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. 2011 Sep 26;589(Pt 23):5691–5699. doi: 10.1113/jphysiol.2011.218347

The role of sensory experience in presynaptic development is cortical area specific

Claire E J Cheetham 1, Kevin Fox 1
PMCID: PMC3249043  PMID: 21946850

Non-technical summary

The visual cortex and the somatosensory whisker ‘barrel’ cortex are widely studied model systems of experience-dependent plasticity, which enables the brain to adapt to changes in the environment and is required for recovery in conditions such as stroke. It is known that presynaptic development of excitatory synapses in the cortex involves a decrease in the probability of neurotransmitter release, and a change in the dynamic properties of synapses during repetitive stimulation. These changes enable synapses in the mature brain to perform more complex functions. However, it is not known whether these developmental changes are dependent on sensory experience. In this study, we show that sensory experience is required for normal presynaptic development in barrel cortex, but not in visual cortex. Therefore, the role of sensory experience in synaptic development varies between different cortical areas. These findings are important for understanding how experience shapes neuronal circuitry during development and in disease.

Abstract

Abstract

The postsynaptic response to a stimulus is dependent on the history of previous activity at that synapse. This short-term plasticity (STP) is a key determinant of neural network function. During postnatal development, many excitatory intracortical synapses switch from strong depression during early postnatal life, to weaker depression and in some cases facilitation in adulthood. However, it is not known whether this developmental switch is an innate feature of synaptic maturation, or whether it requires activity. We investigated this question in the barrel and visual cortex, two widely studied models of experience-dependent plasticity. We have previously defined the time course over which presynaptic development occurs in these two cortical areas, enabling us to make the first direct comparison of the role of sensory experience during synaptic development. We found that maturation of STP in visual cortex was unaffected by dark rearing from before eye opening. In marked contrast, total whisker deprivation completely blocked the developmental decrease in presynaptic release probability (Pr), and the concomitant increase in paired pulse ratio (PPR), which occur in barrel cortex during the third and fourth postnatal weeks. However, the developmental increase in the steady state response to a train of stimuli was unaffected by whisker deprivation. This supports a mechanistic link between Pr and the PPR, but dissociates Pr from the steady state amplitude during repetitive stimulation. Our findings indicate that sensory experience plays a greater role in presynaptic development at L4 to L2/3 excitatory synapses in the barrel cortex than in the visual cortex.

Introduction

Synaptic transmission between neurons is dynamic, and depends on the history of previous activity at a given synapse (Tsodyks & Markram, 1997). During a stimulus train, the amplitude of postsynaptic responses can facilitate and/or depress over a timescale of milliseconds to seconds (Dobrunz & Stevens, 1997; Zucker & Regehr, 2002). These changes result from the interaction of multiple forms of STP, which can operate in parallel even at individual synapses (Markram et al. 1998; Abbott & Regehr, 2004). STP determines both the content and gain of the information transmitted by a synapse during repetitive stimulation, and hence the range of coding strategies that are available (Tsodyks & Markram, 1997).

The visual cortex and the barrel cortex are widely studied models of experience-dependent plasticity, and sensory input is readily manipulable in both systems (Fox & Wong, 2005). Studies of the cellular, synaptic and molecular mechanisms underlying cortical development and plasticity have revealed many similarities between these two sensory systems (Fox & Wong, 2005). Nevertheless, important differences between these two cortical areas have also been discovered, such as the timing of developmental critical periods (Gordon & Stryker, 1996; Stern et al. 2001) and the rates of dendritic spine motility (Majewska et al. 2006) and turnover (Holtmaat et al. 2005). The balance between STP processes is developmentally regulated at many excitatory synapses in primary somatosensory, visual (V1) and auditory cortex: these synapses undergo a ‘developmental switch’ from strong depression to weaker depression and/or facilitation during postnatal development (Reyes & Sakmann, 1999; Feldmeyer & Radnikow, 2009; Cheetham & Fox, 2010). We have shown recently that this developmental switch occurs contemporaneously with a reduction in Pr (Cheetham & Fox, 2010). Maturation of both Pr and STP at layer (L)4 to L2/3 excitatory synapses occurs later in visual cortex than in barrel cortex (Cheetham & Fox, 2010). What remains unclear is whether these developmental changes are intrinsic features of synaptic maturation, or are dependent on sensory experience.

