Abstract

Excitatory neurons undergo dendritic spine remodeling in response to different stimuli. However, there is scarce information about this type of plasticity in interneurons. The polysialylated form of the neural cell adhesion molecule (PSA-NCAM) is a good candidate to mediate this plasticity as it participates in neuronal remodeling and is expressed by some mature cortical interneurons, which have reduced dendritic arborization, spine density, and synaptic input. To study the connectivity of the dendritic spines of interneurons and the influence of PSA-NCAM on their dynamics, we have analyzed these structures in a subpopulation of fluorescent spiny interneurons in the hippocampus of glutamic acid decarboxylase-enhanced green fluorescent protein transgenic mice. Our results show that these spines receive excitatory synapses. The depletion of PSA in vivo using the enzyme Endo-Neuraminidase-N (Endo-N) increases spine density when analyzed 2 days after, but decreases it 7 days after. The dendritic spine turnover was also analyzed in real time using organotypic hippocampal cultures: 24 h after the addition of EndoN, we observed an increase in the apparition rate of spines. These results indicate that dendritic spines are important structures in the control of the synaptic input of hippocampal interneurons and suggest that PSA-NCAM is relevant in the regulation of their morphology and connectivity.

Introduction

Excitatory neurons experience structural remodeling under different conditions, involving changes in the length and complexity of dendritic arbors and the density or morphology of their spines (Fu and Zuo 2011). In contrast, despite the important role of inhibitory networks in central nervous system (CNS) physiology, there is little information on the structural plasticity of interneurons. Only some recent studies have shown that inhibitory neurons undergo dendritic remodeling during normal conditions (Chen, Flanders, et al. 2011) and after visual deprivation (Chen, Lin, et al. 2011) or chronic stress (Gilabert-Juan et al. 2011, 2012).

Although dendritic spines were thought to be a distinctive feature of principal neurons, different studies have shown that some interneuronal subpopulations also display these postsynaptic specializations (Freund and Buzsáki 1996). However, there is scarce information on the dynamics of interneuron spines and on the nature of their synaptic inputs. Only a recent study has described that, in the visual cortex, they receive mainly a glutamatergic input, and that the removal of visual input decreases their density (Keck et al. 2011).

Different molecules may mediate the structural plasticity of interneurons, particularly those involved in cell adhesion or cytoskeletal dynamics. In this regard, the polysialylated form of the neural cell adhesion molecule (PSA-NCAM), a molecule involved in synaptogenesis and neurite remodeling (Rutishauser 2008), is a specially promising candidate because it is expressed in a subpopulation of cortical interneurons (Nacher et al. 2002; Gómez-Climent et al. 2010). A previous report from our laboratory showed that pyramidal neurons in the hippocampus, the medial prefrontal cortex, and the paleocortex do not express PSA-NCAM in their somata or proximal dendrites (Gómez-Climent et al. 2010), and the same situation is found in the rest of neocortical regions (Nacher et al. unpublished results). However, it is possible that some PSA-NCAM-expressing structures in the neuropil of the cornus ammonis 1 (CA1) and CA3 regions of the hippocampus belong to excitatory neurons (Gómez-Climent et al. 2010), which would be in consonance with previous reports suggesting the presence of this molecule associated with structures belonging to hippocampal pyramidal neurons (Muller et al. 1996; Schuster et al. 2001). Although NCAM is the major carrier of PSA in the CNS, this sugar has also been detected in other proteins (Hildebrandt et al. 2008; Vitureira et al. 2010). However, NCAM is the only carrier of PSA in interneurons of the adult cerebral cortex (Gómez-Climent et al. 2010). PSA-NCAM-expressing interneurons receive less synaptic contacts than those lacking this molecule and have reduced dendritic arborization and spine density (Gómez-Climent et al. 2010), suggesting that PSA-NCAM plays a role in the regulation of interneuronal structure. PSA can be selectively removed from NCAM by the enzyme Endo-Neuraminidase-N (Endo-N; Vimr et al. 1984), and experiments using this enzyme have demonstrated that PSA is involved in several aspects of neural plasticity (Di Cristo et al. 2007; Rutishauser 2008; Castillo-Gómez et al. 2011).

The objective of this work was to explore the nature of the synaptic input to the dendritic spines of hippocampal interneurons and to study whether their plasticity is influenced by the PSA-NCAM. Consequently, we have used mice-expressing enhanced green fluorescent protein (eGFP) under the glutamic acid decarboxylase promoter (Oliva et al. 2000), which allowed us to observe the structure of a subpopulation of spiny interneurons. We have analyzed the neurochemical phenotype of these interneurons in the CA1 region to know in which interneuronal subpopulation we were performing the morphometric analysis. We have also analyzed the expression of synaptic markers in the proximity of their spines and their ultrastructure in order to study their synaptic input. Finally, we have analyzed the effects of PSA removal on spine density in vivo, and on the turnover and density of spines of CA1 interneurons in organotypic cultures to examine its effects with time-lapse imaging.

