Deprivation-induced strengthening of presynaptic and postsynaptic inhibitory transmission in layer 4 of visual cortex during the critical period

Abstract

Inhibition from fast-spiking (FS) interneurons plays a crucial role in shaping cortical response properties and gating developmental periods of activity-dependent plasticity, yet the expression mechanisms underlying FS inhibitory plasticity remain largely unexplored. In layer 4 of visual cortex (V1), monocular deprivation (MD) induces either depression or potentiation of FS to star pyramidal neuron (FS→SP) synapses, depending on the age of onset (Maffei et al., 2004, 2006). This reversal in the sign (- to +) of plasticity occurs on the cusp of the canonical critical period (CP). To investigate the expression locus behind this switch in sign of inhibitory plasticity, mice underwent MD during the pre-CP [eye-opening to postnatal day (p)17] or CP (p22-p25), and FS→SP synaptic strength within layer 4 was assessed using confocal and immunoelectron microscopy, as well as optogenetic activation of FS cells to probe quantal amplitude at FS→SP synapses. Brief MD before p17 or p25 did not alter the density of FS→SP contacts. However, at the ultrastructural level, FS→SP synapses in deprived hemispheres during the CP, but not the pre-CP or in GAD65 knock-out mice, had larger synapses and increased docked vesicle density compared with synapses from the nondeprived control hemispheres. Moreover, FS→SP evoked miniature IPSCs increased in deprived hemispheres when MD was initiated during the CP, accompanied by an increase in the density of postsynaptic GABAA receptors at FS→SP synapses. These coordinated changes in FS→SP synaptic strength define an expression pathway modulating excitatory output during CP plasticity in visual cortex.

Keywords: LTP; electron microscopy; fast-spiking; monocular deprivation; optogenetic; pyramidal.

Figure 1.

Brief MD does not affect FS→SP contact density. A, Time course for developmental and MD analyses used for all experiments. B, Three-dimensional projection of a confocal stack, showing a single PV-ir FS interneuron (green) contacting NeuN-labeled pyramidal somata (red) in layer 4 of V1m. Inset shows zoomed-in z-projection of boxed pyramidal cell displaying basket-like PV-ir putative synaptic contacts. C, Single optical sections of a NeuN-labeled pyramidal neuron (left), and the same neuron with superimposed PV-ir thresholded labeling used for contact density quantification (right). D, Development of perisomatic PV-ir FS contact density with pyramidal cells in layer 4. E, FS→SP contact density during the pre-CP (p17) and CP (p25), in control (C) and deprived (D) hemispheres. Scale bars: B, inset, 5 μm; B, C, 15 μm. *p < 0.05.

Figure 2.

Examination of FS→SP synaptic ultrastructure. A, Low-magnification electron micrograph showing a layer 4 pyramidal soma (white outline) contacted by eight PV-ir perisomatic synaptic terminals (asterisks; silver-enhanced gold labeling). B, Representative images of PV-ir FS→SP synapses at EM resolution in control and deprived hemispheres at p17 (wild-type) and p25 (wild-type and GAD₆₅KO). Lower “colorized” panels are duplicates of those above, highlighting FS boutons (green), postsynaptic densities (red), and docked vesicles (blue). Note that the PV-ir silver-enhanced gold particles within each bouton do not prevent analyses of synaptic ultrastructure. C, Serial-section three-dimensional reconstruction of a synaptic PV-ir perisomatic bouton. C₁, PV-ir bouton (green) displaying both undocked (red) and docked vesicles (blue), and forming two synapses (red) with a pyramidal soma (data not shown). C₂, Same bouton as in C₁, displaying only docked vesicles. Scale bars: A, 2 μm; B, 200 nm; C, 300 nm.

Figure 3.

