Article Figures & Data
Figures
- Figure 1.
The excitatory and inhibitory perisomatic innervation on parvalbumin interneurons in the dentate gyrus. Triple immunostaining for parvalbumin (blue), gephyrin (red), and PSD95 (green) postsynaptic markers for inhibitory and excitatory synapses, respectively. A, Confocal image showing the soma and a proximal dendrite of a parvalbumin interneuron located in the inner molecular layer (iml). Note the high density of PSD95 puncta in this area. B, Confocal image showing the soma and dendritic trunk of a parvalbumin interneuron with the cell body sitting at the border between the granule cell layer (gcl) and the hilus (h), and the dendritic trunk traveling toward the molecular layer. As expected, the labeling of PSD95 was scarce in the granule cell layer but abundant in the h. C, Higher magnification of the inset shown in A. The number of gephyrin (arrowheads) and PSD95 puncta (arrows) was approximately even for all the cells analyzed. D, Higher magnification of the inset shown in B. The number of gephyrin (arrowheads) at the parvalbumin interneuron surface is lower than PSD95 puncta (arrows) for all the cells analyzed. E, Graph showing the percentage of PSD95 puncta on the sampled parvalbumin cells located either in the granule cell layer or in the inner molecular layer. Vertical bars represent SEM. Asterisks show significance of the statistical analysis for the predominance of PSD95 puncta; ***p < 0.001. Geph, gephyrin; PV, parvalbumin. Scale bar: 20 μm.
- Figure 2.
Perisomatic innervation of parvalbumin interneurons by mossy cells detected by confocal microscopy. Confocal analysis of the calretinin-positive elements (red) found in apposition to parvalbumin-positive interneurons (blue) and their co-expression of synaptic markers (green). A, Maximal intensity projection image taken from a parvalbumin interneuron in the granule cell layer near to calretinin-expressing fibers of mossy cells that run through the granule cell layer (gcl) and sprout densely into the inner molecular layer (iml). B–D, Colocalization of synaptophysin (green) and calretinin (red) elements in close apposition to somata (B, C) and inner molecular layer dendrites (D) of different parvalbumin-positive cells (blue). The presence of double immunoreactive elements on parvalbumin cells (open arrows) indicates that some fibers from mossy cells can establish synaptic contacts with parvalbumin cells. E, F, Colocalization of PSD95 (green) and calretinin (red) puncta in close apposition to the soma (E) and to a proximal dendrite (F) of a parvalbumin-positive interneuron (blue). Although rarely, some apposition of calretinin and PSD95 can be found on parvalbumin somata (open arrows), indicating potential perisomatic contacts of mossy cells with parvalbumin cells. G, H, Graphs showing the proportion of synaptophysin puncta that were calretinin immunoreactive in apposition to parvalbumin elements (G) and the proportion calretinin immunoreactive boutons facing PSD95 puncta in apposition to parvalbumin elements (H) sampled on the somata and dendrites in the hilus, granule cell layer, and inner molecular layer. Vertical bars represent SEM CR, calretinin; h, hilus; oml, outer molecular layer; PV, parvalbumin; Syn, synaptophysin. Scale bar: 20 μm (A) and 5 μm (B–F).
- Figure 3.
Appositions from calretinin-containing mossy fiber axons on parvalbumin cells rarely make synaptic contacts when confirmed by electron microscopy. Example of a single parvalbumin cell (DAB) with apposed calretinin fibers (DAB-Ni) studied by correlative light and electron microscopy. A, Soma and apical dendrite of the parvalbumin cell (asterisk) under light microscopy apparently contacted by calretinin fibers. The boutons on the cell (arrows) were analyzed for connectivity by electron microscopy (see the corresponding images indicated by letters). B, C, Low-magnification electron microscopic images taken of the proximal dendritic segment (B) and soma (C, asterisk) of the cell. The boxes contain calretinin elements apposed to the cell that are shown enlarged in D–F. D–F, Calretinin elements tested for contacts with the parvalbumin cell were analyzed in all consecutive sections. Calretinin varicosities (asterisks) often did not establish synaptic specializations (D, E), however, a few of them (F, open arrow) presented a small postsynaptic density and synaptic cleft, although the asymmetric nature of the contact is dubious. As a reference, the asymmetrical contact is clearly seen on a neighboring soma shown in F (arrow). PV-Den, parvalbumin dendrite; PV-Som, parvalbumin soma. Scale bar: 2 μm (B, C) and 400 nm (D–F).
