to increase from 1.02 ± 0.12 in the youngest neonates, to 2.12 ± 0.13 in young adults.
; Isaac et al. 1995; Liao et al. 1995). If this strategy were employed, one would expect that over development, the number of synaptic AMPARs would increase relative to the number of synaptic NMDARs.
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FIG. 7.
Relative contribution of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors (AMPARs and NMDARs, respectively), as well as NMDAR kinetics, changes over development. A: AMPAR and NMDAR EPSCs recorded at +50 mV are shown for 3 developmental stages. A1: striking example from a 2-day-old animal, in which a 44-pA NMDAR component had no accompanying AMPAR component. A2: EPSCs from a 4-day-old, 8-day-old, and 2-mo-old animal are superimposed and scaled such that the AMPAR components are of equal size, to illustrate the correspondingly larger NMDAR components in neonatal animals. B: summary graph of AMPA/NMDA ratios as a function of age. The relative contribution of AMPARs is small in neonates, and increases over development. C: plot of the time to half-decay of the NMDAR EPSC, showing that there is a transient increase in the decay time during the 2nd week of age followed by a decrease to a time shorter than that recorded at the earliest age.
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FIG. 8.
Aged animals, when compared to young adults, show similar circuit properties. A1: summary graph of the multiplicity in aged animals versus young adults. A2: average EPSCs from a representative experiment, demonstrating that TTX application in aged animals causes a decrease in spontaneous EPSC amplitude comparable to that seen in young adults. B: PPF is similar in young adult and aged animals for both normal and elevated Ca2+/Mg2+ ratios. C: neither AMPA/NMDA ratios nor NMDAR EPSC kinetics reveal significant differences between young adult and aged animals. D: mEPSC amplitude (D1) and frequency (D2) are also not significantly different.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Dumas and Foster 1995; Liao and Malinow 1996), suggesting that the association between the CA3 and CA1 stages of the hippocampal circuit strengthens over development. To determine the mechanisms responsible for this change, we examined the three variables, n, Pr, and q, that control the size of synaptic responses. Changes in Pr or q did not occur over development, leaving changes in n as the likely cause for the observed strengthening of field responses. Consistent with this hypothesis, mEPSC frequency increases fourfold, and TTX-sensitive, spontaneous EPSCs double in amplitude over development. Together these observations suggest an increase in the number of excitatory synapses onto CA1 cells over development, which is expressed in part by an increase in the coupling between individual CA3-CA1 cell pairs.
), ~5% of which connect with any one CA1 cell (Bolshakov and Siegelbaum 1995; Sayer et al. 1990). Thus by studying spontaneous EPSCs resulting from random action potentials in individual CA3 cells, we were able to perform, in a single experiment, the equivalent of on the order of hundreds of single-fiber stimulation experiments. The average amplitude of these TTX-sensitive spontaneous EPSCs, when divided by the average mEPSC amplitude, yields the average multiplicity of CA3 inputs: the average number of release sites per CA3 cell that simultaneously release transmitter onto the recorded CA1 cell. While mEPSCs likely arise from a larger population of synapses than those driven by action potentials in CA3 cells (e.g., synapses disconnected from CA3 cell bodies during slice preparation), these events do provide a useful measure of quantal size. The multiplicity values we obtained are conservative estimates of nij, the number of functional synapses between cell pairs, because although Pr was high in these experiments, it was less than one. Thus many of the spontaneous TTX-sensitive events were not a consequence of simultaneous release from all synapses between individual CA3-CA1 cell pairs. Although multiplicity values will likelyunderestimate nij, changes in the multiplicity will directly correlate with changes in cell coupling. Using this approach, we observed a twofold increase in the multiplicity between CA3-CA1 cell pairs from neonatal to adult ages. A functional implication of the enhanced coupling in adult animals is that fewer CA3 cells need to be activated to cause a CA1 cell to fire. The intensification of existing CA3-CA1 connections that we describe likely occurs in tandem with an additional developmental strategy for strengthening the association between CA3 and CA1, which is the increase in the number of target CA1 cells per CA3 cell.
; Stevens and Wang 1995). As both of these studies were performed in young animals (<1 mo old), they are in basic agreement with our findings. An independent study in <1-mo-old animals compared spontaneous EPSCs in the absence of TTX with events evoked in TTX by hyperosmotic media or ruthenium red (Raastad et al. 1992). It was found that events in the absence of TTX were similar in amplitude to the TTX-insensitive events. While also in fundamental agreement with our findings, no effort was made in that study to increase Pr; the Ca2+/Mg2+ ratio was in fact lowered to 1 mM Ca2+/1 mM Mg2+. As we stress in Fig. 3A, it is critical to maximize Pr to increase the likelihood of simultaneous release; by doing so we were able to resolve small increases in multiplicity during the first few weeks of development (Fig. 6E). Experiments involving stimulation of single CA3 inputs have not been performed in adults. However, an anatomic study in adult animals, which counted the number of crossings of individual CA3 axons with the dendritic arbors of single CA1 cells, suggests that approximately one-half of the axons of CA3 cells make multiple contacts onto a single CA1 cell (Sorra and Harris 1993). Although neonatal animals were not examined, this study does offer an anatomic substrate for our physiological finding of multiple synapses between CA3-CA1 cell pairs in the adult.