Materials and Methods

Animal Treatments

Twenty-four GFP-expressing inhibitory neurons (GINs, Tg(GadGFP)45704Swn) male mice (Oliva et al. 2000), 3 month old, were used for the analysis of dendritic spines in fixed tissue. They were placed, under deep anesthesia (5 mg/g xylazine and 0.5 mL/kg ketamine intraperitoneally), in a stereotaxic instrument (David Kopf Instruments) and received an intracranial injection (using a Flexifil tapertip syringe; World Precision Instruments) of EndoN (0.7 U/μL in glycerol; AbCys) or the vehicle solution (1 μL; saline and glycerol 1:1) in the primary somatosensory cortex (bregma −1.7 mm, lateral ±1.5 mm, deep –0.6 mm; Paxinos and Franklin 2001). EndoN is a phage enzyme that specifically cleaves α-2,8-linked N-acetylneuraminic acid polymers with a minimum chain length of 8. The needle was left in position for 1 min and then 1 μL of the enzyme EndoN was injected during another minute into one hemisphere. After the injection was completed, the needle was left in place for 2 min to reduce reflux of the solution into the track of the injection needle and then withdrawn.

Mice were separated in 4 groups (6 animals per group): 2 groups were injected with the enzyme EndoN and the other 2 were injected with vehicle. Half of the animals from each group were perfused 2 days after the injection, whereas the other half was perfused 7 days after.

Ten GIN pups were used to obtain transverse hippocampal organotypic slice cultures (300 µm thick). The cultures were prepared from P7 mice and maintained for 11–15 days in vitro as described before (Stoppini et al. 1991). To facilitate the transfer of slices to recording conditions, we used a second small membrane confetti (Millipore) placed on the top of Millipore inserts. The cultures were stored in a humid atmosphere at 33 °C in 5% CO2.

All animal experimentation was conducted in accordance with the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes and was approved by the Committee on Bioethics of the Universitat de València.

Histological Procedures

Adult mice were perfused transcardially under deep chloral hydrate anesthesia, with saline and then 4% paraformaldehyde in sodium phosphate buffer 0.1 M, pH 7.4 (PB). The brain was then cut, alternating 150-µm thick sections for the study of interneuron morphology and 50-µm sections for immunohistochemistry, with a vibratome (Leica VT 1000E, Leica).

Immunohistochemistry

Tissue sections were processed free-floating as follows: briefly, sections were incubated for 1 min in an antigen unmasking solution (0.01 M citrate buffer, pH 6) at 100 °C. After cooling down the sections to room temperature, the endogenous peroxidase was blocked with 10 min incubation in a solution with 3% H2O2 in phosphate buffered saline (PBS). Afterwards, slices were incubated with 10% normal donkey serum (NDS; AbCys) in PBS with 0.2% Triton-X 100 (Sigma) for 1 h; then, they were incubated overnight at room temperature with mouse monoclonal anti-PSA (1:700; AbCys). After washing, sections were incubated for 30 min with biotinylated donkey antimouse IgM (1:200; Jackson Immunoresearch), followed by an avidin–biotin–peroxidase complex (ABC, Vector Laboratories) for 30 min in PBS. Color development was achieved by incubating with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma) for 4 min.

For the hippocampal organotypic cultures, after 1 h fixation with paraformaldehyde 4% in PB, fluorescence immunohistochemistry was performed. The procedure was similar to that described above, but without the endogenous peroxidase inhibition. Slices were incubated overnight at room temperature with mouse monoclonal anti-PSA (1:700; AbCys). They were also incubated for 1 h with donkey antimouse IgM Dylight 549 (1:200; Jackson Immunoresearch).

For the analysis of the phenotype of eGFP-expressing interneurons and the puncta in apposition to their spines, double-fluorescence immunohistochemistry was performed. The procedure was similar to that used for the organotypic cultures, but using the primary antibodies listed in Table 1, and incubating afterwards with donkey antimouse IgM, donkey antirabbit IgG, or donkey antiguinea pig secondary antibodies conjugated with Dylight 549 or Dylight 649 (1:200; Jackson Immunoresearch).