- Figure 4.
Most contacts on the perisomatic region of parvalbumin interneurons are not from mossy cells. Example of a single parvalbumin cell (DAB) with apposed calretinin fibers (DAB-Ni) studied by correlative light and electron microscopy. A, Soma of the parvalbumin cell (asterisk) under light microscopy apparently contacted by calretinin fibers. B, Low-magnification electron microscopic image of this soma (asterisk). A mossy cell bouton can be seen apposed to the plasma membrane (arrow), but did not form a synaptic contact. The boxes contain other presynaptic elements apposed to the cell that are shown enlarged in E, F. C, D, The soma was contacted by numerous small boutons filled with round clear vesicles making large asymmetric synapses (arrows). These boutons were not immunoreactive for calretinin. E, A bouton visualized by DAB forms a symmetric synapse on the parvalbumin cell (open arrow) and shows the hallmarks of parvalbumin basket cell boutons with clear pleomorphic vesicles and large mitochondria. F, A hilar juxtagranular parvalbumin dendrite (likely belonging to other cell based on its higher level of immunoreactivity) is covered by several non-immunoreactive small boutons making large asymmetrical synaptic contacts (arrows) similar to the soma analyzed. PV-Den, parvalbumin dendrite; PV-Som, parvalbumin soma. Scale bar: 2 μm (B, C) and 400 nm (D–F).
- Figure 5.
Fibers from the supramammillary nucleus do not contact soma and proximal dendrites of parvalbumin interneurons. We studied the fibers from the supramammillary nucleus using either VGLUT2 as a marker or anterograde labeling using BDA 10 kDa. A, Triple immunostaining for VGLUT2 (red), bassoon (green), and parvalbumin (blue) failed to show clear boutons of subcortical origin (open arrows) with presynaptic release sites (arrows) apposing parvalbumin interneurons located in the granule cell of the dentate gyrus. B, Using anterograde labeling with BDA 10 kDa from supramammillary nucleus (inset for injection site), we selected putative candidates that presented fiber apposition to parvalbumin cells (asterisk) in the dentate gyrus (inset), followed by analyzing the boutons using electron microscopy to test whether the boutons contacted the cells. Open arrows label some of the boutons from which electron micrographs are shown. Parvalbumin-expressing structures have been colored for easy identification. Bouton in panel B (arrow) approached the parvalbumin cell, but did not contact it. C–E, Three different boutons were followed through the sections until synaptic contacts formed by them were identified. In all three cases, the axon terminals (asterisks) made asymmetric synapses only with granule cell somata (arrows). gcl, granule cell layer; GC, granule cell; PV, parvalbumin. Scale bar: 10 μm (A), 2 μm (B), and 500 nm (C, D).
- Figure 6.
SGCs contacting parvalbumin interneurons resemble the innervation by Timm-stained boutons. A, A parvalbumin cell (asterisk) in the granule cell layer contacted by Timm-stained boutons (arrows). Because of the nature of the Timm staining only the axonal varicosities are visible and the origin of them cannot be resolved. The image was reconstructed from several optical planes. B, An intracellularly filled SGC (white asterisk) in the inner molecular layer gives rise to an axon (arrowhead) that descends in apposition to a parvalbumin cell (black asterisk) in its way to the hilus. The axonal varicosities (arrows) formed close apposition with the surface of the parvalbumin cell. gcl, granule cell layer; h, hilus; iml, inner molecular layer; oml, outer molecular layer. Scale bar: 20 μm.
- Figure 7.
