Experience and activity-dependent maturation of perisomatic GABAergic innervation in primary visual cortex during a postnatal critical period

Abstract

The neocortical GABAergic network consists of diverse interneuron cell types that display distinct physiological properties and target their innervations to subcellular compartments of principal neurons. Inhibition directed toward the soma and proximal dendrites is crucial in regulating the output of pyramidal neurons, but the development of perisomatic innervation is poorly understood because of the lack of specific synaptic markers. In the primary visual cortex, for example, it is unknown whether, and to what extent, the formation and maturation of perisomatic synapses are intrinsic to cortical circuits or are regulated by sensory experience. Using bacterial artificial chromosome transgenic mice that label a defined class of perisomatic synapses with green fluorescent protein, here we show that perisomatic innervation developed during a protracted postnatal period after eye opening. Maturation of perisomatic innervation was significantly retarded by visual deprivation during the third, but not the fifth, postnatal week, implicating an important role for sensory input. To examine the role of cortical intrinsic mechanisms, we developed a method to visualize perisomatic synapses from single basket interneurons in cortical organotypic cultures. Characteristic perisomatic synapses formed through a stereotyped process, involving the extension of distinct terminal branches and proliferation of perisomatic boutons. Neuronal spiking in organotypic cultures was necessary for the proliferation of boutons and the extension, but not the maintenance, of terminal branches. Together, our results suggest that although the formation of perisomatic synapses is intrinsic to the cortex, visual experience can influence the maturation and pattern of perisomatic innervation during a postnatal critical period by modulating the level of neural activity within cortical circuits.

Figure 4.

High-resolution characterization of the development of basket interneurons in organotypic cultures. A, A DNA segment containing a 10 kb region of the GAD67 promoter (P_G67) followed by the EGFP cassette from our modified GAD67-GFP BAC clone (see Fig. 1 A) was taken to replace the EGFP coding region in the expression vector pEGFP-1 (Clontech), through gap repair-mediated BAC engineering. B, Biolistic transfection of P_G67-GFP directs GFP expression in Pv-positive GABA interneurons in cortical organotypic cultures. The arrow points to a GFP-expressing interneuron (middle), which is also positive for Pv immunofluorescence (red in left panel). Scale bar, 50 μm. C, Whole-cell current-clamp recording shows that GFP-expressing neurons are fast spiking, nonadapting, and have large AHPs. Bottom trace, 400 pA current step; top trace, 650 pA current step. Calibration: 20 mV, 100 msec. Synaptic activity is evident, as expected in organotypic cultures (Echevarria and Albus, 2000). D-G, Basket interneurons develop their elaborate dendritic and axon arbors and synaptic boutons in vitro. Higher-magnification images in the bottom panels show details of terminal branches and presynaptic boutons. D, At EP11, basket interneurons display relatively simple dendritic trees and sparse axon branches (arrows; bottom) with very few boutons (arrowheads; bottom). E, At EP18, the dendrite and axon arbors appear to have reached near mature sizes. The axons are branched extensively, yet the axon terminals are still rather immature, characterized by filopodial-like protrusions (arrow; bottom), in addition to distinct boutons (arrowheads; bottom). F, At EP24, axon branches are more exuberant, and highly distinct terminal forks emerge (asterisk; bottom) with strings of synaptic boutons (arrowheads; bottom). G, At EP28, axon terminal forks become more complex (asterisk; bottom), with larger and more boutons (arrowheads; bottom). Scale bars: top, 50 μm; bottom, 5 μm. H, A basket cell axon terminal fork (asterisk; middle) surrounds a cell soma (large asterisk; right) with synaptic boutons positive for GAD65 immunoreactivity (red; arrowheads) at EP28. Note that the thin axon segments between boutons are devoid of GAD65 immunoreactivity (arrows). Scale bar, 2 μm. I, A basket bouton containing dark GFP precipitate was confirmed to be the presynaptic terminal of a symmetric synapse (arrow) by immunoelectron microscopy. The postsynaptic dendritic target is outlined by segmented lines. Scale bar, 0.7 μm.

Figure 2.