Table 1

List of primary antibodies and lectins

Primary antibody Abbreviation Dilution Company 
Polyclonal rabbit antiparvalbumin Anti-PV 1:2000 SWANT 
Polyclonal rabbit anticalbindin-D28K Anti-CB 1:2000 SWANT 
Polyclonal rabbit anticalretinin Anti-CR 1:2000 SWANT 
Polyclonal rabbit antisomatostatin Anti-SOM 1:200 Provided by Dr T. J. Gorcs 
Polyclonal rabbit antivasoactive intestinal peptide Anti-VIP 1:1000 Provided by Dr T. J. Gorcs 
Monoclonal mouse anticholecystokinin Anti-CCK 1:1000 CURE 
Monoclonal mouse anti-PSA-NCAM Anti-PSA 1:1400 Abcys 
Polyclonal rabbit antivesicular GABA transporter Anti-VGAT 1:1000 Synaptic systems 
Polyclonal guinea pig antivesicular glutamate transporter 1 Anti-VGluT1 1:2000 Millipore 
Polyclonal rabbit anti-GFAP Anti-GFAP 1:1000 Sigma-Aldrich 
Tomato lectin  1:50 Sigma-Aldrich 
Polyclonal chicken anti-GFP Anti-GFP 1:1000 Millipore 
Primary antibody Abbreviation Dilution Company 
Polyclonal rabbit antiparvalbumin Anti-PV 1:2000 SWANT 
Polyclonal rabbit anticalbindin-D28K Anti-CB 1:2000 SWANT 
Polyclonal rabbit anticalretinin Anti-CR 1:2000 SWANT 
Polyclonal rabbit antisomatostatin Anti-SOM 1:200 Provided by Dr T. J. Gorcs 
Polyclonal rabbit antivasoactive intestinal peptide Anti-VIP 1:1000 Provided by Dr T. J. Gorcs 
Monoclonal mouse anticholecystokinin Anti-CCK 1:1000 CURE 
Monoclonal mouse anti-PSA-NCAM Anti-PSA 1:1400 Abcys 
Polyclonal rabbit antivesicular GABA transporter Anti-VGAT 1:1000 Synaptic systems 
Polyclonal guinea pig antivesicular glutamate transporter 1 Anti-VGluT1 1:2000 Millipore 
Polyclonal rabbit anti-GFAP Anti-GFAP 1:1000 Sigma-Aldrich 
Tomato lectin  1:50 Sigma-Aldrich 
Polyclonal chicken anti-GFP Anti-GFP 1:1000 Millipore 

Regarding the analysis of gliosis in the somatosensory cortex and the hippocampus, we have also used a procedure similar to that used for the organotypic cultures, using a combination of an anti-GFAP primary antibody, to label astrocytes, and biotinylated tomato lectin (Sigma-Aldrich), to label microglial cells (see dilutions in Table 1). The next day, sections were incubated with donkey antirabbit IgG conjugated with Dylight 649 and streptavidin conjugated with Dylight 549 (1:200; Jackson Immunoresearch).

To find evidences of degeneration in the population of interneurons in which we performed the morphometric analysis, a set of sections was stained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA) in a concentration of 2 μg/mL during 10 min. This was used for the detection of degenerated or abnormal nuclei in the CA1 region of the hippocampus in general, and particularly in the eGFP-expressing interneurons.

Confocal Imaging and Analysis

Laser scanning microscopy was performed with a Leica TCS SPE confocal microscope. For the analysis of fixed tissue, a ×63 oil immersion objective was used to analyze eGFP-expressing dendrites. A ×3.5 additional digital zoom was used to observe the first 180 µm of dendrites in segments of 60 µm (Z-step size of 0.8 µm). Six dendrites from 6 different neurons within the CA1 region of the hippocampus were analyzed per animal. Dendrites within GFP-positive interneurons were chosen at random, but they had to meet different criteria to be included in the study: (1) They should measure at least 180 µm from the soma and (2) no other dendrites should be found crossing their trajectory.

To analyze the juxtaposition between GFP-positive spine and vesicular glutamate transporter (VGluT) or GABA vesicular transporter (VGAT) puncta, confocal planes were solved as average from 3 images of the same plane. We admitted colocalization of a juxtaposed presynaptic element with a size between 0.01 and 0.2 µm2 and shared any number of pixels with the protrusion. Those percentages were averaged per dendrite (we took samples of 10 spines per dendrite in 5 different dendrites). The analysis was done with the images coded and blindly to the experimental condition. The colocalization of VGAT or VGluT1 with the dendritic spines was also blindly assessed with the eGFP channel rotated 90°, in order to indicate the level of chance colocalization, and was compared with the level of colocalization observed in the nonrotated analysis.

For the analysis in real time of organotypic cultures, short imaging sessions (10–15 min) were carried out with a ×40 water immersion objective. A ×10 additional digital zoom was used to analyze dendritic segments of about 35 µm in length, located between 100 and 150 µm from the soma (Z-step size of 0.5 µm). Acquisition conditions were maintained mostly unchanged over the different days of observation. We used culture medium at the same temperature than that of the cultures (medium was placed inside the incubator 10 min before its usage). After adding the medium, the slices were placed under the confocal microscope. Then, we used the minimum observable intensity, that is, the laser intensity was chosen between a range previously determined with pilot experiments, between 1% and 7%. We always tried to minimize both the time in which the slices were outside the incubator and the intensity of the laser (if spines could be observed at 2%, we did not use a higher percentage). However, since the expression of GFP is variable among interneurons, we had to use slightly different laser intensities for different cells. With these conditions, we obtained images at different zoom levels (with ×4 and ×40 immersion objectives) to render a “map” of the culture. Using this map, we were able to localize the same neuron and the same dendritic segment rapidly in each time point. Control experiments showed that this procedure did not produce any deleterious effect on cell viability, such as cell death or dendritic beadings. One dendrite from one neuron was analyzed per organotypic culture slice, and 6 slices from 3 different animals were analyzed. All the interneurons analyzed were located within the CA1 region of the hippocampus. Spines were defined as any kind of protrusion found in a dendrite. Spine density was calculated in the entire stack of confocal planes covering the whole volume of the dendritic segment analyzed.

