Developmental presence and disappearance of postsynaptically silent synapses on dendritic spines of rat layer 2/3 pyramidal neurons

Abstract

Silent synapses are synapses whose activation evokes NMDA-type glutamate receptor (NMDAR) but not AMPA-type glutamate receptor (AMPAR) mediated currents. Silent synapses are prominent early in postnatal development and are thought to play a role in the activity- and sensory-dependent refinement of neuronal circuits. The mechanisms that account for their silent nature have been controversial, and both presynaptic and postsynaptic mechanisms have been proposed. Here, we use two-photon laser uncaging of glutamate to directly activate glutamate receptors and measure AMPAR- and NMDAR-dependent currents on individual dendritic spines of rat somatosensory cortical layer 2/3 pyramidal neurons. We find that dendritic spines lacking functional surface AMPARs are commonly found before postnatal day 12 (P12) but are absent in older animals. Furthermore, AMPAR-lacking spines are contacted by release-competent presynaptic terminals. After P12, the AMPAR/NMDAR current ratio at individual spines continues to increase, consistent with continued addition of AMPARs to postsynaptic terminals. Our results confirm the existence of postsynaptically silent synapses and demonstrate that the morphology of the spine is not strongly predictive of its AMPAR content.

Figure 1. 2PLGU selectively activates individual dendritic spines

A, left panel, image of a layer 2/3 pyramidal neuron from a P16 rat. The asterisk and arrow highlight, respectively, the recording electrode and the spiny basal dendrite shown at higher magnification in the right panel. B, image of a spine and its parent dendrite. Asterisks indicate uncaging locations that yielded the largest (‘best’) and a secondary (‘sec.’) uEPSCs. The insets show the average uEPSC_AMPAR at each spot. C, uEPSC_AMPAR from ‘best’ and ‘secondary’ spots in 21 spines. D, uEPSCs from a P16 spine at holding potentials of −70 and +40 mV; 5 individual trials (grey) and the corresponding averages (black) are shown. E, uEPSC_NMDAR from individual dendritic spines of P14–17 animals plotted versus uEPSC_AMPAR (n = 37 spines).

Figure 2. AMPAR-lacking spines are found in immature pyramidal neurons

A, left panel, image of a layer 2/3 pyramidal neuron from a P9 rat highlighting the recording electrode (*). Right panel, higher magnification image of the basal dendrite indicated by the arrow in the left panel, showing mature spines (arrowheads) and a filopodium (#). B, uEPSCs from a P9 spine at holding potentials of −70 and +40 mV; 5 individual trials (grey) and the corresponding averages (black) are shown. C, uEPSC_NMDAR plotted versus uEPSC_AMPAR in control conditions (○, n = 72 spines, P9–13) or in the presence of NBQX (▴, n = 13 spines, P9–15). The dashed line marks the position expected for spines lacking AMPARs. Many control spines fall along this line and show uEPSC_AMPAR similar to that recorded in the presence of NBQX.

Figure 4. Spines lacking AMPARs are associated with functional presynaptic terminals

A, the rate of mEPSCs detected at −70 mV is increased by puffs of hypertonic solution (right panel) relative to baseline levels (left panel) (age P9). B, image of a parent dendrite (dn) and spine (sp.) that showed no uEPSCs at −70 mV and large currents at +40 mV (white traces) that are blocked by the NMDAR antagonist CPP (red trace) (age P10). C, green fluorescence collected in the line scan indicated by the dashed line in (B). Inset: quantification of the green fluorescence transient (ΔG/R) in the spine head (sp) and neighbouring dendrite (dn) indicating that Ca²⁺ influx was limited to the spine. D, quantification of fluorescence transients in the spine (sp, green trace) and dendrite (dn, black trace) on a longer time scale than C, displaying several evoked Ca²⁺ transients in the spine. Over this longer time scale, evidence of Ca²⁺ diffusion in the parent dendrite is seen. The asterisk (*) marks the event shown in (C). Ca²⁺ influx into the spine was blocked by CPP (red trace). E, uEPSC_NMDAR plotted versus uEPSC_AMPAR for spines that were exposed to hypertonic ACSF. In some cases a Ca²⁺ increase restricted to the spine was evoked (green circle, n = 9), whereas in others ΔCa²⁺ was not detectable or was not restricted to the spine (•, n = 8) (age P9–15). A red contour marks AMPAR-lacking spines.

Figure 3. Spines lacking or expressing AMPARs are morphologically indistinguishable

A, images of spines from relatively immature (left panel) and mature (right panel) rats. B, mean length (○, left ordinate) and width (•, right ordinate) of spines analysed electrophysiologically as a function of animal age. The numbers of analysed spines at each age are indicated. C, uEPSC_AMPAR plotted versus spine length (n = 74 spines). Dashed line marks the predicted position of AMPAR-lacking spines. D, percentage of AMPAR-lacking spines as a function of animal age. E, developmental curve of uEPSC_AMPAR (•) and uEPSC_NMDAR (○). The average uEPSCs calculated from the subset of spines that are non-silent in P9–11 animals are shown by grey circles. The numbers of analysed spines contributing to each data point are also given.