The morphology of SGCs differs from normal granule cells. A, Specimen of normal granule cell filled with biocytin. One dendrite arises from the soma, the axon arises vertically and goes directly toward the hilus (arrow). B, Example of SGC. Several dendrites arise from the soma, whereas the axon arises horizontally and enters the granule cell layer after running in the inner molecular layer (arrow). C, D, Example of the morphology of two semilunar cells with axons (open arrows) in apposition to parvalbumin interneurons. Insets show some of the biocytin-filled boutons (arrows) in close apposition to parvalbumin profiles. E–I, Morphologic analysis shows that SGCs, including those that make contacts onto parvalbumin interneurons in the granule cell layer, give rise to more dendrites close to the soma as shown with the Sholl analysis (E, black symbols label differences between granule cells and SGCs, khaki symbols label significance between granule cells and semilunar cells innervating parvalbumin interneurons), a larger number of primary dendrites (F), and a wider spread of the dendritic arbor (G) than granule cells. SGCs also have a larger total dendritic length (H) and larger somatic horizontal diameter (I) than granule cells, though this difference was not statistically different for semilunar cells innervating parvalbumin interneurons. Asterisks show significance of the statistical analysis; *p < 0.05, **p < 0.01, ***p < 0.001; gcl, granule cell layer; h, hilus; iml, inner molecular layer; ml, molecular layer; n.s., not significant. Scale bar: 50 μm.
- Figure 8.
Axons of SGCs form asymmetric synapses with the perisomatic region of parvalbumin cells located in the granule cell layer. A SGC was filled with biocytin using patch-clamp technique. The section was then fixed in a fixative containing glutaraldehyde and subjected to parvalbumin immunostaining before be processed for electron microscopy. A, An axon from this SGC (DAB-Ni) running in the inner molecular layer (arrowheads) sends a collateral toward the hilus delineating a parvalbumin cell labeled with DAB. In the inset, the relative position of the SGC in the inner molecular layer (asterisk) and the parvalbumin cell (arrow) is shown. The boxes show axonal varicosities of the SGC apposing the distinct portions of the parvalbumin cell in the inner molecular layer (B) the granule cell layer (C) and the hilus (D). B, SGC axon giving rise to varicosities (arrows) along a thin parvalbumin dendrite originating from the parvalbumin cell in the inner molecular layer (left panel). The same string of boutons is presented on an electron micrograph shown in the right panel. B1–B3, Higher magnification of boutons shown in B at the level where they form large asymmetric synapses on the parvalbumin dendrite (arrowheads). The boutons are filled with round vesicles as well as many dense core vesicles (open arrows). C, A correlative electron microscopic image taken from the granule cell layer (asterisks label granule cells) shown in A. A large bouton from the SGC (open arrow) contacting the parvalbumin cell. C1, This large bouton (asterisk) makes an asymmetric synapse (arrowheads) on the parvalbumin cell and is filled with round vesicles and dense core vesicles (open arrows). D1, Bouton (asterisk) from the SGC in the juxtagranular hilus makes asymmetric synapse on a proximal dendrite of the parvalbumin interneuron (arrowhead). Complementary results are presented in Extended Data Figure 8-1. gcl, granule cell layer; h, hilus; iml, inner molecular layer; oml, outer molecular layer. Scale bar: 20 μm (A) and 500 nm (B–D).
- Figure 9.
Diagram of the connectivity involving the excitatory perisomatic innervation of dentate gyrus parvalbumin interneurons. Dentate gyrus parvalbumin interneurons (PV) receive perisomatic excitatory input mainly from SGCs. Input from mossy cells is rare in the granule cell layer (gcl) but occurs in the inner molecular layer (iml). Supramammillary fibers (SuMM) do not seem to contact parvalbumin cells on the soma. When SGCs fire, they could activate mossy cells with which they engage in repeating firing known as hilar up states (1), at the same time SGCs will excite parvalbumin interneurons (2) that would control large populations of granule cells (3) synchronizing their firing when excited by the perforant pathway. At the same time, mossy cells likely project back to SGCs, although this projection has not been specifically described (question mark). GC, granule cells; h, hilus; MC, mossy cells; oml, outer molecular layer; PP, perforant pathway; PV, parvalbumin interneurons; SuMM, projection from the supramammillary nucleus.
