Developmental changes in presynaptic Ca²⁺ clearance kinetics and synaptic plasticity in mouse Schaffer collateral terminals

Abstract

Presynaptic Ca²⁺ influx pathways, cytoplasmic Ca²⁺ buffering proteins and Ca²⁺ extrusion processes undergo considerable change during the first postnatal month in rodent neurons. These changes may be critical in establishing short-term plasticity at maturing presynaptic terminals where neurotransmitter release is directly dependent on the dynamics of cytoplasmic residual Ca²⁺ ([Ca²⁺]_res). In particular, the robust paired-pulse facilitation characteristic of adult neurons is almost entirely lacking in newborns. To examine developmental changes in processes controlling [Ca²⁺]_res, we measured the timecourse of [Ca²⁺]_res decay in presynaptic terminals of Schaffer collateral to CA1 synapses in acute hippocampal slices following single and paired orthodromic stimuli in the stratum radiatum. Developmental changes were observed in both the rise time and slow exponential decay components of the response to single stimuli such that this decay was larger and faster in the adult. Furthermore, we observed a greater caffeine-sensitive basal Ca²⁺ store, which was differentially affected when active uptake into the endoplasmic reticulum was blocked, in the presynaptic fields of the Schaffer collateral to CA1 terminals of P6 and younger mice when compared to adults. These transitions in [Ca²⁺]_res dynamics occurred gradually over the first weeks of postnatal life and correlated with changes in short-term plasticity.

Introduction

Marked developmental changes occur in the rodent hippocampus during the first 3 weeks after birth, with consequences that affect a wide range of processes extending from the expression and regulation of proteins to the determination of cell morphology and phenotype (Dumas, 2005; Danglot et al., 2006). This early postnatal period of rodent brain development correlates with a parallel developmental period in humans during which the onset of many neurological disorders including obsessive–compulsive disorder, attention-deficit hyperactivity disorder and schizophrenia are believed to occur (Anderson, 2002; Dumas, 2005).

Paired-pulse facilitation (PPF) is a measure of short-term plasticity in which the efficacy of the second of paired synaptic responses is increased in amplitude over that of the first response. It is generally accepted that PPF results from presynaptic residual Ca²⁺ ([Ca²⁺]_res) and its downstream effects (Katz & Miledi, 1968; Wu & Saggau, 1994; Cabezas & Buno, 2006; Schiess et al., 2006) and this suggests that changes in [Ca²⁺]_res clearance after stimulation should play an important role in the kinetics of PPF. PPF in the Schaffer collateral to CA1 pyramidal neuron (SC-CA1) synapse exhibits significant change during the early postnatal period of development, during which time there is an increase in the magnitude of PPF and a predicted change in [Ca²⁺]_res regulation (Wasling et al., 2004). Thus the initiation of PPF can provide a criterion that distinguishes the transition between immature and mature synaptic transmission.

Significant changes in the kinetics of presynaptic Ca²⁺ dynamics at SC-CA1 synapses during early postnatal periods are likely to be crucial to the maturation of synaptic plasticity and brain function; however, little is known about the development of presynaptic components of synaptic plasticity at this synapse (Hanse et al., 2009). In adult rodents, the presynaptic [Ca²⁺]_res clearance in SC-CA1 terminals has been accurately described as a double exponential process (Schiess et al., 2006), which suggests contributions from more than one underlying mechanism. These mechanisms could include buffering, diffusion, uptake into or release from internal stores, and extrusion (Neher & Augustine, 1992; Koester & Sakmann, 2000; Emptage et al., 2001; Carter et al., 2002; Cabezas & Buno, 2006). The [Ca²⁺]_res clearance in SC-CA1 presynaptic terminals of immature animals may be even more complex. While [Ca²⁺]_res clearance is only minimally sensitive to Ca²⁺ buffer saturation in rodent SC-CA1 terminals, immature terminals are somewhat more sensitive than are adult terminals (Blatow et al., 2003). This small change in buffer saturation, however, does not appear to account for the large developmental change in short-term synaptic plasticity (Dekay et al., 2006). One attractive alternative possibility, which could contribute significantly to [Ca²⁺]_res kinetics but has not been analyzed in this synapse, is developmental changes in the process of Ca²⁺ uptake into, and release from, internal stores. In this study, we have addressed this question and we report modifications in [Ca²⁺]_res clearance kinetic processes that correlate well with developmental changes in short-term synaptic plasticity.

Materials and methods

Slice preparation

Experiments were performed in coronal hippocampal slices prepared from C57/Bl6 mice at ages described in each experiment; adults were used between 60 and 120 days, average age 86 days. Animals were deeply anaesthetized by i.p. injection of 250 mg kg⁻¹ ketamine (Fort Dodge Animal Health, Fort Dodge, IA, USA), brains were rapidly removed, and slices were cut at 300–400 μm with a vibroslicer (Pelco 101, St Louis, MO, USA) in an ice bath with a cutting solution containing (in mM): sucrose, 220; KCl, 3; NaH₂PO₄, 1.2; NaHCO₃, 26; MgSO₄, 12; CaCl₂, 0.2; glucose, 10; and ketamine, 0.01 mg mL⁻¹; equilibrated with 95%O₂–5%CO₂. Slices were then transferred to a bath containing artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 1 MgSO₄, 26 NaHCO₃, 2.5 CaCl₂ and 10 glucose equilibrated with 95%O₂–5%CO₂ at 30°C for 1 h and then maintained at room temperature until transfer to a temperature-controlled recording chamber (Warner Instruments, Hamden, CT, USA or Scientific Systems Design, Mercerville, NJ, USA), which was maintained at 32°C and continuously perfused at 2 ml min⁻¹ with ACSF saturated with 95%O₂–5%CO₂. All experiments were approved by the Institutional Animal Care and Use Committee at the University of New Mexico Health Sciences Center and conformed with NIH guidelines.