Spines were classified according to the length of the protrusion and the diameters of their head and neck. Three different categories were established: (1) stubby, when the length of the protrusion was <1.5 µm; (2) mushroom, when a clear head could be observed (maximum diameter of the head should be at least 1.5 times the average length of the neck) and the total length of the protrusion was <3 µm; and (3) filopodial, when the length of the protrusion was >3 µm, or when this length was between 1.5 and 3 µm and a clear head could not be distinguished.

For the statistical analysis of spine turnover, 2 different controls were used: An internal control referring to measures taken from the cultures before the addition of EndoN (2 U/μL in glycerol), between t0 and t24, and an external control taken from the data of a separate group of slices, where 1 μL of vehicle was added instead of EndoN.

For the study on fixed adult tissue, all the preparations were coded after extraction of the brain. For the in vitro cultures, all the images were coded prior to analysis. All the images were analyzed using the ImageJ software (National Institutes of Health). All the statistics are given with the standard error of the mean. Two-way analysis of variance (ANOVA) for repeated measures followed by Fisher's protected least significant difference (LSD) was performed to analyze dendritic spine turnover in the organotypic cultures. The interneuron spine density in fixed tissue was analyzed using 2-way ANOVA with Fisher's protected LSD. For all tests, α was set to 5%.

Electron Microscopy

For electron microscopy, 3 GINs (Tg(GadGFP) 45704Swn) mice were perfused with a fixative containing 0.5% glutaraldehyde and 4% paraformaldehyde in phosphate buffer containing 15% saturated picric acid. Brains were removed from the skull, and 60-µm-thick coronal sections were cut with a vibratome and washed in PB; then, sections were cryoprotected in a solution of 10% glycerol and 30% sucrose in 0.01 M PB for 30 min and freeze-thawed in an aluminum foil boat over liquid nitrogen 3 times to enhance the penetration of antisera. Sections were washed extensively and treated with 1% sodium borohydride for 30 min. Endogenous peroxidase activity was suppressed by 1% H2O2 for 30 min. Then, unspecific unions were blocked using 10% NDS. GFP was detected using chicken (IgY) anti-GFP 1:500, (Table 1) at 4 °C for 2 days. Donkey antichicken IgY biotinylated antibody, 1:200, 2 h (AP194B, Chemicon) was used as a second layer. Biotin labeling was made evident using ABC (Vector, 1:100, 2 h) and DAB as chromogen. The sections were then treated with 1% OsO4, 7% glucose in PB for 45 min, and stained with 1% uranyl acetate in sodium maleate buffer (0.05 M, pH 5.2, 1 h), dehydrated in ascending ethanol series, cleared in propylene oxide, and then embedded in Durcupan (ACM, Fluka). Selected section showing spiny immunoreactive dendrites in the stratum oriens was re-embedded. Then, series of 50-nm-thick ultrathin sections were cut with an ultramicrotome and mounted on Formvar-coated single-slot nickel grids. They were also stained with uranyl acetate and lead citrate and observed under transmission electron microscopy. Spines were followed in the series to assess their identity and the types of synapses that they received.

Results

Phenotype of eGFP-Expressing Inhibitory Neurons

To understand in which subpopulation of interneurons we were performing the morphometric analysis, we have analyzed the neurochemical phenotype of eGFP-expressing interneurons in the CA1 region (Table 2). Previous reports have demonstrated that, in the mice strain used in our study, most eGFP-expressing interneurons in the hippocampus correspond to the subpopulation-expressing somatostatin (Oliva et al. 2000). We have confirmed this result (Fig. 1C) and have found that these interneurons did not coexpress other neuropeptides, such as cholecystokinin (Fig. 1B) or vasoactive intestinal peptide (Fig. 1D). Regarding calcium-binding proteins, eGFP-expressing interneurons did not express parvalbumin (Fig. 1A), whereas some of them expressed calbindin (Fig. 1C) or, in a much lower proportion, calretinin (Fig. 1D). In addition, we have found that all these interneurons display spines on their dendrites.

Table 2

Neurochemical phenotype of GFP-expressing interneurons

Marker Mean ± SE (%) 
PV 
CB 33.9 ± 0.9 
CR 4 ± 1 
SOM 70.7 ± 7.7 
CCK 
VIP 
Marker Mean ± SE (%) 
PV 
CB 33.9 ± 0.9 
CR 4 ± 1 
SOM 70.7 ± 7.7 
CCK 
VIP 
Figure 1.

Expression of different interneuronal markers in the eGFP-expressing cells of the hippocampal CA1 region. (A) Lack of colocalization with parvalbumin (PV). (B) Lack of colocalization with cholecystokinin (CCK). (C) Colocalization with calbindin (CB) and somatostatin (SOM). (D) Colocalization with calretinin (CR), but not with vasoactive intestinal peptide (VIP). All images are single confocal planes. Scale bar: 20 μm.

Figure 1.