Tables
- Table 1
Statistical analysis of the morphologic characteristics of normal granule cells, SGCs, and SGCs contacting dentate parvalbumin interneurons
GCs SGCs SGC-PVs MW (GCs vs SGCs) MW (GCs vs SGCs-PV) MW (SGCs vs SGCs-PV) Number of primary dendrites 1.5 ± 0.22
(n = 10)4.0 ± 0.32
(n = 18)3.75 ± 0.56
(n = 8)p < 0.001 p = 0.003 p = 0.797 Dendritic spread angle (°) 73.4 ± 8.5
(n = 10)120.9 ± 5.0
(n = 18)107.3 ± 11.0
(n = 8)p < 0.001 p = 0.037 p = 0.389 Total dendritic length (μm) 1575 ± 114
(n = 10)2055 ± 103
(n = 20)1779 ± 212
(n = 4)p = 0.009 p = 0.358 p = 0.296 Somatic horizontal diameter (μm) 11.3 ± 0.59
(n = 10)13.6 ± 0.79
(n = 17)11.9 ± 1.03
(n = 8)p = 0.039 p = 0.894 p = 0.210 Sholl analysis Intersections at 20 μm 2.4 ± 0.52
(n = 10)5.55 ± 0.38
(n = 20)5.29 ± 0.68
(n = 7)p < 0.001 p = 0.010 p = 0.865 Intersections at 40 μm 4.5 ± 0.45
(n = 10)7.0 ± 0.33 7.43 ± 0.61
(n = 7)p < 0.001 p = 0.005 p = 0.571 Intersections at 60 μm 6.3 ± 0.54
(n = 10)8.2 ± 0.45
(n = 20)8.29 ± 0.80
(n = 7)p = 0.022 p = 0.068 p = 0.845 Intersections at 80 μm 8.2 ± 0.80
(n = 10)9.1 ± 0.39
(n = 20)9.0 ± 1.41
(n = 7)p = 0.392 p = 0.730 p = 0.843 Intersections at 100 μm 8.1 ± 0.82
(n = 10)9.35 ± 0.54
(n = 20)8.86 ± 1.64
(n = 7)p = 0.436 p = 0.883 p = 0.285 Intersections at 120 μm 9.2 ± 0.83
(n = 10)10.1 ± 0.57
(n = 20)9.17 ± 1.99
(n = 6)p = 0.463 p = 0.620 p = 0.104 Intersections at 140 μm 8.2 ± 1.33
(n = 10)8.65 ± 0.73
(n = 20)7.67 ± 1.86
(n = 6)p = 0.773 p = 0.785 p = 0.409 Intersections at 160 μm 5.6 ± 1.27
(n = 10)6.5 ± 0.67
(n = 20)3.83 ± 1.19
(n = 6)p = 0.506 p = 0.412 p = 0.061 Intersections at 180 μm 3.5 ± 1.06
(n = 10)3.8 ± 0.61
(n = 20)2.33 ± 1.23
(n = 6)p = 0.876 p = 0.532 p = 0.268 Intersections at 200 μm 1.3 ± 0.54
(n = 10)2.2 ± 0.47
(n = 20)1.83 ± 0.91
(n = 6)p = 0.283 p = 0.727 p = 0.683 Intersections at 220 μm 0.4 ± 0.31
(n = 10)1.2 ± 0.28
(n = 20)1.0 ± 0.51
(n = 6)p = 0.087 p = 0.261 p = 0.846 Data are shown as mean ± SEM. For the comparison of the results obtained by morphometric analysis, the Mann–Whitney test was used.
Extended Data Figure 8-1
SGC establishing synaptic contacts with a parvalbumin cell have characteristics of granule cells. A, The same intracellularly filled SGC as in Figure 8 visualized with DAB-Ni, while parvalbumin was developed by DAB. A1, Panoramic view of the dendritic arbor of the intracellularly filled SGC (asterisk). The main axon runs along the inner molecular layer (arrows), entering into the granule cell layer and reaching the hilus, where it gives rise to collaterals and varicosities. A2, Higher magnification of the dendrites of the intracellularly filled SGC shown in A1. Spine morphology is similar to that of typical granule cells. A3, The soma of the cell shown in A1. The cell body was sitting at the border between the inner molecular layer and granule cell layer (asterisk). The axon protruding from a proximal dendrite ran along the inner molecular layer (arrow), where it could be followed in the section represented in A1. A4, Electron microscopy of a mossy fiber collateral originated from the SGC shown in A. The fiber forms mossy boutons (asterisk) that made asymmetric synaptic contacts (arrowheads) the thorny excrescences of hilar mossy cells. B, Soma and dendritic arbor of the intracellularly filled SGC whose innervation is shown in Figure 6B. gcl, granule cell layer; h, hilus; iml, inner molecular layer. Scale bars: 50 μm (A1, B), 20 μm (A2, A3), and 1 μm (A4). Download Figure 8-1, TIF file.
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