Presynaptic Ca²⁺ imaging

Presynaptic fibers were filled with the Ca²⁺ fluorophore, Mg Green AM or Fura2 AM (Molecular Probes, Eugene, OR, USA) using an established technique that measures a spatial and temporal average of [Ca²⁺] in the presynaptic terminal (Regehr & Tank, 1991; Wu & Saggau, 1994; Atluri & Regehr, 1996; Sinha et al., 1997; Kamiya & Ozawa, 1999). This technique provides simultaneous measurements of presynapic calcium and postsynaptic fEPSP in localized populations of synaptic contacts. In a small number of instances either the presynaptic or postsynaptic recording was not successful, but the data from the successful component were still included. To minimize the effect of exogenous buffers we used Mg Green, with a Ca²⁺ binding K_D = 6 μM, for experiments in which we measured the timecourse of ΔF/F₀ decay (Regehr & Atluri, 1995; Atluri & Regehr, 1996); however, in some experiments where the rapid decay timecourse was not being assessed, we improved the sensitivity of the ΔF/F₀ measurements by using the higher affinity fluorophore Fura2. Briefly, an ejection electrode (tip diameter 5–10 μm) containing the fluorophore (0.9mM Mg Green AM, 10% DMSO, 1% pluronic acid in ACSF or 1 mM Fura2 AM, 10% DMSO, 1% pluronic acid in ACSF) was lowered into the fiber pathway between the stimulating electrode and the presynaptic terminal field to be investigated. While observing the emission image following excitation (490 nm Mg Green AM or 350 nm Fura2 AM) an air pressure pulse was applied with a syringe to the ejection electrode until a small bright spot (≈1 μL) was observed in the fiber pathway.

The slice was then maintained with a 2 mL min⁻¹ flow of oxygenated ACSF at 32°C for 1 h to allow intracellular diffusion of the dye to the presynaptic imaging site 500 μm away from the ejection site. The excitation light was then reduced to a 100- to 200-μm-diameter spot with a diaphragm in the epi-illumination path, and the emitted light was measured with a photomultiplier tube. This spot included or was immediately adjacent to the area electrically summed by the field potential recording. A single stimulus or pairs of stimuli were delivered orthodromically at 0.05 or 0.067 Hz by a Master 8 pulse generator (AMPI Instruments, Jerusalem, Israel) under control of the imaging system (TILL Photonics, Pleasanton, CA, USA). For Mg Green studies, fluorescence responses are reported as the ratio of the change in fluorescence to the pre-stimulus fluorescence (ΔF/F₀). For Fura-2 studies, we first determined the 350–380 nm fluorescent ratio and reported the response as the ratio of the change in this ratio to the pre-stimulus ratio (ΔR/R₀). The ΔF/F₀ and ΔR/R₀ signals were corrected for bleaching by subtraction of a linear baseline slope and were inverted so that increasing presynaptic [Ca²⁺]_i produced an upward deflection. To diminish noise inherent with the low-affinity Ca²⁺ indicator, it was necessary to average five fluorescence responses and to filter the photomultiplier tube signal at 1 kHz.

We used two tests to demonstrate that the measured ΔF/F₀ signal was consistent with a [Ca²⁺] response predominately from the presynaptic Schaffer collateral axons and axon terminals (Wu & Saggau, 1994; Atluri & Regehr, 1996; Sinha et al., 1997; Kamiya & Ozawa, 1999). First, in the presence of 10 μM CNQX, 25 μM D-AP5 and 20 μM bicuculline, the fEPSP was blocked while the presynaptic fiber volley and ΔF/F₀ signal were left unchanged; the ΔF/F₀ signal was thus not a postsynaptic response. Second, subsequent addition of 600 nM TTX blocked both the presynaptic fiber volley and the ΔF/F₀ signal, arguing against direct stimulation of inadvertently filled postsynaptic dendrites.

Field potential recordings

We used standard electrophysiological techniques for slice field excitatory postsynaptic potential (fEPSP) recordings in the SC-CA1 pyramidal neuron synapse in the stratum pyramidale in hippocampal slices (Schiess et al., 2006). Briefly, fEPSPs were recorded with an Axoclamp 2B or Multiclamp 700B amplifier (both from Axon Instruments, Union City, CA, USA) and a Digidata 1322A interface using PCLAMP 9.2 or 10 software (Axon Instruments) for experimental control and data analysis. fEPSPs were digitized at 20 kHz and filtered at 2 kHz. Presynaptic constant-current pulses (150 μs duration) were applied to Schaffer collateral fibers with an Iso-Flex constant current stimulator (API Instruments, Jerusalem, Israel) through a concentric bipolar electrode (FHC, Bowdoinham, ME, USA), at a current which was adjusted to produce 40–60% of the maximum fEPSP amplitude. The paired-pulse ratio (PPR) of the fEPSP signal was calculated as the ratio of the slope of the second fEPSP at a 50-ms interpulse interval to that of the initial fEPSP. The relationship between [Ca²⁺]_res and PPR was determined using concurrent ΔF/F₀ and fEPSP recordings of paired pulses at interpulse intervals of 50, 100, 150, 200, 300 and 500 ms. The [Ca²⁺]_res was calculated by measuring the amplitude of the ΔF/F₀ immediately before the second pulse of each pair.

Data analysis

Combining data, even for single developmental days, from the period during which there are rapid changes in [Ca²⁺]_res dynamics is expected to introduce variability into measured parameters. Indeed, our data from early developmental points varied substantially and this variability decreased with increasing age. We have interpreted this to be an indication of the narrow temporal window over which some of these maturation processes occur.