Expression of different interneuronal markers in the eGFP-expressing cells of the hippocampal CA1 region. (A) Lack of colocalization with parvalbumin (PV). (B) Lack of colocalization with cholecystokinin (CCK). (C) Colocalization with calbindin (CB) and somatostatin (SOM). (D) Colocalization with calretinin (CR), but not with vasoactive intestinal peptide (VIP). All images are single confocal planes. Scale bar: 20 μm.

Excitatory and Inhibitory Inputs on eGFP-Expressing Dendritic Spines

To explore the connectivity of the dendritic spines of hippocampal eGFP-expressing interneurons, we analyzed with confocal microscopy the presence of putative synaptic inputs in the close vicinity of these spines (Fig. 2). We found that many of them (46 ± 4%) were juxtaposed to VGluT1-expressing puncta, while 39 ± 6% were juxtaposed to VGAT-expressing puncta. We also found a 14 ± 4% of spines associated with both VGluT1- and VGAT-expressing puncta. Finally, we found a 27 ± 2% of spines not associated with any excitatory or inhibitory markers (Fig. 2F). We found that spines were juxtaposed to VGlut1-expressing puncta at a level much higher than chance, but not to those expressing VGAT. Taken together, these data suggest that the majority of spines on the dendrites of eGFP-expressing interneurons in the CA1 region have synaptic inputs, and that many of them appear to belong to glutamatergic presynaptic neurons.

Figure 2.

Expression of presynaptic markers in the proximity of dendritic spines of interneurons. (A) Reconstruction of a dendritic segment of a CA1 hippocampal interneuron carrying spines on its dendrites. (B and D) Single confocal planes showing puncta expressing the vesicular GABA transporter (labeled in red, B2, D2) in close apposition to spines from an eGFP-expressing interneuron (labeled in green, B4, D4). (C and E) Single confocal plane showing a dendritic spine from an interneuron (C1, E1) in close apposition to puncta-expressing vesicular glutamate transporter-1 (labeled in blue, C3, E3). (F) Graphs showing the percentage of juxtaposition between presynaptic markers and dendritic spines of interneurons in original images compared with images in which the eGFP channel was rotated 90°. Scale bar: 2 μm in A and 1.1 μm in BE.

Figure 2.

Expression of presynaptic markers in the proximity of dendritic spines of interneurons. (A) Reconstruction of a dendritic segment of a CA1 hippocampal interneuron carrying spines on its dendrites. (B and D) Single confocal planes showing puncta expressing the vesicular GABA transporter (labeled in red, B2, D2) in close apposition to spines from an eGFP-expressing interneuron (labeled in green, B4, D4). (C and E) Single confocal plane showing a dendritic spine from an interneuron (C1, E1) in close apposition to puncta-expressing vesicular glutamate transporter-1 (labeled in blue, C3, E3). (F) Graphs showing the percentage of juxtaposition between presynaptic markers and dendritic spines of interneurons in original images compared with images in which the eGFP channel was rotated 90°. Scale bar: 2 μm in A and 1.1 μm in BE.

Ten spines of eGFP-expressing interneurons in CA1 were observed under transmission electron microscopy (Fig. 3); none of them received putative GABAergic symmetrical contacts, although they did receive asymmetrical ones (Fig. 3D). On dendritic shafts, there was also a preponderance of asymmetrical synapses, although occasionally large boutons making symmetrical perforated contacts could be observed (Fig. 3E). Asymmetrical boutons were small and had few mitochondria, resembling boutons arising from collaterals from CA1 pyramidal cells. Symmetrical boutons were larger and richer in mitochondria.

Figure 3.

Synaptic contacts on dendritic shafts and spines of spiny immunoreactive interneurons in the stratum oriens of CA1. (A) Field survey showing the location of eGFP-immunoreactive cells and spiny dendrites in CA1. (B) Enlargement of the stratum oriens showing the dendrite shown below at ultrastructural level. (C) Microphotograph of the trimmed block previous to cut the ultrathin sections, showing the slender sparsely spines on the selected dendrites. (D) Reconstructed spine through 50 nm thick sections. This thin spine that arises from the dendritic shaft (DS) is about 1 μm long and 200 nm thick. It receives a single asymmetrical contact on the head (arrowheads) from a small bouton (asterisk). D6 is an enlarged view of the squared region in D5. (E) Two consecutive sections (E1 and E2) of a dendritic shaft (DS) receiving a large symmetrical contact. E3 is an enlarged view of the squared region in E2. The bouton (asterisk) makes a large perforated synaptic contact (arrowheads point to active places). a: alveus; pl: pyramidal layer; DS: dendritic shaft; so: stratum oriens; sr: stratum radiatum. Scale bar: A: 300 μm; D1–D5: 700 nm; D6: 210 nm; E1–E2: 600 nm; E3: 170 nm.

Figure 3.