Previous studies of experience-dependent changes in STP have typically focused on the effects of brief periods of activity imbalance (Finnerty et al. 1999; Bender et al. 2006; Maffei & Turrigiano, 2008). Here, we took a different approach, using manipulations designed to reduce sensory input uniformly in the cortical area of interest in order to test the role of sensory experience in presynaptic development of the L4 to L2/3 excitatory synapse. We found that patterned visual activity was not required for maturation of STP in V1. In contrast, total whisker deprivation prevented developmental changes in short-term facilitation in barrel cortex.

Methods

Ethical approval

All procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986, and were approved by the Cardiff University Ethics Committee.

Animals

C57BL/6JOlaHsd mice (Harlan) at postnatal days 12 to 13 (P12–13) [designated P12], P26–30 [designated P28] or P40–44 [designated P42] were used. The mean ages given in square brackets are quoted elsewhere in the text for clarity. Eye opening was carefully checked, and had not occurred in any of the recorded P12–13 mice, or in the P12 mice used for dark rearing. Males and females were approximately equally represented in all experimental groups. Recordings were made from 136 neurons from 47 mice. Data from control mice have been published previously (Cheetham & Fox, 2010). One neuron per slice was recorded. Mice were normally kept on a 12 h light/12 h dark cycle (light 06.00–18.00 h) with unrestricted access to food and water.

Dark rearing

To eliminate patterned visual input, litters of mice were dark reared from P12 (i.e. before eye opening) until P40–44. Night vision goggles were used for animal care. On the experimental day, mice were anaesthetised with isoflurane (4% in O2 by inhalation) in the dark prior to brain slice preparation.

Whisker deprivation

We used total bilateral whisker deprivation to reduce sensory input to barrel cortex. Whisker deprivation began at P12 and continued until P26–30, and was performed under isoflurane anaesthesia (4% in O2 by inhalation). Whiskers were plucked under gentle tension using visual guidance, and whisker re-growth was removed every other day.

Brain slice preparation and whole cell recording

Mice were killed by cervical dislocation and decapitated. Coronal slices, 400 μm thick, containing either primary visual cortex or a continuous band of barrels were cut in ice-cold dissection buffer (in mm: 108 choline chloride, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 25 d-glucose, 3 sodium pyruvate, 1 CaCl2, 6 MgSO4, 285 mosmol kg-1) bubbled with 95% O2/5% CO2. Brain slices were incubated in artificial cerebrospinal fluid (ACSF; in mm: 119 NaCl, 3.5 KCl, 1 NaH2PO4, 10 d-glucose, 2 CaCl2, 1 MgSO4, 300 mosmol kg-1) bubbled with 95% O2/5% CO2, at 32°C for 45 min, then at room temperature (21–23°C) until recording. L2/3 pyramidal neurons were identified under infrared differential interference contrast. There was no difference in the distance of recorded neurons from the pia across experimental groups (P = 0.33, F5,68 = 1.18; one-way ANOVA;n = 10–20 neurons per group). Whole cell voltage recordings were made at 35–37°C. Recording pipettes (5–8 MΩ) contained (in mm: 130 KMeSO4, 8 NaCl, 2 KH2PO4, 2 d-glucose, 10 Hepes, 4 Mg-ATP, 7 phosphocreatine, 0.3 GTP, 0.5 ADP, pH 7.30, 285 mosmol kg-1). The identity of pyramidal neurons was confirmed by their regular spiking behaviour in response to depolarising current injection.

A 0.5 MΩ tungsten monopolar extracellular stimulating electrode was positioned vertically above the recorded neuron in L4. It is possible that axons other than those of L4 excitatory neurons could have been stimulated; however, this is unlikely to have made a significant contribution to the recorded responses (Lefort et al. 2009; Cheetham & Fox, 2010), and the type of axon stimulated is unlikely to differ between barrel cortex and V1. Paired recordings were not feasible due to the low connectivity of long-range intracortical pathways. Extracellular stimuli consisted of 1 ms current pulses with stimulation intensity (0.8–4.0 V) set to produce a 3–6 mV monosynaptic EPSP in the postsynaptic neuron. Peaks of monosynaptic EPSPs were within 5 ms of stimulus onset.