Prolonged postnatal maturation of perisomatic innervation by basket interneurons in primary visual cortex. A-D, Time course of the maturation of perisomatic innervation in the primary visual cortex of G42 mice. Each image in the top panel is a single confocal plane, whereas each image in the bottom panel represents a projection of a z-stack across a single pyramidal cell soma. Higher-magnification images in the bottom panel show arrangement of GFP-labeled presynaptic terminals around pyramidal cell somata, which are immunostained with NeuN antibody (red). A, At P14, there is no evidence of perisomatic bouton rings (top; asterisks mark cell body positions). Basket cell presynaptic boutons are small and poorly developed around pyramidal cell soma (bottom; arrowhead indicates a small bouton). B, At P18, a marked increase of basket cell axon terminal density in the neuropil and around pyramidal cell somata is observed (top). However, presynaptic boutons are still poorly organized around pyramidal cell soma and far from forming a complete perisomatic bouton ring (bottom). C, At P24, mature perisomatic bouton rings start to emerge (top), and the number of boutons around each pyramidal cell soma increases significantly (bottom). D, At P28, highly prominent perisomatic bouton rings are observed (top), which often enclose a large portion of the pyramidal cell soma surface (bottom). Asterisks indicate cell somata, and the arrowheads show boutons from basket interneurons. Scale bars: top, 20 μm; bottom, 5 μm. E, Quantification of the developmental increase of the density of GFP-positive basket cell boutons around NeuN-positive pyramidal cell somata. The graph shows the number of boutons per pyramidal cell soma quantified from single confocal sections (boutons/soma section; see Materials and Methods) at different ages. Bouton density increases significantly between P18, P24, and P28 (one-way ANOVA, post hoc Dunn's test; p < 0.05).

Figure 8.

Quantification of activity-dependent maturation of perisomatic innervation in cortical organotypic culture. A, TTX treatment between EP18 and EP24 and EP24 and EP28 prevents the increase of bouton density around pyramidal cell soma (Mann-Whitney U test; p < 0.001). Right, Effects of TTX treatment normalized to age-matched control. B, The increase of basket axon bouton size between EP18 and EP24 is prevented by TTX treatment (Mann-Whitney U test; p < 0.001). Right, Effects of TTX treatment normalized to age-matched control. C-E, Reverse Three-Dimension Sholl analysis of basket axon terminal complexity around pyramidal cell soma in TTX-treated slices compared with age-matched controls. C1-E1, Pyramidal cells are grouped according to the average number of intersections between a 7 μm Sholl sphere from the center of their nuclei and basket axon branches surrounding them. The developmental increase in the percentage of pyramidal cell somata surrounded by more complex basket axon terminal branches is blocked by TTX treatment between EP18 and EP24 (χ² test; p < 0.001). However, TTX treatment between EP24 and EP28 has no effect (χ² test; p = 0.9), suggesting that, once extended, basket terminal branches can be maintained around pyramidal cell soma despite reduced level of activity. C2-E2, An alternative method to quantify basket axon terminal complexity around pyramidal cell soma, by plotting of the number of intersections between basket axons and Sholl spheres with increasing radius from the center of a pyramidal nucleus. TTX treatment between EP18 and EP24 dramatically reduces the number of intersections and therefore the complexity of basket axon branches close to pyramidal cell soma.

Figure 1.