Synaptic contacts on dendritic shafts and spines of spiny immunoreactive interneurons in the stratum oriens of CA1. (A) Field survey showing the location of eGFP-immunoreactive cells and spiny dendrites in CA1. (B) Enlargement of the stratum oriens showing the dendrite shown below at ultrastructural level. (C) Microphotograph of the trimmed block previous to cut the ultrathin sections, showing the slender sparsely spines on the selected dendrites. (D) Reconstructed spine through 50 nm thick sections. This thin spine that arises from the dendritic shaft (DS) is about 1 μm long and 200 nm thick. It receives a single asymmetrical contact on the head (arrowheads) from a small bouton (asterisk). D6 is an enlarged view of the squared region in D5. (E) Two consecutive sections (E1 and E2) of a dendritic shaft (DS) receiving a large symmetrical contact. E3 is an enlarged view of the squared region in E2. The bouton (asterisk) makes a large perforated synaptic contact (arrowheads point to active places). a: alveus; pl: pyramidal layer; DS: dendritic shaft; so: stratum oriens; sr: stratum radiatum. Scale bar: A: 300 μm; D1–D5: 700 nm; D6: 210 nm; E1–E2: 600 nm; E3: 170 nm.

Dendritic Spine Density 2 and 7 Days After Intracerebral EndoN Injection

To know whether the presence of PSA-NCAM influenced the dendritic spines of eGFP interneurons, we studied their dendritic spine density in the hippocampal CA1 region, 2 and 7 days after an intracranial injection of the EndoN enzyme. Since EndoN can diffuse long distances in the brain parenchyma, the enzyme was injected in the somatosensory cortex, not in the hippocampus, in order to avoid lesion or edema caused by the intracerebral injection. Consequently, as shown in Figure 4AC, PSA was depleted from the hippocampus even considering that the injection of EndoN was performed in a distant region. Analysis with an anti-GFAP antibody and tomato lectin has revealed the presence of reactive astrocytes and microglia in the somatosensory cortex, surrounding the EndoN injection site. In contrast, the appearance of these cells in the hippocampus appeared normal and was indistinguishable from the uninjected hemisphere (Supplementary Fig. 1). None of the interneurons analyzed in the hippocampal CA1 region showed signs of degeneration, neither in their somata, nor in their dendrites. No evidence of abnormal or degenerated nuclei was found either in the CA1 region of the hippocampus or particularly in the eGFP-expressing interneurons of any of the group of animals studied.

In all interneurons analyzed, the spine density increased with the distance from the soma to each segment (Fig. 4F,H; 2-way ANOVA; 2 days: P < 0.0001; 7 days: P < 0.0001).

Figure 4.

Effects of PSA removal on the hippocampus of GINs mice. (AC) Micrographs showing the expression of PSA in the hippocampus of mice 2 days after intracraneal injections of vehicle (A), EndoN (B), and 7 days after intracranial injections of EndoN (C). (D and E) Z-projections of the distal portion of interneuron dendrites in CA1 showing the difference in spine density between animals injected with the vehicle (D1) or EndoN (D2) sacrificed 2 days after the injection and between those injected with the vehicle (E1) or EndoN (E2) sacrificed 7 days after the injection. (FI) Graphs representing the spine density of interneuron dendrites between vehicle- and EndoN-injected animals per 60 μm segments (F: 2 days after the injection; H: 7 days after the injection) and the total spine density (G: 2 days after the injection; I: 7 days after the injection) at 2 and 7 days, respectively. White bars represent vehicle-injected animals and black bars represent EndoN-injected animals. Statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001) 2-way ANOVA. Scale bar: 500 μm for AC and 15 μm for D1, D2, E1, and E2.

Figure 4.

Effects of PSA removal on the hippocampus of GINs mice. (AC) Micrographs showing the expression of PSA in the hippocampus of mice 2 days after intracraneal injections of vehicle (A), EndoN (B), and 7 days after intracranial injections of EndoN (C). (D and E) Z-projections of the distal portion of interneuron dendrites in CA1 showing the difference in spine density between animals injected with the vehicle (D1) or EndoN (D2) sacrificed 2 days after the injection and between those injected with the vehicle (E1) or EndoN (E2) sacrificed 7 days after the injection. (FI) Graphs representing the spine density of interneuron dendrites between vehicle- and EndoN-injected animals per 60 μm segments (F: 2 days after the injection; H: 7 days after the injection) and the total spine density (G: 2 days after the injection; I: 7 days after the injection) at 2 and 7 days, respectively. White bars represent vehicle-injected animals and black bars represent EndoN-injected animals. Statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001) 2-way ANOVA. Scale bar: 500 μm for AC and 15 μm for D1, D2, E1, and E2.

When analyzing the different 60-µm segments 2 days after the injection, we did not observe differences in dendritic spine density between the control and the treated animals, neither in the first (protected LSD P = 0.6659) nor in the second segment, where there was a trend toward an increase (P = 0.0706). However, we observed a significant increase in the distal segment (P = 0.0205) in the animals injected with EndoN compared with the controls (Fig. 4D,F). We also observed a significant increase (t-test, P = 0.0256) when considering the total number of spines in the dendrite between the animals injected with EndoN and those injected with the vehicle (Fig. 4G). When considering the different types of spines, we found that there was a significant increase in the number of filopodial protrusions (P = 0.003) in animals injected with EndoN (Supplementary Fig. 2A).