The ΔF/F₀ signal was digitally filtered with a five-point center-weighted filter and fit using a least-squares regression routine to a single exponential decay beginning 50 ms after the stimulus initiation. In addition, the ΔF/F₀ signal was numerically integrated before (∫ΔF/F₀) or after being normalized to the ΔF/F₀ peak value (∫νΔF/F₀). The rise time was measured by subtracting the time of the ΔF/F₀ peak value and the time of stimulation. Fitting and statistical analysis were carried out with MATLAB 7.0 (Mathworks, Natick, MA, USA), PROSTAT 4.0 (Polysoft International, Pearl River, NY, USA) or PRISM (GraphPad Software, La Jolla, CA, USA). Coefficient of determination (COD) was used as a measure of goodness of fit for linear regressions. Average data are presented as means ± SEM, and statistical significance was determined at P < 0.05.

Drugs

Drugs were stored frozen in aliquots and diluted to the appropriate concentration in ACSF on the day of the experiment. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX), d-(1)-2-amino-5-phosphonopentanoic acid (d-AP5), bicuculline, thapsigargin and tetrodotoxin (TTX) were obtained from Tocris (Ellisville, MO, USA) and all other drugs and reagents were obtained from Sigma-Aldrich (St Louis, MO, USA). Drugs were applied through a bath perfusion system for a minimum of 10 min (> 2.5 bath exchanges) before recording commenced. The timecourse experiments were continuously recorded during bath exchange and the time for complete response was expected to result from a combination of the bath exchange time constant (70 s) and the time necessary for drug permeation into the slice.

Results

Previous studies have shown that there is a developmental increase in hippocampal short-term facilitation during the first few postnatal weeks in rats (Wasling et al., 2004). To determine whether a similar developmental change occurs in mice we measured the PPR of fEPSPs in response to paired stimuli at 50-ms interpulse intervals in hippocampal slice preparations from P1 to adult (> P60; Fig. 1A). Similar to the findings in rats, we found a developmental increase in PPR at the SC-CA1 synapse in the mouse between P6 and P9 (Fig. 1B); however, no significant additional change was observed between P15 and adult animals.

Fig 1.

Developmental increase in short-term synaptic plasticity. (A) Representative traces of fEPSP slopes at half maximum at two developmental stages during paired stimulation at 50-ms interpulse interval, average of five traces [A1, immature (P6); A2, adult (P114)]. (B) PPR at different developmental ages (ANOVA with Fisher’s post hoc comparison of significance of PPR vs. age between groups, F_4,25 = 8.71, P = 0.0002; P1, n = 4; P3, n = 6; P6, n = 11; P9, n = 6; P15, n = 4; adult, n = 3 animals). **P < 0.01 and ***P < 0.001.

As PPF is believed to be dependent on [Ca²⁺]_res, we next chose to measure the kinetics of [Ca²⁺]_res during this developmental period. To characterize the developmental changes of presynaptic Ca²⁺ kinetics in the SC-CA1 synapse, we used Ca²⁺-sensitive fluorescent dyes to measure presynaptic [Ca²⁺]_res following stimuli in the stratum radiatum in brain slices from animals aged between P1 and P120. For this purpose, presynaptic terminals were loaded with AM-derivatives of either Mg Green for millisecond kinetic analysis or Fura-2 for more sensitive analysis of [Ca²⁺]_pre on a timescale of seconds (see Materials and Methods). Previous investigations in adult hippocampus in our lab (Schiess et al., 2006) and other labs (Regehr & Tank, 1991; Wu & Saggau, 1994) have demonstrated that this method reports almost exclusively Ca²⁺ dynamics of presynaptic terminals (see Materials and Methods, and Discussion). We evaluated quantitatively the changes in [Ca²⁺]_res kinetics by first measuring the change in fluorophore fluorescence of the Mg Green signal (ΔF/F₀) in response to a single stimulus. To obtain a measure of [Ca²⁺]_res timecourse that made no assumptions about the specific underlying kinetic processes we calculated the integral of ΔF/F₀ after it had been normalized to its peak; this provides a measure of the overall kinetic behavior of the [Ca²⁺]_res transient (Fig. 2, A1 and A2; ∫νΔF/F₀). We found a significant age-dependent decrease in the ∫νΔF/F₀ (Fig. 2B), which is consistent with a more rapid [Ca²⁺]_res clearance kinetics in older animals.

Fig 2.

Developmental changes in presynaptic ∫νΔF/F₀. (A) Representative traces of ∫νΔF/F₀ at two developmental ages (A1, P6; A2, Adult (P85); Mg Green) (B) Summary data of ∫νΔF/F₀ vs. age. ANOVA with Fisher’s post hoc comparison of significance of ∫νΔF/F₀ vs. age between groups: F_40,7 = 6.65; P = 0.00001; P1–P3, n = 8; P6, n = 12; P9, n = 5; P12, n = 4; P15, n = 3; P21–P27, n = 9; P30, n = 3; adult, n = 4 animals. (C) Concurrent recordings of fEPSP PPF and ∫νΔF/F₀ (P1–P6, grey open squares, n = 20 animals; P9–adult, black circles, n = 15 animals) fit by least-squares linear regression. *P < 0.05, **P < 0.01 and ***P < 0.001.