) it was concluded that Pr decreases over development, from 0.9 at 4-8 days, to 0.5 at 14-21 days. At 4-8 days, PPF was found to be completely absent, in contrast with our finding of substantial PPF (~150%) at this age. While some of the difference in absolute PPF values may be due to a 30% difference in the Ca2+/Mg2+ ratio between their normal external solution and our own, the important finding in the present study is that PPF remains constant over development. Two other studies have studied PPF at different stages of development and have found it to either increase (Muller et al. 1989) or decrease (Dumas and Foster 1995); these results are difficult to interpret, however, because inhibition was not blocked. In terms of our measurements of multiplicity, a developmental decrease in Pr would only enhance our conclusion of increasing multiplicity with age. For if Pr is truly near the ceiling value of one in very young animals, then further increases in Pr could not account for the more than twofold increase in multiplicity we observed over development.
; Katz and Shatz 1996). One proposal is that early in development, a significant proportion of synapses may be functionally silent because they contain only NMDARs, and that over development these silent synapses become functional through an LTP-like mechanism that involves the appearance of AMPARs. Indeed, in a number of different preparations, synaptic responses mediated solely by NMDARs have been observed in greatest abundance early in postnatal development (Durand et al. 1996; Isaac et al. 1997; Wu et al. 1996). Consistent with this hypothesis, we found that the ratio of AMPAR- to NMDAR-mediated synaptic currents increases over development. However, this change could also reflect changes in the relative number and/or conductance of AMPARs and NMDARs at individual synapses, or a developmental change in the degree of glutamate spillover from one synapse to another, which has been proposed to account for synaptic responses mediated only by NMDARs (Kullmann et al. 1996). It should be noted that an increase in the AMPA/NMDA ratio was not observed in a previous developmental study (Liao and Malinow 1996), perhaps because the age range examined was relatively narrow (4 to 14 days, vs. 2 days to 2 mo in our study). Over the range of ages examined in this study, there is increasing dendritic arborization (Pokorny and Yamamoto 1981), which will cause increased cable filtering of EPSCs. Because the amplitude of faster AMPAR EPSCs will be attenuated more than NMDAR EPSCs, filtering will lead to an underestimation of the actual AMPA/NMDA ratio. However, because the AMPA/NMDA ratio will be increasingly underestimated as a function of age, consideration of filtering effects only strengthens our conclusion of an increasing AMPA/NMDA ratio over development.
; Crair and Malenka 1995; Hestrin 1992; Kirson and Yaari 1996). Such a change may be a result of decreased NMDAR2B mRNA expression relative to NMDAR2A in adults (Flint et al. 1997; Monyer et al. 1994). The finding that the NMDAR response is slower during the second week of development than the first (Fig. 7C) has not been described, perhaps because our study included younger animals than studied previously (Kirson and Yaari 1996; Liao and Malinow 1996). A possible explanation for this peak in decay time is provided by the finding that NMDAR2B-containing heteromers are in fact maximally expressed during the second week in cortex (Sheng et al. 1994).
; Dumas and Foster 1995), we did not observe a developmental change in quantal size, as assayed by measuring mEPSCs. Because recordings were made from the cell soma, there is the possibility that increased dendritic arborization over development could mask a developmental increase in the current generated at single synaptic sites. But if quantal size were increasing uniformly across synapses over development, we would still expect to observe an increase in the size of the largest mEPSCs, which presumably correspond to minimally filtered, proximal events. However, we did not observe such an increase. Although we could not detect a developmental change in quantal size, we did observe a twofold decrease in the coefficient of variance of mEPSC amplitudes between neonatal and adult ages. This decrease in variance cannot be explained by a developmental increase in dendritic arborization, because attenuation of distant events would be expected to increase the variance of events recorded at the soma. Either a decreased variance in the number of AMPA receptors from synapse to synapse, or a decreased variance in transmitter concentration per vesicle could account for the decreased variance in quantal size in the adult. Whatever the mechanism, this change suggests that there is a tuning process that occurs over development to achieve greater uniformity of quantal responses.
; deToledo-Morrell et al. 1988; Grady et al. 1995). Changes in neuron number in the pyramidal cell layers of hippocampus are unlikely to account for aging-related cognitive changes, because the number of CA3 and CA1 cells appears to remain constant from before birth (when cell division ceases) (Bayer 1980) to very advanced ages (Gallagher et al. 1996; Rapp and Gallagher 1996). We therefore addressed whether changes occur in the circuitry between existing cells.
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ACKNOWLEDGEMENTS |
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We thank D. Copenhagen, M. Frerking, L. Jan, and M. Scanziani for valuable comments. We also thank D. Selig for providing software for on-line data acquisition and analysis, S. Nicola for providing computer programs for off-line detection and analysis of spontaneous EPSCs and H. Czerwonka for secretarial assistance.
This work was supported by an Office of Naval Research predoctoral fellowship to A. Y. Hsia, grants to R. C. Malenka and R. A. Nicoll from the National Institutes of Health and a grant to R. C. Malenka from the Human Frontier Science Program. R. C. Malenka is a member of the Center for Neurobiology and Psychiatry and the Center for the Neurobiology of Addiction. R. A. Nicoll is a member of the Keck Center for Integrative Neuroscience and the Silvio Conte Center for Neuroscience Research.
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FOOTNOTES |
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Address for reprint requests: R. A. Nicoll, Box 0450, Dept. of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143-0450.
Received 25 August 1997; accepted in final form 9 December 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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