Access resistance and pipette capacitance were compensated and data were amplified (Multiclamp 700B, Molecular Devices Co.). Postsynaptic responses were low-pass filtered at 6 kHz, digitised at 10–20 kHz and recorded using Signal v1.85/v4.01 (Cambridge Electronic Design).

STP

Neurons were stimulated with ≥20 trains of eight pulses (Master8; AMPI) delivered at 20 Hz, with 15 s between trials. Monosynaptic EPSP amplitudes were measured using Signal, as the voltage difference between baseline (before response onset) and peak membrane potentials. We found no effect of isoflurane on STP during repetitive stimulation at 20 Hz (P = 0.94, F1,119 = 0.005; two-way ANOVA;n = 9 neurons from isoflurane-anaesthetised mice, n = 11 neurons from control mice). Therefore, we pooled data from control and isoflurane-anaesthetised P42 mice.

Rate of use-dependent blockade of NMDA receptors by MK-801

NMDA receptor (NMDAR)-mediated responses were isolated in Mg2+-free ACSF containing 20 μm CNQX (6-cyano-7-nitroquinoxaline-2,3-dione). Single presynaptic stimuli were delivered at 0.1 Hz. Stable baseline responses (∼5 mV) were recorded before MK-801 application. Stimulation was halted for 10 min during 10 μm MK-801 wash-on, then resumed at 0.1 Hz. Response amplitudes to successive stimuli were normalised to the first response after MK-801 application.

Statistics

For each data set, the mean value for each neuron was calculated, and the grand mean ± SEM was then calculated for each mouse. Data were analysed using one- and two-way ANOVAs with Bonferroni corrections for multiple comparisons. Distributions of data from individual neurons were compared using Kolmogorov–Smirnov (KS) tests. All tests were two-tailed with α = 0.05.

Results

Dark rearing does not affect the maturation of STP in V1

We began by investigating the effect of dark rearing on developmental changes in STP at L4 to L2/3 excitatory synapses in mouse V1. Control mice were reared on a standard light/dark cycle. Dark-reared mice were housed in a light-tight room from P12, before eye opening had occurred, until P42, after the developmental shift in STP has occurred in V1 (Cheetham & Fox, 2010). We prepared acute coronal brain slices containing the binocular zone of V1 from control mice at P12, and from control and dark-reared mice at P42, and made whole cell voltage recordings of L2/3 pyramidal neurons at 35–37°C. Axons in L4, radially aligned with the recorded neuron, were stimulated with eight-pulse trains at 20 Hz (Fig. 1A).

Figure 1. Dark rearing does not affect maturation of STP in V1.

Figure 1

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A, example responses of single L2/3 pyramidal neurons to 20 Hz stimulus trains in L4 for P12, P42 control and P42 dark-reared (DR) mice. Traces are averages of 20 trials. Scale bars: 1 mV; 50 ms.B, responses to 20 Hz stimulus train for P12, P42 and P42DR mice (n = 5 per group).C, effect of dark rearing on maturation of PPR. CON: control.P = 0.010, F2,12 = 6.92, one-way ANOVA. *P< 0.05.D, effect of dark rearing on maturation of steady state response amplitude.P = 0.001, F2,12 = 12.54, one-way ANOVA. **P< 0.01.E, distribution of PPR for individual neurons.F, distribution of normalised steady state amplitude for individual neurons.

We found that dark rearing did not affect the maturation of STP in V1: normalised EPSP amplitude throughout 20 Hz stimulus trains was indistinguishable in control and dark-reared mice at P42 (Fig. 1B, n = 5 mice per group). We used two measures to quantify STP: the PPR and the normalised steady state amplitude (Cheetham & Fox, 2010). PPR was significantly larger in P42 control (1.08 ± 0.04, P = 0.026, t = 3.14) and P42 dark-reared (1.07 ± 0.02, P = 0.025, t = 3.15) mice than in P12 mice (0.90 ± 0.05, one-way ANOVA with Bonferroni correction). Normalised steady state amplitude was also significantly larger in P42 control (0.84 ± 0.05, P = 0.001, t = 4.82) and P42 dark-reared (0.78 ± 0.03, P = 0.003, t = 4.23) mice than in P12 mice (0.51 ± 0.06, one-way ANOVA with Bonferroni correction). Furthermore, there were no differences in either PPR (Fig. 1C;P = 1.00, t = 0.18) or normalised steady state amplitude (Fig. 1D;P = 1.00, t = 0.81) between control and dark-reared mice at P42.