MD during the CP increases FS presynaptic docked vesicle density. A, Synapse sizes, as measured by mean postsynaptic density cross-section length, in control (C) and deprived (D) hemispheres from wild-type (p17 and p25) and GAD₆₅KO (p25) animals. B, Mean docked vesicles per terminal in control versus deprived hemispheres. C, Average readily releasable pool in control versus deprived hemispheres. D, Cumulative percentage plot of docked vesicle density in control (gray line) and deprived (black line) hemispheres at p25. Inset shows data from cumulative histogram of average docked vesicle density. *p < 0.05.

Figure 4.

Evoked FS→SP mIPSCs. A, Collage of a three-dimensional projection from a confocal stack, showing a single FS interneuron double-labeled for EYFP-tagged ChR2 (green) and PV (red), and the merge of these two images. Arrow points to basket-like puncta. B, Single optical section showing colocalization of EYFP and PV-ir labels in basket-like perisomatic puncta. C, Diagram of the experimental paradigm used for ChR2-evoked FS→SP mIPSC recordings. D, Representative trace showing evoked spikes in a FS interneuron following 5 ms laser pulses. E, Representative trace of evoked mIPSCs (asterisks) recorded in strontium from a single star pyramidal neuron following a 5 ms laser pulse. Note that the large evoked inward current is followed by desynchronized and temporally separable mIPSCs. F, Average spontaneous (diamonds) and evoked (triangles) mIPSC frequency from pyramidal cells in control hemispheres. G, Spontaneous and evoked mean mIPSC frequency from control hemispheres. H, Spontaneous and evoked mean mIPSC 10–90% rise time in control hemispheres. I, Spontaneous and evoked mean mIPSC decay time in control hemispheres. J, Spontaneous and evoked mean mIPSC amplitude in control hemispheres. K, Cumulative percentage and histogram (inset) plots of spontaneous and evoked mIPSC amplitude in control hemispheres. Inset, Dotted and solid lines are running average fits with a period = 2. Scale bars: A, 10 μm; B, 5 μm. *p < 0.05.

Figure 5.

Evoked FS→SP mIPSC amplitude increases following MD during the CP. A, Diagram of the experimental paradigm used for ChR2-evoked FS→SP mIPSC recordings. B, Average spontaneous (diamonds) and evoked (triangles) mIPSC frequency from pyramidal cells in control (white) and deprived (black) hemispheres. C, Average mIPSC evoked frequency for control and deprived conditions. D, Average evoked mIPSC 10–90% rise time for control and deprived hemispheres. E, Average evoked mIPSC decay time for control and deprived hemispheres. F, Average evoked mIPSC amplitude for control and deprived hemispheres. G, Cumulative percentage and histogram (inset) plots of evoked mIPSC amplitude in control and deprived hemispheres. Inset, Dotted and solid lines are running average fits with a period = 2. H, Average evoked mIPSC waveforms for control (gray) and deprived (black) hemispheres. *p < 0.05.

Figure 6.

GABA_AR density at FS→SP synapses increases following MD during the CP. A, Representative image of a FS→SP synapse from a p25 control hemisphere showing preembedding labeling for PV (large silver-enhanced gold particles) and postembedding labeling for presynaptic (blue arrowheads) and postsynaptic (red arrowheads) GABA_A γ2-subunits (5 nm gold particles). B, GABA_ARs at a p25 FS→SP synapse from a deprived hemisphere. C, Axo-somatic (presynaptic vs postsynaptic) distribution of GABA_A γ2-gold labeling in control and deprived hemispheres. Red line and arrowhead indicate centroid of synaptic cleft. Dotted and solid lines are running average fits with a period = 2. D, Radial distribution (normalized to synapse length) of GABA_A γ2-gold labeling in control and deprived hemispheres. E, Cumulative percentage histogram and average bar plot (inset) for presynaptic GABA_A γ2-gold particle density (see Materials and Methods, and Results). F, Cumulative percentage histogram and average bar plot (inset) for postsynaptic GABA_A γ2-gold particle density. Scale bar: (in B) A, B, 100 nm. *p < 0.05.