To assess the relationship between presynaptic Ca²⁺ kinetics and facilitation we plotted the ∫νΔF/F₀, following a single pulse against the PPR observed in concurrent postsynaptic recordings (Fig. 2C). Because there appeared to be a distinct transition in PPR between P6 and P9, we grouped the data into two groups: P1–P6 and P9–adult. We found a negative slope in the P9–adult group, consistent with the dependence of facilitation on ∫νΔF/F₀ with decreased probability of release and enhanced facilitation (slope = −1.36 × 10⁻⁵, COD = 0.29). In contrast, however, the P1–P6 age group did not exhibit a negative slope and the data were more poorly correlated (slope = 0.521 × 10⁻⁵, COD = 0.14). This suggests a developmental transition between P6 and P9 during which presynaptic [Ca²⁺]_res assumes a role in short-term facilitation. Typically, increased [Ca²⁺]_res correlates with increased facilitation and depletion of vesicles correlates with decreased facilitation (Liley & North, 1953; Takeuchi, 1958; Elmqvist & Quastel, 1965; Thies, 1965; Betz, 1970; Zucker & Regehr, 2002). However, the negative correlation between ∫νΔF/F₀ and PPR suggests [Ca²⁺]_res removal mechanisms are important in the amount of facilitation. Interestingly, a more rapid [Ca²⁺]_res removal is reflected by decreased ∫νΔF/F₀ and this is correlated with increased facilitation. To determine the processes that contribute to the regulation of [Ca²⁺]_res during this important developmental period, we further investigated the kinetics of [Ca²⁺]_res clearance.

As shown in Fig. 3A, there were distinct age-dependent differences in the decay of the Mg Green fluorescence signal, indicative of a change in the rate of [Ca²⁺]_res clearance between P6 and adult mice. We first measured the kinetic components responsible for Ca²⁺ accumulation, peak amplitude of ΔF/F₀, and time to peak of ΔF/F₀. There was an approximate doubling of the peak amplitude of ΔF/F₀ from P1 to adult (Fig. 3B). Because this could have resulted in part from a difference in Ca²⁺ influx or in the kinetics of an internal store contribution, we also compared the time at which the peak ΔF/F₀ occurred at the different ages. This analysis revealed a dramatic monotonic 20-fold decrease in the time to peak ΔF/F₀ from P1 to adult (Fig. 3C). Taken together, the age-dependent decrease in the ∫νΔF/F₀ signal must therefore reflect an overall increase in the rate of [Ca²⁺]_res clearance, as there was a simultaneous increase in the peak ΔF/F₀ amplitude.

Fig 3.

Developmental changes in presynaptic [Ca²⁺]_res kinetics. (A) Representative traces of ΔF/F₀ during single stimulus pulses at two developmental ages (top, P6; bottom, P114; Mg Green). (B) Summary data of maximum ΔF/F₀ vs. age. ANOVA with Fisher’s post hoc comparison of significance of peak ΔF/F₀ between age groups: F_40,7 = 4.12, P = 0.0017. (C) Time to peak ΔF/F₀ vs. age. ANOVA with Fisher’s post hoc comparison of significance of time to peak between age groups: F_40,7 = 4.21, P = 0.0016. P1–P3, n = 8; P6, n = 12; P9, n = 5; P12, n = 4; P15, n = 3; P21–P27, n = 9; P30, n = 3; adult, n = 4 animals. Further analysis of this data is shown in Figs 2 and 4. (D) Relationship of PPR vs. [Ca²⁺]_res measured as % ΔF/F₀ before stimulation compared to the PPR at IPIs of 50, 100, 150, 200, 300 and 500 ms; least-squares linear regression yields a nonsignificant difference in slope between groups (slope= 0.7756; F_99,3 = 0.18, P = 0.91; P3–P6, black squares, black line, n = 4; P9, gray squares, gray line, n = 4; P15, gray circles, dashed gray line, n = 4; P30, black diamonds, dashed black lines, n = 4 animals) (E) Y-intercept vs. age from the linear regression of PPR vs. [Ca²⁺]_res; significant difference of age vs. intercept (F_102,3 = 17.97, P < 0.0001). *P < 0.05, **P < 0.01 and ***P < 0.001.

As the [Ca²⁺]_res has been associated with the degree of PPF, we next compared the relationship between [Ca²⁺]_res and the simultaneously measured PPF in the different aged groups by changing the interpulse interval while keeping the stimulation strength constant (Fig. 3D). Analysis of these data demonstrated there was a significant positive slope of this relationship in slices from older animals: P9 (slope=0.96 PPR/%ΔF/F₀, COD = 0.23, F_1,25 = 7.47, P = 0.011), P15 (slope = 0.97 PPR/%ΔF/F₀, COD = 0.27, F_1,24 = 8.68, P = 0.0071) and adult mice (slope = 1.17 PPR/%ΔF/F₀, COD = 0.1752, F_1,25 = 5.31, P = 0.023). While the slopes of this relationship were not significantly different (Fig. 3D, P = 0.93) among all age groups, the intercept was found to increase significantly with age (Fig. 3E). By contrast, the slope of this relationship for slices in the younger P3 and P6 mice, which do not exhibit significant PPF, did not differ significantly from zero (P3–6 slope = −0.23, COD = 0.06, F_1,26 = 1.78, P = 0.19). This suggests that the mechanisms that underlie the [Ca²⁺]_res-dependent facilitation are not estabilised or are disengaged until the second postnatal week of hippocampal development (see Disscusion).