The relationship between stimulus voltage and the amplitude of the first response to the stimulus train (EPSP1 amplitude) was similar for all three experimental groups (P12, 0.48 ± 0.07 V mV−1; P42 control, 0.54 ± 0.06 V mV−1; P42 dark-reared, 0.41 ± 0.02 V mV−1;P = 0.46, F2,12 = 0.84, n = 5 mice per group, one-way ANOVA). Dark rearing did not affect either the hyperpolarisation of resting membrane potential or the decrease in input resistance of L2/3 pyramidal neurons between P12 and P42 (Table 1). We considered the possibility that dark rearing might have a greater effect if initiated immediately before presynaptic development occurs, but found no significant effect of dark rearing from P26 to P42 on STP in V1 (P = 0.56, t = 0.61 for PPR;P = 0.29, t = 1.15 for steady state amplitude;ttests, n = 5 mice per group; data not shown).

Table 1.

Dark rearing and total whisker deprivation do not affect developmental changes in resting membrane potential or input resistance

Resting membrane potential (mV) Input resistance (MΩ)
Group (number of mice) Grand mean ± SEM Statisticsvs. P12 Grand mean ± SEM Statisticsvs. P12
P12 visual cortex (5) −64 ± 1 — 159 ± 10 —
P42 visual cortex (5) −74 ± 1 P< 0.001t = 5.71 42 ± 3 P< 0.001t = 10.62
P42DR visual cortex (5) −77 ± 1 P< 0.001t = 7.79 44 ± 2 P< 0.001t = 11.18
P12 barrel cortex (7) −64 ± 1 — 121 ± 7 —
P28 barrel cortex (8) −75 ± 0 P< 0.001t = 11.64 42 ± 3 P< 0.001t = 16.49
P28DEP barrel cortex (5) −75 ± 1 P< 0.001t = 11.07 48 ± 4 P< 0.001t = 13.36
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Groups were compared using one-way ANOVAs with Bonferroni correction for multiple comparisons. Overall test statistics: visual cortex resting membrane potential: P< 0.001, F2,12 = 34.57; visual cortex input resistance: P< 0.001, F2,12 = 85.69; barrel cortex resting membrane potential: P< 0.001, F2,17 = 85.16; barrel cortex input resistance P< 0.001, F2,17 = 75.11. DR, dark reared; DEP, whisker deprived.

We further analysed the effects of dark rearing by comparing PPR in individual neurons from the three groups (Fig. 1E). The distribution of PPRs was shifted towards larger values for P42 control neurons (n = 20) relative to P12 neurons (n = 10, P = 0.008, D = 0.63, KS test), indicating a uniform increase in PPR during postnatal development. The distribution of PPRs in P42 dark-reared neurons (n = 21) was also shifted towards larger values relative to P12 neurons (P = 0.004, D = 0.67, KS test), and was indistinguishable from that of P42 control neurons (P = 0.93, D = 0.16, KS test). We found the same pattern of changes for steady state amplitudes (Fig. 1F): the distribution was shifted towards larger values for both P42 control (P< 0.001, D = 0.75, KS test) and P42 dark-reared (P = 0.007, D = 0.61, KS test) neurons relative to P12, and again there was no difference between the steady state amplitude distributions in P42 control and dark-reared mice (P = 0.82, D = 0.19, KS test). Therefore, we concluded that patterned visual activity is not required for normal presynaptic development of L4 to L2/3 excitatory synapses in V1.

Total whisker deprivation affects developmental changes in PPR but not steady state amplitude in barrel cortex

We next investigated whether the maturation of STP at L4 to L2/3 excitatory synapses in barrel cortex is dependent on sensory experience. We used total bilateral whisker deprivation to reduce sensory input to barrel cortex: whiskers were plucked every other day from P12 until the day of recording (P26–30), the time period over which STP matures in barrel cortex (Cheetham & Fox, 2010). We then cut brain slices containing barrel cortex from control mice at P12, and from control and whisker-deprived mice at P28, and recorded responses to 20 Hz stimulation of L4 to L2/3 excitatory synapses within barrel columns (Fig. 2A).