Figure 5.

Effects of PSA removal on organotypic hippocampal cultures. (A and B) Single confocal planes showing the expression of PSA in the cultures 24 h after the addition of vehicle (A) or EndoN (B) in the culture medium. Insets in A and B are low magnification views of the whole cultures. (C and D) Z-projections showing the complete morphology of interneurons from slices treated with the vehicle (C) or EndoN (D). (E and F) Turnover of interneuronal spines: external control between 0 (E1) and 24 h (E2) and 24 h after the addition of vehicle (E3). Internal control between 0 (F1) and 24 h (F2) and 24 h after the addition of EndoN (F3). White arrowheads indicate stable spines, red arrowheads disappeared spines and blue arrowheads newly appeared spines. (GI) Graphs representing the apparition rate of new spines (G), the disapparition rate (H), and the stability rate of preexisting spines (I) during the internal control (t0–t24) and the treatment with either vehicle or EndoN (t24–t48). (J) Graph representing the spine density relative to the original spine density at 0, 24 and 48 h. White bars represent vehicle-injected animals and black bars represent EndoN-injected animals. Statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001) 2-way ANOVA for repeated measures. Scale bar: 125 μm for A and B (700 μm for their insets), 75 μm for C and D and 5 μm for E1, E2, E3, F1, F2 and F3.

Figure 5.

Effects of PSA removal on organotypic hippocampal cultures. (A and B) Single confocal planes showing the expression of PSA in the cultures 24 h after the addition of vehicle (A) or EndoN (B) in the culture medium. Insets in A and B are low magnification views of the whole cultures. (C and D) Z-projections showing the complete morphology of interneurons from slices treated with the vehicle (C) or EndoN (D). (E and F) Turnover of interneuronal spines: external control between 0 (E1) and 24 h (E2) and 24 h after the addition of vehicle (E3). Internal control between 0 (F1) and 24 h (F2) and 24 h after the addition of EndoN (F3). White arrowheads indicate stable spines, red arrowheads disappeared spines and blue arrowheads newly appeared spines. (GI) Graphs representing the apparition rate of new spines (G), the disapparition rate (H), and the stability rate of preexisting spines (I) during the internal control (t0–t24) and the treatment with either vehicle or EndoN (t24–t48). (J) Graph representing the spine density relative to the original spine density at 0, 24 and 48 h. White bars represent vehicle-injected animals and black bars represent EndoN-injected animals. Statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001) 2-way ANOVA for repeated measures. Scale bar: 125 μm for A and B (700 μm for their insets), 75 μm for C and D and 5 μm for E1, E2, E3, F1, F2 and F3.

Comparing the control and the EndoN-treated animals 7 days after the injection in the different dendritic segments analyzed, we found a decrease in spine density in the distal segment (P = 0.0484) of the animals injected with EndoN compared with those injected with the vehicle (Fig. 4E,H), but we did not find significant changes in the proximal (P = 0.7516) or the intermediate segments (P = 0.1766). When analyzing the total density of spines, we only found a tendency toward a decrease in the EndoN-injected group (Fig. 4I; P = 0.0892). This decrease in the number of protrusions was mainly due to a significantly reduced density of mushroom spines (P = 0.018) in animals injected with EndoN (Supplementary Fig. 2B).

Effects of EndoN Treatment on Spine Dynamics Analyzed in Real Time on Organotypic Hippocampal Cultures

To know whether the spines of interneurons, whose density is influenced by PSA depletion in vivo, show a dynamic behavior when observed in real time, we have monitored the stability and the apparition rate of these postsynaptic structures in hippocampal organotypic cultures.

In control conditions (see Materials and methods section), the stability rate of CA1 hippocampal interneuronal spines in a 24-h time-lapse experiment represented between 67 ± 5% (t0–t24) and 68 ± 5% (t24–t48) of the total preexisting spines, whereas the appearance rate of new interneuronal spines represented between 35 ± 7% (t0–t24) and 32 ± 4% (t24–t48) of the total of preexisting spines (Fig. 5A,C,E,G).

Under experimental conditions (Fig. 5B,D,F), the apparition rate of new spines was significantly increased 24 h after the addition of EndoN to the culture medium (88 ± 7%; t0–t24), when compared with either the internal (P = 0.0024) or the external control (P = 0.0063; Fig. 5G). However, there was no effect of EndoN on the disappearance rate (Fig. 5H) or the stability of preexisting spines (Fig. 5I).

When analyzing the relative variation of the spine density, we observed a strong increase in the spine density in the slices treated with EndoN, when compared with the internal (P = 0.0003) and external controls (P = 0.0008; Fig. 5J).

Discussion

In the current study, we have described that the dendritic spines of hippocampal interneurons appear to receive mostly excitatory inputs. Using hippocampal organotypic slices and intracerebral injections of the enzyme EndoN, we have shown that these spines have a basal turnover, and that PSA removal influences some of their structural features.