As we reported previously for presynaptic SC-CA1 terminals in adult rats (Schiess et al., 2006), the ΔF/F₀ decay in these terminals in adult mice followed a double exponential timecourse whose time constants differed by ~ 10-fold (not shown). In contrast, the presynaptic ΔF/F₀ decay in younger mice could not be accurately fit with the sum of two decaying exponential processes and we attributed this to the significantly delayed ΔF/F₀ peak time in the younger animals. In adult mice, the fast ΔF/F₀ clearance process had a time constant of ~20 ms. Consequently, the significantly slower rising kinetic component in the younger animals (time to peak = 30.95 ± 8.31 ms) markedly overlapped this portion of [Ca²⁺]_res clearance (Fig. 3). Therefore, in order to assess developmental changes in [Ca²⁺]_res clearance kinetics independently of the initial Ca²⁺ accumulation, we measured the decay of ΔF/F₀ beginning 50 ms after its peak when the fast rise and decay contributions were minimal. We reasoned that changes in either the rate or the proportion of this slower component of Ca²⁺ decay would have considerable impact on the overall response. To obtain an estimate of this kinetic component, we fit the slow component of the remaining ΔF/F₀ decay as a single exponential process (Fig. 4A). The rate of this slow decay component was then extrapolated to the time of the peak of ΔF/F₀ in order to obtain the value indicated as ‘b’ in the rise time in Fig. 4A. The ratio of this value to the actual peak (‘a’ in Fig. 4A) provided an estimate of the contribution of the slow component to the peak ΔF/F₀ signal. We found an age-dependent decrease in this ratio (b/a, Fig. 4C) indicating an age-dependent increase in the relative contribution of a process with a fast time constant. Interestingly, the increased peak ΔF/F₀ (Fig. 3B) and decreased time to peak (Fig. 3C) offset the decreased slow time constant (Fig. 4B) to minimize any difference in ∫νΔF/F₀ between the P1–P3 and P6 age groups (Fig. 2B). Thus, the most dramatic reduction in ∫νΔF/F₀ occurred between P6 and P9 (Fig. 2B). These data further indicate that the most dramatic change in [Ca²⁺]_res timecourse occurred over the developmental period of P6 to P9, strongly suggesting a significant transition in the underlying mechanisms of [Ca²⁺]_res clearance during this period. Importantly, the developmental changes that led to a decrease in total [Ca²⁺]_res, as represented by the ∫νΔF/F₀, coincided with the simultaneously recorded increase in the amount of PPF (see Discussion).

Fig 4.

Kinetic components of [Ca²⁺]_res evaluated from fits to ΔF/F₀. (A) Example ΔF/F₀ trace (P9); ‘a’ is the measured peak and ‘b’ the predicted peak from extrapolated single exponential fit to ΔF/F₀ data beginning 50 ms after the peak (Mg Green). (B) Slow time constant (τ_slow) measured by exponential fit to ΔF/F₀ trace beginning 50 ms after the peak. ANOVA with Fisher’s post hoc comparison of significance of slow time constant between age groups: F_40,7 = 3.03, P = 0.012. (C) Ratio of the extrapolated peak of τ_slow to the peak amplitude of the ΔF/F₀ signal. ANOVA with Fisher’s post hoc comparison of significance of between ratio of predicted peak to actual peak between age groups: F_40,7 = 3.38, P = 0.0067. P1–P3, n = 8; P6, n = 12; P9, n = 5; P12, n = 4; P15, n = 3; P21–P27, n = 9; P30, n = 3; adult, n = 4 animals; *P < 0.05, **P < 0.01 and ***P < 0.001.

We next focused on the possibility that the mechanism that drives the developmental decrease in the [Ca²⁺]_res clearance timecourse could be linked to changes in either release from, or uptake into, internal Ca²⁺ stores. A contribution from Ca²⁺ stores would be consistent with the observation that internal stores have a contrasting developmental role in silent and active synapses and that the endoplasmic reticular SERCA (sarco/endoplasmic reticulum Ca²⁺-ATPase) pump plays an important role in PPF (Cabezas & Buno, 2006).

To investigate the possibility that Ca²⁺ stores play an important role in the regulation of presynaptic [Ca²⁺]_res, we used thapsigargin to block the uptake of Ca²⁺ into the endoplasmic reticulum (ER) by the SERCA pump and measured the effect of SERCA block on the ∫ΔF/F₀ signal following stimulation. To compare the timecourse of ∫ΔF/F₀ at different ages, we normalized the ∫ΔF/F₀ in 3 μM thapsigargin to the response in the same slice in ACSF (Fig. 5). Interestingly, at P3 thapsigargin caused a decrease in ∫ΔF/F₀ while at P5–P7 it caused no change in ∫ΔF/F₀, and it clearly enhanced the ∫ΔF/F₀ of adults (Fig. 5A). To further test whether SERCA clearance is involved in plasticity, we recorded the PPR of fEPSPs and found that application of thapsigargin had no significant effect in the P5–P7 age group, and a trend toward a decrease in the P3 mice. However, application of thapsigargin did cause an increase in the PPR of the adult mice (Fig. 5C). This suggests that during this time period (P5–P7) there is a rapid transition in the contribution from ER Ca²⁺ stores at synaptic terminals. Furthermore, either release from stores or uptake by the SERCA pump may be reflected in the slow time constant of the [Ca²⁺]_res clearance.

Fig 5.

Developmental changes in the effect of thapsigargin on [Ca²⁺]_pre clearance. ∫ΔF/F₀ following a single pulse in 3 μM thapsigargin normalized to the ∫ΔF/F₀ response in ACSF. (A) Representative traces (left, P3 and right, adult; gray, ACSF and black, thapsigargin) normalized to the respective ACSF traces. (B) Summary data. P3, n = 3; P5–P7, n = 4; adult, n = 5 animals; Student’s t-test, against theoretical mean of 1. (C) Effect of application of thapsigargin on PPR vs. age (P3, n = 4, P = 0.11; P5–P7, n = 6, P = 0.34; adult, n = 5 animals); *P < 0.05 and **P < 0.01, Student’s t-test against a theoretical mean of 1.