Figure 2. Total whisker deprivation affects developmental changes in PPR but not steady state response amplitude in barrel cortex.

Figure 2

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A, example responses of single L2/3 pyramidal neurons to 20 Hz stimulus trains in L4 for P12, P28 control and P28 whisker-deprived (DEP) mice. Traces are averages of 20 trials. Scale bars: 2 mV; 50 ms.B, average responses to 20 Hz stimulus trains for P12, P28 and P28DEP mice (n = 5 per group).C, effect of total whisker deprivation on maturation of PPR.P = 0.041, F2,12 = 4.22, n = 5 per group, one-way ANOVA. *P< 0.05.D, effect of total whisker deprivation on maturation of steady state response amplitude.P = 0.009, F2,12 = 6.95; one-way ANOVA. *P< 0.05.E, distribution of PPR for individual neurons.F, distribution of normalised steady state amplitude for individual neurons.

The effect of whisker deprivation was greatest early in the stimulus train (Fig. 2B, n = 5 mice per group). Further analysis revealed that whisker deprivation prevented the developmental increase in PPR that normally occurs between P12 and P28 (Fig. 2C): PPR was significantly lower in P12 control mice (0.88 ± 0.11) than in P28 control mice (1.08 ± 0.09, P = 0.040, t = 2.90), but not P28 whisker-deprived mice (0.92 ± 0.03, P = 0.74, t = 1.22, one-way ANOVA with Bonferroni correction). In contrast, the normalised steady state amplitude was unaffected by whisker deprivation from P12 to P28 (Fig. 2D). Steady state amplitude was significantly greater in both P28 control mice (0.76 ± 0.10, P = 0.013, t = 3.43) and P28 whisker-deprived mice (0.72 ± 0.03, P = 0.037, t = 2.69) than in P12 mice (0.46 ± 0.05, one-way ANOVA with Bonferroni correction). The relationship between stimulus voltage and response amplitude was very similar between experimental groups (P12, 0.55 ± 0.10 V mV−1; P28 control, 0.63 ± 0.18 V mV−1; P28 deprived, 0.51 ± 0.08 V mV−1;P = 0.87, F2,12 = 0.14, n = 5 mice per group, one-way ANOVA). Whisker deprivation did not affect either the developmental hyperpolarisation of resting membrane potential or the decrease in input resistance exhibited by L2/3 pyramidal neurons that occurs between P12 and P28 (Table 1).

To investigate further the experience dependence of the developmental reduction in PPR in barrel cortex, we compared the distributions of PPRs recorded in individual neurons from P12, P28 control and P28 whisker-deprived mice (Fig. 2E). The distribution of PPRs was shifted towards larger values at P28 (n = 12) than at P12 (n = 11, P = 0.017, D = 0.62, KS test), indicating a uniform increase in PPR for all neurons during postnatal development. In contrast, the distribution of PPRs for neurons in the P28 whisker-deprived group (n = 10) exhibited a partial shift, and was significantly different to both the P12 (P = 0.031, D = 0.60, KS test) and P28 control (P = 0.024, D = 0.61, KS test) distributions. Further analysis indicated that 50% of neurons at P12 exhibited PPRs <0.80, whereas no neurons in P28 control or whisker-deprived mice had mean PPRs <0.80. Nevertheless, the proportion of neurons showing paired-pulse facilitation (PPR > 1.00) was the same in the P12 (20%) and P28 whisker-deprived (20%) groups, but much higher in the P28 control group (67%, P = 0.031, χ2 = 6.97, χ2 test). The distribution of steady state amplitudes was shifted uniformly towards larger values for both P28 control (P = 0.023, D = 0.58, KS test) and P28 whisker-deprived (P = 0.005, D = 0.71, KS test) groups relative to neurons from P12 mice (Fig. 2F), and there was no difference between the distributions of steady state amplitudes between P28 control and P28 whisker-deprived groups (P = 0.56, D = 0.32, KS test). Taken together, our results indicate that maturation of short-term facilitation requires sensory experience, whereas developmental changes in short-term depression are experience-independent in barrel cortex.