GFP BAC transgenic mice allow high-resolution labeling of Pv-positive basket interneurons and their soma-targeted presynaptic boutons. A, Scheme of GAD67 BAC engineering. Top, Diagram of overlapping BAC clones containing the mouse GAD67 gene. Bottom, Strategy for the modification of BAC clone 56I19 with an EGFP recombination vector. An EGFP expression cassette was inserted at the translation initiation site in the second exon of the GAD67 gene. The GAD67 genomic structure is not drawn to scale. Open box, Noncoding exons; shaded box, coding exons; pA, phosphoglycerate kinase polyadenylation signal. Note that a gene of interest can be inserted into the recombination vector as a PacI fragment. B, GFP expression pattern in the visual cortex of a P28 GAD67-GFP mouse (G42 line). Cortical layers are indicated to the left. Scale bar, 100 μm. C, GFP-expressing cells are Pv immunopositive (PV; red) but do not express SOM (red) or CCK (red). Scale bar, 20 μm. D, Basket cell presynaptic boutons (arrowheads) surround pyramidal cell somata (asterisk) and contain GAD65 immunoreactivity (red) in mature primary visual cortex (P36). Scale bar, 5 μm. E, Left, Perisomatic boutons (arrowheads) visualized by GFP immunohistochemistry and light microscopy. The asterisks indicate neuronal soma. Scale bar, 2 μm. Right, A basket cell bouton (outlined by dashed lines) containing dark DAB precipitate, indicating the presence of GFP, is confirmed to be a presynaptic terminal of a symmetric synapse (arrow) on a cell soma (outlined by arrowheads) by immunoelectron microscopy. Scale bar, 0.5 μm.

Figure 5.

Visualization of perisomatic innervation and perisomatic synapses by a single basket interneuron with its pyramidal cell targets. A, Slice cultures from emx-nlslacZ-ires-cre knock-in mice are transfected biolistically with P_G67-GFP. Sparsely labeled basket interneurons (green; arrow) are visualized in the background of all pyramidal cell nuclei labeled by lacZ immunofluorescence (red dots) at EP22. Cortical layers are indicated. B, Projection of a confocal stack showing pyramidal neurons (red) within the axon arbor of a single basket interneuron (green). C, Higher-magnification view of the region marked in B. Many pyramidal cell nuclei are in close proximity to basket axon terminals and are likely innervated (arrows). D, Higher-magnification view of the region marked in C. A pyramidal nucleus is surrounded by a terminal fork (asterisk) from a basket interneuron, with strings of boutons (arrowheads). Individual optical sections comprising the confocal stack of this projection show the nucleus positioned between the two terminal branches (supplemental material, available at www.jneurosci.org). Sholl spheres for Reverse Three-Dimensional Sholl analysis are shown (see Materials and Methods). Scale bars: A, 100 μm; B, 50 μm; C, 10 μm; D, 2 μm.

Figure 3.

Intraocular TTX injection decreases the maturation of perisomatic GABAergic innervation in visual cortex during the third, but not the fifth, postnatal week. A, Diagram showing monocular TTX injection paradigm. B, C, Daily monocular TTX injection between P20 and P24 reduces perisomatic innervation of pyramidal neurons in binocular primary visual cortex. In the cortex ipsilateral to TTX injection (nondeprived; B), pyramidal cell somata (asterisks) are consistently surrounded by basket interneuron terminal branches and perisomatic bouton rings (arrowheads; note similarity to Fig. 2C). In the contralateral deprived cortex (C), however, perisomatic innervation is significantly reduced. Many pyramidal cell somata (asterisks) are poorly surrounded by basket axon terminals and boutons (arrows), with no apparent perisomatic bouton rings. Pyramidal neurons in B and C are labeled with NeuN immunofluorescence (red). D, E, Monocular TTX injection between P32 and P36 does not affect perisomatic innervation of pyramidal neurons in the visual cortex. Both the ipsilateral (D) and controlateral (E) cortex show prominent perisomatic bouton rings (arrowheads) around pyramidal cell somata (red). Asterisks mark pyramidal cell soma. Scale bar, 20 μm. F, G, Quantification of perisomatic bouton density around pyramidal neurons in the visual cortex of mice with monocular TTX injection. F1, In the P20-P24 injected group, the contralateral (deprived) cortex shows a significant reduction in perisomatic bouton density compared with the ipsilateral (nondeprived) cortex (Mann-Whitney U test; p < 0.01). F2, In the P24-P28 injected group, the contralateral (deprived) cortex shows a trend toward a reduction in perisomatic bouton density compared with the ipsilateral (nondeprived) cortex, but this reduction is significant only in two of five animals (asterisk; Mann-Whitney U test; p < 0.05). F3, In the P32-P36 injected group, there was no statistical difference in perisomatic bouton density between the contralateral (deprived) and the ispilateral (nondeprived) cortex (Mann-Whitney U test; p > 0.4). Values from the ipsilateral and contralateral cortices are shown for each animal. G, Effects of monocular TTX injection on perisomatic innervation in the contralateral (deprived) cortex normalized to ipsilateral (nondeprived) cortex. Values from each animal are represented by open circles. The average values are indicated by histogram.