In many cell types, thapsigargin unmasks a basal leak of Ca²⁺ from intracellular stores (Verkhratsky, 2005). We reasoned that thapsigargin would produce an increase in the cytoplasmic Ca²⁺ that would be measurable with the high affinity indicator Fura2 (ΔR/R₀ signal) if the SERCA pump normally counteracted a significant leak from Ca²⁺ stores. This could be relevant to our findings because such a leak might contribute to the difference in [Ca²⁺]_res timecourse which we observed between immature and adult SC-CA1 terminals. Importantly, we did not find a significant increase in ΔR/R₀ following thapsigargin application in nonstimulated slices at any age (data not shown). This observation is consistent with the idea that the activity of SERCA does not continually oppose a measurable Ca²⁺ leak from intracellular Ca²⁺ stores in presynaptic terminals from either young or adult animals.

We next examined whether changes in the participation of Ca²⁺-dependent release from presynaptic Ca²⁺ stores could contribute to the observed age-dependent differences in the [Ca²⁺]_res clearance time-course. Thus, we tested the contribution of these internal stores to [Ca²⁺]_res in SC-CA1 terminals from both young and adult mice by measuring the relative change in ΔR/R₀ in response to bath application of 10 mM caffeine. Caffeine increases the Ca²⁺ sensitivity of ryanodine receptors and thereby initiates release of Ca²⁺ from Ca²⁺-sensitive internal stores into the cytoplasm (Verkhratsky, 2005). As shown in Fig. 6A, there was a consistent, significant increase in ΔR/R₀ following application of caffeine in SC-CA1 terminals of young mice (P2–P6) but no equivalent increase in ΔR/R₀ in adults (Fig. 6B). Furthermore, the normalized change in the effectiveness of caffeine occurred during the narrow developmental time period between P6 and P9 (Fig. 6C). Both the thapsigargin and caffeine data suggest that some of the developmental decrease in the timecourse of [Ca²⁺]_res clearance results from a decreasing effectiveness of internal stores involvement during this process.

Fig 6.

Age-dependent differences in basal loading state of Ca²⁺ stores. (A) Timecourse of ΔR/R₀ using Fura-2 in adult (black) and young (grey) mice. (B) Average non-normalized ΔR/R₀ in ACSF (four samples, between 375 and 435 s) vs. ΔR/R₀ in 10 mM caffeine (four samples between 525 and 585 s). Left, young mice (P2–P6, n = 5 animals); right, adult mice (> P60, n = 5 animals; P = 0.45; paired Student’s-t test. (C) Caffeine-induced change in ΔR/R₀ normalized to internal control in ACSF (at 375–435 s): P2–P6, n = 5; P9, n = 5; P15, n = 5; adult, n = 5; paired Student’s t-test. There was no significant change in baseline ΔR/R₀ at any age without application of caffeine (P2–P6, P = 0.38, n = 3; P9, P = 0.22, n = 3; P15, P = 0.38, n = 3; adult, P = 0.30, n = 3); *P < 0.05 and ***P < 0.001, paired Student’s t-test.

To investigate the contribution of Ca²⁺ clearance to short-term plasticity, we measured the ΔF/F₀ response during paired pulses at a 50-ms interpulse interval (Fig. 7A). We computed the ratio of the paired-pulse ∫ΔF/F₀ to the single-pulse ∫ΔF/F₀ in order to compare the changes in [Ca²⁺]_res between the first and second of paired stimuli. We have previously shown in SC-CA1 terminals from adult rats that, after subtracting the residual ΔF/F₀ that remains following the first of paired pulses, there is an increase in ∫ΔF/F₀ resulting from the second pulse (Schiess et al., 2006). This increase would lead us to anticipate a ratio > 2 of the paired-pulse ∫ΔF/F₀ to the single-pulse ∫ΔF/F₀. As expected, the ∫ΔF/F₀ ratio was significantly greater than two in the adult mouse, indicating a supralinear increase in [Ca²⁺]_res during the second pulse (Fig. 7B). Surprisingly, however, we found that at P1 the ∫ΔF/F₀ ratio was significantly less than two and that between P3 and P9 the ∫ΔF/F₀ ratio did not differ from two. Thus the short-term effect of [Ca²⁺]_res on subsequent Ca²⁺ clearance reflects unique characteristics at P1 and the adult phenotype is not established until ~ P15.

Fig 7.

Developmental increase in [Ca²⁺]_res during paired pulses. (A) Representative traces of ΔF/F₀ during paired stimulation [A1, P6; A2, adult (P85); Mg Green]. (B) Ratio of the ∫ΔF/F₀ for paired stimuli vs. ∫ΔF/F₀ for single stimulus at different developmental ages (P1, n = 4; P3, n = 6; P6, n = 11; P9, n = 6; P15, n = 4; adult, n = 5 animals); *P < 0.05, Student’s t-test against a theoretical mean of 2.

Discussion

It has been proposed that developmental changes in short-term facilitation are correlated with changes in presynaptic Ca²⁺ (Wasling et al., 2004). We demonstrate here that there is a developmental threshold for the involvement of presynaptic [Ca²⁺]_res in the regulation of short-term synaptic facilitation. This temporal change occurs between P6 and P9, at which time there is a marked alteration in the [Ca²⁺]_res clearance, in the amount of [Ca²⁺]_res following a single pulse and in the accumulation of Ca²⁺ in intracellular stores. This was apparent in the incremental change in the general shape of the [Ca²⁺]_i response (Fig. 3A), in the peak amplitude of this response (Fig. 3B) and in the time that it takes to reach the peak (Fig. 3C). The developmental decrease in the ∫νΔF/F₀ (Fig. 2) further indicates that there are important age-dependent changes in the time course of [Ca²⁺]_res clearance and hence the total amount of Ca²⁺ available in the presynaptic terminal.