Total whisker deprivation prevents the developmental reduction in Pr

The developmental increase in PPR is related to a developmental decrease in Pr (Cheetham & Fox, 2010). Therefore, we next analysed the effect of total whisker deprivation on Pr, using the rate of use-dependent blockade of NMDARs by MK-801 as an assay (Fig. 3A). The rate of use-dependent blockade at L4 to L2/3 excitatory synapses in barrel cortex decreases between P12 and P28, indicating that Pr decreases during this time period (Cheetham & Fox, 2010). We found that use-dependent blockade occurred significantly faster in whisker-deprived mice (n = 4) than in control mice (n = 7) at P28 (Fig. 3B;P< 0.001, t = 7.75; 2-way ANOVA with Bonferroni-corrected pairwise comparison), indicating that whisker deprivation prevented the developmental decrease in Pr.

Figure 3. Total whisker deprivation prevents the developmental reduction in Pr in barrel cortex.

Figure 3

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A, use-dependent blockade of NMDARs by MK-801 for an example neuron from a P28 whisker-deprived mouse. Inset: NMDAR-mediated responses to stimulus numbers 1, 10, 20, 50 and 100 for the same example neuron. Scale bars: 0.5 mV; 10 ms.B, rate of use-dependent blockade of NMDARs for P28 control and whisker-deprived mice.C, percentage blockade of NMDARs by MK-801 after 100 trials. *P = 0.018, t = 2.79, P12vs. P28 control mice, Bonferroni-corrected pairwise comparison. †P< 0.05, P28 controlvs. whisker-deprived mice.D, decay time constant of NMDAR-mediated responses recorded in ACSF with or without 10 μm MK-801 for P28 control and whisker-deprived mice.

We used a simple and model-independent measure of the rate of use-dependent blockade, the percentage blockade of the NMDAR-mediated response after 100 trials, to compare experimental groups further. Use-dependent blockade occurred significantly more slowly in P28 control mice (73.5 ± 4.4% block after 100 trials) than in P28 whisker-deprived mice (83.8 ± 2.8% block after 100 trials, P = 0.011, t = 2.96; Fig. 3C, one-way ANOVA with Bonferroni correction). Furthermore, the rate of use-dependent blockade was similar in P12 mice (82.1 ± 1.7% block after 100 trials, n = 4) and P28 whisker-deprived mice (P = 1.00, t = 0.17; Fig. 3C). Whisker deprivation did not affect the decay kinetics of NMDAR-mediated responses (Fig. 3D;P = 0.99, F1,18 < 0.001, 2-way ANOVA); hence, differences in NMDAR subunit composition did not underlie the effect of whisker deprivation on use-dependent blockade of NMDAR-mediated responses. Therefore, our findings indicate that total whisker deprivation prevents the developmental reduction in Pr that occurs between P12 and P28 at L4 to L2/3 excitatory synapses in barrel cortex.

Discussion

The major finding of this study is that presynaptic development of the excitatory L4 to L2/3 pathway requires sensory experience in barrel cortex, but not in visual cortex. We found no effect of either dark rearing or total whisker deprivation on developmental changes in the passive membrane properties of L2/3 pyramidal neurons. This is in agreement with previous studies in both barrel and visual cortex (Finnerty et al. 1999; Desai et al. 2002; Maravall et al. 2004), and rules out the possibility that differences in passive membrane properties between control and sensory-deprived mice could have affected our measurements of STP.

Experience dependence of presynaptic development in barrel and visual cortex

We found that dark rearing from P12 did not affect presynaptic development in V1. This is surprising, given that dark rearing delays the maturation of cortical inhibition, and hence the critical period for ocular dominance plasticity (Hensch, 2005). There are three possible explanations for the lack of effect of dark rearing on maturation of STP. First, presynaptic maturation in V1 is an intrinsic feature of postnatal development, and hence is unaffected by changes in cortical activity. Second, although dark rearing prevented patterned visual input to V1, the remaining cortical activity (resulting from spontaneous activity and/or the cortical activity evoked by the retinal photoreceptor dark current) was sufficient to drive normal presynaptic development. Third, dark rearing alters the type and quality of input to V1, but overall neuronal activity levels are maintained. Indeed, dark rearing increases both the photoreceptor dark current (Hagins et al. 1970) and spontaneous firing rates in V1 (Gianfranceschi et al. 2003), which could compensate for the loss of patterned visual input.