Figure 6.

Formation of perisomatic synapses proceeds in cortical organotypic cultures. A-C, At EP14, many axon branches have reached and surrounded pyramidal cell somata but contain only one or two putative boutons around each soma (arrowheads). Note long segment of axons devoid of boutons (arrow). D-F, At EP18, more pyramidal cell somata are likely innervated (D), but basket axon terminal branch complexity and bouton density around each pyramidal cell soma (arrowheads in E and F) are similar to those at EP14. G-I, At EP24, one or more axonal branches extend around a pyramidal cell soma to form a terminal fork (asterisk), along with an increase in the number and size of boutons (arrowheads in H and I). J-L, At EP28, there is an additional increase in bouton number (arrowheads) within terminal forks, which in some cases resemble a basket (asterisk). Scale bar: (top) A, D, G, J, 15 μm; (middle) B, E, H, K, 10 μm; (bottom) C, F, I, L, 2 μm. M, A schematic summarizing the stereotyped process of perisomatic synapse formation. N, Quantification of the developmental increase of basket cell presynaptic boutons density around pyramidal cell somata. The bouton number around each pyramidal cell soma is quantified by Reverse Three-Dimensional Sholl analysis within a 7 μm Sholl sphere from the center of its nucleus. Bouton density increases significantly between EP18 and EP28 (1-way ANOVA, post hoc Dunn's test; p < 0.05). O, Average bouton diameter, measured perpendicular to the axon, at different developmental ages. Bouton size increases significantly between EP18 and EP24 (one-way ANOVA, post hoc Dunn's test; p < 0.05). P, Developmental increase of basket axon terminal branch complexity around pyramidal cell somata. Pyramidal cells at different ages are grouped according to the average number of intersections between a 7 μm Sholl sphere from the center of their nuclei and basket axon branches surrounding them. At EP24 and EP28, pyramidal cell somata are surrounded by more complex terminal branches than cells from EP18 and EP14, as indicated by the appearance of pyramidal cell groups showing a higher number of intersections.

Figure 7.

Formation of perisomatic synapses is regulated by neuronal spiking activity within cortical organotypic culture. A-D, TTX treatment from EP14 to E18 does not significantly affect the maintenance of already formed perisomatic synapses. A, B, Overall axon arbor and major axon branches of TTX-treated basket cells are very similar to age-matched controls (compare with Figs. 4E, 6D, E). In addition, pyramidal cell somata are usually contacted by a single basket axon branch (arrows) containing only a few boutons (arrowheads) from TTX-treated slices (C, D1-D3), which is indistinguishable from control slices (D4). E-H, TTX treatment from EP18 to EP24 prevents the progression of perisomatic synapse formation. The overall axon arbor morphology (E) was not notably affected by TTX treatment compared with age-matched controls (see Figs. 4E,6G). However, TTX-treated basket cell axons (arrows in G and H) appear more wavy, do not extend as many terminal branches, and appear to have fewer boutons (arrowheads) around pyramidal cell somata (H1-H3) compared with the control group (H4; asterisk represents terminal fork). I-L, TTX treatment from EP24 to EP28 does not affect the already established terminal forks but prevents the additional increase of bouton density around pyramidal cell somata. The overall axon arbor morphology (I) was not notably affected by TTX treatment compared with age-matched controls (see Figs. 4G, 6J). Basket axons terminal forks are present around pyramidal cell soma (K, L1-L3; asterisk) with distinct boutons (arrowhead), similar to the age-matched control (L4). However, bouton density around pyramidal cell soma appears to be lower compared with the control group (quantified in Fig. 8A). Scale bars: A, E, I, 50 μm; B, F, J, 30 μm; C, G, K, 15 μm; D, H, L, 2 μm.