PPF at a 50-ms interpulse interval undergoes a significant developmental increase after the first postnatal week (Fig. 1). Other studies have shown an additional decrease in the PPR between juvenile (P13–P18) and young adult (P28–P42) rat SC-CA1 terminals (Speed & Dobrunz, 2008). Although we observed a small decline in the PPR between P15 and adult, this decrease was not significant. The difference in these observations could be a result of the broader range of ages of our adult animals, or of species differences between mice and rats. Additionally, while we consistently placed our stimulating electrode in the stratum radiatum, where we would expect to stimulate only Schaffer collateral fibers, it is possible that in some instances the stimulus current could have spread to temporoammonic fibers in the stratum lacunosum–moleculare. In contrast to the decrease in PPF between juvenile and adult Schaffer collateral terminals, PPF in temporoammonic fiber terminals increases during this developmental period (Speed & Dobrunz, 2009). It is conceivable, therefore, that current spread to stratum lacunosum–moleculare in some of our slices could have counteracted the expected age-dependent decrease in the SC-CA1 terminals.

Wasling et al., (2004) have shown that there is a developmental change in release probability of the first of paired responses that is indicative of this change in facilitation in rats. While our determination of spatially and temporally averaged ΔF/F₀ does not directly reflect the [Ca²⁺] in the release domain, nor does it typically reflect influences on release proability of the initial pulse, changes in the measured rise time (Fig. 3C) do suggest a developmental change in the [Ca²⁺] that determines release during the first pulse. Although action potentials are a few milliseconds broader in immature than in adult neurons (Lockery & Spitzer, 1992; Gao & Ziskind-Conhaim, 1998), this increased period of depolarization is not expected to increase the duration of Ca²⁺ influx sufficiently to account for the observed slower rise time of [Ca²⁺]_res. A possible explanation for the slower clearance of [Ca²⁺]_res in slices from young animals could be maturational changes from growth cones and silent synapses to mature synapses. Growth cones differ from mature synapses in their morphology and volume, their larger number of L-type Ca²⁺ channels (Ohbayashi et al., 1998), and the role of Ca²⁺-induced Ca²⁺ release (Tojima et al., 2007). Functionally, influx through L-type Ca²⁺ channels has been shown to play a role in activation of presynaptic silent synapses (Yao et al., 2006) and in the change from silent to active synapses (Gasparini et al., 2000; Cabezas & Buno, 2006). Any of these factors might be a significant contributory factor to the observed maturational differences in [Ca²⁺]_res clearance. However, these alterations in [Ca²⁺]_res kinetics would not be expected to contribute to the observed changes in PPF as short-term synaptic plasticity must depend on mechanisms specific to presynaptic terminals of functional synapses.

In presynaptic terminals of the adult rodent brain, [Ca²⁺]_res decay kinetics are accurately reflected by the sum of two exponentials (Schiess et al., 2006), which indicates either two separate populations with first-order kinetics or a multi-step process. A likely candidate for such a multi-step process is the presynaptic Ca²⁺ clearance by the endoplasmic reticular SERCA pump (Higgins et al., 2006). The decay kinetics for [Ca²⁺]_res in presynaptic terminals of immature SC-CA1 synapses is slower, and is not accurately fit with a double exponential. We thus chose to analyze the timecourse of [Ca²⁺]_res decay in immature synapses by dividing it into the temporally distinct components of peak time, slow time constant, and the ratio of the peak slow component to the peak amplitude. As shown in Figs 3C, 4A and 4B, each of these components showed an age-dependent decrease. This indicates a developmental change in the underlying kinetic processes and, consequently, marked differences in the mechanisms of [Ca²⁺]_res clearance in immature and mature synapses that are coincident with the establishment of the adult phenotype of short-term plasticity (Wasling et al., 2004; Dumas, 2005). Importantly, expression levels and isoform expression of proteins with demonstrated roles in synaptic plasticity and Ca²⁺ sensitivity have been shown to change during this maturational period, including, for example, adenylyl cyclases 1 and 8 (Conti et al., 2007), CDK1/4 (Li et al., 2007), and SNAP-25 (Bark et al., 1995).

As noted above, the multi-step kinetics of [Ca²⁺]_res clearance are consistent with the established multi-step kinetics of SERCA sequestration, making changes in this pathway likely candidates for the mechanism for our observations. There are conflicting findings regarding the role of Ca²⁺-induced Ca²⁺ release from internal stores in short-term plasticity (Emptage et al., 2001; Carter et al., 2002; Cabezas & Buno, 2006). Furthermore, age-dependent changes in ryanodine receptor expression levels have been shown to play a role in endogenous cannabinod mobilization (Isokawa & Alger, 2006) and the maintenance of presynaptic forms of LTP (Martin & Buno, 2003). We therefore reasoned that developmental changes in the ability to sequester Ca²⁺ in intracellular stores could affect the timecourse of [Ca²⁺]_res clearance directly through [Ca²⁺]_res uptake from the cytoplasm, or indirectly because of a counteracting Ca²⁺ release into the cytoplasm. We found that blocking SERCA-dependent Ca²⁺ sequestration increased the ∫ΔF/F₀ in adult presynaptic terminals while causing a decrease at P3 and no difference in the P5–P6 age group. Even with the more sensitive Fura2 measurements we were unable to discern a passive Ca²⁺ release from stores induced by thapsigargin in slices from either young or adult animals; however, thapsigargin slowed [Ca²⁺]_res clearance in slices from animals older than P3, but not in slices from those that were younger than P3. Additionally, we found that caffeine, which can release Ca²⁺ from ryanodine-sensitive stores, caused an increase in presynaptic [Ca²⁺]_i in the younger, but not in more mature, mice. These results suggest that, while SERCA uptake and Ca²⁺ -induced Ca²⁺ release are effective at all ages, there are developmental differences in the passive leak from stores and in the processes that participate in [Ca²⁺]_res clearance. Taken together, these data suggest that the basal filling state of intracellular Ca²⁺ stores could account for much of the developmental differences in [Ca²⁺]_res clearance. Although SERCA sequestration is effective in both immature and mature terminals, a more effective passive leak in mature neurons would yield a lower basal store of Ca²⁺ in these terminals. As a consequence of the minimal store filling state, SERCA uptake would play a more significant role in speeding [Ca²⁺]_res clearance in mature terminals; yet the more effectively filled stores of immature terminals would allow Ca²⁺-induced Ca²⁺ release to effectively slow [Ca²⁺]_res clearance. This interpretation is consistent with observations of the involvement of Ca²⁺-induced Ca²⁺ release in organotypic slice cultures from P8 animals (Emptage et al., 2001) and P15 animals (Cabezas & Buno, 2006) and the contrasting minimal effectiveness of Ca²⁺-sensitive stores in adult synapses (Carter et al., 2002).