In barrel cortex, developmental changes in Pr and PPR were largely prevented by whisker deprivation. This suggests that maturation of these synaptic properties is dependent on neuronal activity levels in the barrel cortex, rather than being an innate feature of synaptic development. In barrel cortex, whisker-evoked responses and receptive fields in L4 are mature by P12, and spontaneous firing and whisker-evoked activity in L2/3 neurons emerge between P12 and P14 (Stern et al. 2001). Furthermore, although the whiskers are present from before birth, adult-like exploratory whisking behaviour emerges around P11–13 (Landers & Philip Zeigler, 2006). Whisker deprivation from P12 will therefore significantly alter neuronal activity in barrel cortex. Consequently, reductions in sensory input, and/or the overall neuronal activity level in deprived cortex, are likely to underlie the effects of whisker deprivation on Pr and PPR. Manipulations of sensory experience have also been shown to increase axonal bouton turnover in deprived barrel cortex and V1 (Yamahachi et al. 2009; Marik et al. 2010). Therefore, synapse turnover, as well as activity-dependent structural plasticity of existing boutons, may well contribute to the effects of sensory experience on presynaptic development.

Intrinsic differences in the two systems could explain the greater dependence on sensory activity in barrel cortex. For example, there is no direct equivalent of the retina in the whisker-to-barrel pathway. Furthermore, many neuronal response properties, such as binocularity and orientation selectivity, only emerge in L4 of V1 (Hubel & Wiesel, 1962), whereas most response properties present in L4 of S1 are also present at earlier stages in the whisker-to-barrel pathway (Fox, 2008). In addition, the morphology of mouse L2/3 pyramidal neurons may differ between sensory cortical areas (Benavides-Piccione et al. 2006), which could contribute to the experience dependence of synaptic development.

We employed experimental manipulations of barrel and visual cortex sensory experience that were as similar as possible, avoiding binocular eyelid suture because some patterned visual input occurs through the closed eyelids (Blais et al. 2008). Nevertheless, a caveat to our study is that dark rearing and whisker plucking may not have completely analogous effects on activity in their respective cortical areas. We also cannot rule out the possibility that light exposure through the closed eyelids prior to eye opening is permissive for, or triggers (Mower et al. 1983), cortical synaptic development several weeks later in life. Therefore, it remains to be determined whether more drastic manipulations of visual experience might prevent normal presynaptic development in V1.

Mechanistic dissociation of PPR and steady state response amplitude

In barrel cortex, developmental changes in Pr and PPR required sensory input, whereas the developmental increase in the steady state amplitude did not. This finding strongly suggests that different STP processes predominate early and late in a stimulus train. Our data add further evidence to the view that Pr is a key determinant of PPR (Dobrunz & Stevens, 1997; Zucker & Regehr, 2002; Cheetham & Fox, 2010). Mechanistically, changes in Pr would require maturation of both the presynaptic vesicle release machinery, and presynaptic calcium buffering systems (Zucker & Regehr, 2002). However, our findings suggest that steady state amplitude is determined by other, Pr-independent processes, such as the size of presynaptic vesicle pools and vesicle recycling mechanisms (Zucker & Regehr, 2002; Feldmeyer & Radnikow, 2009). Maturation of such presynaptic properties therefore appears to be independent of sensory experience in both barrel and visual cortex.

At individual synapses in mature cortex, both short-term facilitation and short-term depression contribute to STP during repetitive stimulation (Markram et al. 1998; Abbott & Regehr, 2004). We suggest that the ‘developmental switch’ in STP (Reyes & Sakmann, 1999) comprises two key developmental processes: (1) the emergence of short-term facilitation, which is dependent on the developmental decrease in initial Pr, and results in PPRs greater than unity, and (2) a weakening of short-term depression, which is largely Pr-independent and underlies the increase in steady state amplitude. It is worth noting that both facilitation and depression contribute to the PPR: although whisker deprivation prevented the emergence of short-term facilitation, the strong paired-pulse depression seen in 50% of neurons at P12 was absent in P28 whisker-deprived mice (Fig. 2E).