The [Ca²⁺]_res decay kinetics in the adult rodent SC-CA1 synapse have been shown to be unaffected by Ca²⁺ buffer saturation (Blatow et al., 2003). In contrast, regulation of presynaptic [Ca²⁺]_i in immature SC-CA1 synapses (Dekay et al., 2006) has been reported to be somewhat more sensitive to Ca²⁺ buffer saturation. Our results provide additional support for the notion that the development of mature [Ca²⁺]_res clearance dynamics is not a single mechanistic change, but rather a complex series of modifications that include buffering, SERCA-dependent sequestration and Ca²⁺ dependent release from intracellular Ca²⁺ stores.

We find that although SC-CA1 presynaptic terminals of younger mice have greater total integrated [Ca²⁺]_res than adults (Fig. 2), these synapses exhibit less short-term facilitation (Fig. 1). Interestingly, although slices from mature animals did exhibit an expected positive correlation between PPF and [Ca²⁺]_res (Fig. 3D), we unexpectedly found that the zero [Ca²⁺]_i intercept in this relationship increased with age (Fig. 3E), consistent with the notion that this could reflect a maturation of the expression of a high affinity facilitatory site. Additionally, slices from adult and young animals differed markedly in the relationship between PPF and ∫νΔF/F₀ (Fig. 2C), suggesting a developmental change in the mechanisms that regulate the time course of available Ca²⁺ that influences facilitation.

Clearly, the dependence of short-term facilitation on [Ca²⁺]_res that is characteristic of adult presynaptic terminals is undeveloped before P9. As all of our pre- and postsynaptic data result from population measurements, it is impossible to draw conclusions from them about the maturation of individual synapses. It is likely that the developmental process, which we observe, does not indicate a homogenous transformation of uniform synapses but rather reflects trends in a heterogeneous population. However, regardless of their source, our data show that the developmental change in [Ca²⁺]_res kinetics that lead to the mature phenotype of a positive correlation between PPF and [Ca²⁺]_res is not firmly established before P9. Importantly, the lack of a significant positive correlation between PPF and [Ca²⁺]_res (Fig. 3D) in the P3–P6 mice could be due to a change in the relative contributions of PPD and PPF in the population of synapses.

Several underlying mechanisms can be suggested that may mediate the developmental change in the relationship between [Ca²⁺]_res and facilitation. (i) As discussed above, a varying portion of the [Ca²⁺]_i signal in the slices from immature animals could be from electronically silent growth cones or from silent synapses. (ii) The slowed [Ca²⁺]_res clearance in the younger animals could result in a greater [Ca²⁺] available to deplete the readily releasable pool of vesicles and thus counterbalance a [Ca²⁺]_res-dependent tendency toward increased facilitation (Liley & North, 1953; Takeuchi, 1958; Elmqvist & Quastel, 1965; Thies, 1965; Betz, 1970; Zucker & Regehr, 2002). (iii) Age-dependent changes in Ca²⁺-dependent inhibition of the presynaptic Ca²⁺ currents could contribute to developmental changes in short-term synaptic plasticity (Li et al., 2006; Sullivan, 2007). (iv) Expression of an independent modulatory component of the vesicle release machinery such as the high affinity site, which has been suggested as being necessary for facilitation in SC-CA1 synapses, could mature during this time period (P6–P9) (Atluri & Regehr, 1996; Bark et al., 2004; Schiess et al., 2006). (v) Finally, the change in this relationship could reflect an alteration of the local domain of Ca²⁺ that facilitates release due to a shift of the contribution from internal Ca²⁺ stores. A potential mechanism signaling this change is a difference in the kainate-sensitive glutamate receptor activation, possibly due to BDNF signaling (Lauri et al., 2006; Sallert et al., 2009).

We report here important developmental changes in short-term plasticity and the kinetics of presynaptic [Ca²⁺]_res clearance. These dramatic developmental changes could have important implications for synaptic filtering characteristics and their effects on information processing in the hippocampus (Partridge & Valenzuela, 2002). Understanding the molecular mechanisms that drive these changes will be essential to evaluate the neurological impact of presynaptic Ca²⁺ clearance on diseases that have their onset during the transitional period of the juvenile to adult nervous system.

Abbreviations

ACSF

artificial cerebrospinal fluid

[Ca²⁺]_res

residual presynaptic Ca²⁺ concentration

COD

coefficient of determination

ER

endoplasmic reticulum

ΔF/F₀

change in Mg Green fluorescence divided by the pre-stimulus fluorescence

∫ΔF/F₀

integral of ΔF/F₀

∫νΔF/F₀

integral of ΔF/F₀ normalized to peak ΔF/F₀

ΔR/R₀

change in Fura2 fluorescence ratio divided by the pre-stimulus fluorescence ratio

fEPSP

field excitatory postsynaptic potential

PPF

paired-pulse facilitation

PPR

paired-pulse ratio

SC-CA1

Schaffer collateral to CA1 pyramidal neuron

SERCA

sarco/endoplasmic reticulum Ca²⁺-ATPase