Comparison with previous studies investigating the effects of sensory experience on STP

In this study, we used manipulations that aimed to reduce sensory experience uniformly in the cortical area of interest. In contrast, most previous studies of the effects of sensory experience on STP have used manipulations that create an activity imbalance, generating competition between deprived and spared inputs. For example, monocular eyelid suture from P18 to P21 increased PPR at L4 to L2/3 excitatory synapses in V1 (Maffei & Turrigiano, 2008), and plucking a single row of whiskers for 6–9 days from P12 increased PPR and decreased Pr at L4 to L2/3 excitatory synapses in deprived barrel cortex (Bender et al. 2006). Although these activity-dependent changes are superimposed upon developmental changes in Pr and STP, at least in barrel cortex (Cheetham & Fox, 2010), their magnitude is significantly greater than can be accounted for by developmental changes alone. Notably, an earlier study (Finnerty et al. 1999) found that trimming multiple whisker rows for 10 days from P11–15 did not affect STP of L4 to L2/3 excitatory synapses in deprived cortex (Finnerty et al. 1999). This suggests that the method and specific pattern of whisker deprivation are important in determining experience-dependent changes in STP.

Another study (Finnerty & Connors, 2000), in which all whiskers on one side of the face were trimmed for 10–14 days from P11–15, suggested that a uniform reduction in sensory input did not affect STP at L4 to L2/3 excitatory synapses in deprived barrel cortex. However, recordings from control synapses in the Finnerty & Connors (2000) study showed no evidence of paired-pulse facilitation, suggesting that they were at an earlier developmental stage (Feldmeyer & Radnikow, 2009) than in our study. The reasons for this are unclear, but methodological differences, such as the precise age of animals, whisker trimmingvs. plucking, unilateralvs. bilateral whisker deprivation, species used, slice orientation and recording technique, could all contribute.

Our data indicate that an effect of whisker deprivation on developmental changes in STP would only be expected if assayed after the emergence of short-term facilitation. This highlights the interplay between developmental and experience-dependent changes in synaptic properties during altered sensory experience. Hence, it is important to consider that underlying developmental changes in synaptic physiology, which can continue well into the second postnatal month (Cheetham & Fox, 2010), may interact with activity-dependent changes to neuronal circuits, complicating the interpretation of experimental results.

Conclusions and physiological significance

STP has been suggested as a synaptic mechanism underlying visual response properties (Abbott et al. 1997). Maturation of many visual response properties continues until ∼P45 in rodents (Fagiolini et al. 1994), which coincides with the maturation of presynaptic properties at excitatory L4 to L2/3 synapses (Cheetham & Fox, 2010). However, dark rearing prevents developmental changes in orientation and direction selectivity, visual acuity and receptive field size (Fagiolini et al. 1994; Gianfranceschi et al. 2003), whereas STP matures normally in dark-reared mice. This suggests that presynaptic development at L4 to L2/3 excitatory synapses is not critical for maturation of these visual response properties, which may instead depend on other pathways (Hubel & Wiesel, 1962; Coleman et al. 2010) and/or activity-dependent selection of subsets of synapses (Hensch, 2005).

Our data suggest that total whisker deprivation maintains synapses in barrel cortex in a functionally immature state. These depressing synapses are optimally tuned to encode low frequency activity, producing the strongest responses to single action potentials (Abbott & Regehr, 2004), and can therefore be driven by spontaneous activity, which occurs at a low frequency (Margrie et al. 2002). The effect of whisker deprivation on STP could therefore contribute to systems-level changes in deprived barrel cortex, for example the depression of principal whisker responses in L4 and L2/3 during total whisker deprivation (Glazewski et al. 1998), and the effect of whisker deprivation on maturation of receptive fields in L2/3 (Stern et al. 2001).

Acknowledgments

This study was funded by the NIMH (P50-MH077972) and the MRC. We thank Sam Barnes and Stuart Greenhill for helpful comments.

Glossary

Abbreviations

ACSF

artificial cerebrospinal fluid

DEP

whisker-deprived

DR

dark-reared

KS

Kolmogorov–Smirnov

L

layer

NMDAR

NMDA receptor

P

postnatal day

Pr

presynaptic release probability

PPR

paired-pulse ratio

STP

short-term plasticity

V1

primary visual cortex

Author contributions

C.E.J.C. and K.F. designed the experiments; C.E.J.C. performed the experiments, analysed and interpreted the data; C.E.J.C. and K.F. wrote the paper.

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