A Subpopulation of Hippocampal Interneurons Display Dendritic Spines That Receive Excitatory Inputs

The present study is in accordance with previous studies on GIN mice, which stated that the majority of eGFP-expressing cells in the hippocampal CA1 region were interneurons expressing somatostatin and lacking parvalbumin expression (Oliva et al. 2000; Minneci et al. 2007). This subpopulation of hippocampal interneurons is known to project mainly to the stratum lacunosum moleculare and, in line with our results, to coexpress calbindin (Gulyás et al. 2003).

Although somatostatin-expressing interneurons in the cerebral cortex display dendritic spines (Kawaguchi et al. 2006), nobody, to our knowledge, has explored yet their synaptic input. However, a recent report has shown that the spines of a subpopulation of neocortical interneurons expressing neuropeptide Y receive mostly glutamatergic input (almost 90%), and that spines receiving inhibitory input constitute around 30% (Keck et al. 2011), but the authors found that this number was lower than chance when compared with images in which the eGFP channel was rotated 90°. Our results on the somatostatin-expressing interneurons in the hippocampal CA1 show that >45% of their spines are closely apposed to puncta-expressing VGluT1 and almost a 40% to puncta-expressing VGAT. However, similar to the results found by Keck et al. (2011), we have found that the colocalization with VGAT was lower than that found by chance. These results appear similar to those found in the dendritic spines of excitatory neurons, in which the vast majority of spines receive excitatory synapses (Nimchinsky et al. 2002), while only a minor portion receives an inhibitory input (Knott et al. 2002). Our electron microscopy study confirms the results obtained with confocal microscopy and clearly demonstrates that the spines of these CA1 interneurons receive excitatory input. Future ultrastructural analysis combined with GABA immunohistochemistry should reveal clearly whether inhibitory synapses contact the spines of these interneurons.

Structural Dynamics of Dendritic Spines on Interneurons

The dendritic spines of pyramidal neurons are plastic structures that modify their morphology, density, and dynamics after different paradigms. In these neurons, spines represent the excitatory postsynaptic input, and therefore, changes in spine density and dynamics have been associated with that in neural activity (Yuste and Bonhoeffer 2001). In addition, dendritic spines appear and disappear continuously, although only a small portion of them become stable, suggesting a stabilization mechanism driven by synaptic activity (Muller et al. 2010). Previous studies on organotypic hippocampal cultures have described the turnover of dendritic spines of CA1 pyramidal neurons in conditions similar to those of our experiments, showing a stability rate around 80% and an apparition rate about 20% (De Roo, Klauser, Muller 2008; Mendez, De Roo, et al. 2010). In addition, they have shown how different experimental conditions are able to modify this turnover (De Roo, Klauser, Mendez, et al. 2008; Mendez, Garcia-Segura, et al. 2010). However, studies using in vivo imaging with cranial windows have shown a stability rate over 95% in a daily basis and around 65% for a 1-month period in pyramidal neurons of the somatosensory and visual cortices (Knott and Holtmaat 2008), suggesting that there is a lower degree of spine turnover in vivo in the adult neocortex.

However, to date, very few studies have explored the structural remodeling of interneurons. Using cranial windows in transgenic mice with fluorescent interneurons, different studies by Dr Nedivi's laboratory have shown the elongation/retraction of dendritic branch tips in vivo in different neocortical regions (Lee et al. 2006, 2008; Chen, Flanders, et al. 2011; Chen, Lin, et al. 2011). Moreover, a recent report has studied the turnover of interneuronal dendritic spines in adult mice, showing a stability rate close to 98% in a 24-h time-lapse experiment (Keck et al. 2011). These results are considerably higher than the ones we describe in hippocampal interneurons, where the stability rate is around 65%. These discrepancies may be due to a difference in the stability of different interneuronal subpopulations, the region studied, or more likely, to a higher stability in vivo than in our organotypic cultures, probably due to the immature characteristics of these cultures. In any case, these basal changes in the structure of inhibitory neurons, both at the level of dendrites and spines, may play an important role in the activity-dependent modulation of neuronal connectivity within local cortical circuits, as already has been suggested (Chen and Nedivi 2010).

Effects of PSA Depletion on Dendritic Spine Density and Dynamics

The removal of PSA from the organotypic cultures increased the apparition rate of dendritic spines 24 h after the delivery of EndoN, without affecting the stability of previous spines. Consequently, it increased the relative spine density, which is the same effect that we observe in vivo 2 days after the injection of EndoN. These results suggest an important role for PSA-NCAM in regulating the synaptic input of hippocampal interneurons. It is interesting to note that the increase in the density of protrusions 2 days after EndoN injection in vivo is mainly due to an increase in filopodial structures. These long protrusions are thought to play a role in synaptogenesis and the formation of mature spines (Ziv and Smith 1996; see Knott and Holtmaat 2008 for review). Consequently, the increase in filopodia induced by EndoN may be interpreted as an attempt by interneurons to create new spines and to establish new synaptic contacts. However, the results from the animals sacrificed 7 days after PSA depletion suggest that this was a transitory and failed attempt, since no similar changes in the density of filopodia were found in these animals and the density of mushroom spines (those considered mature or “stable”) not only does not increase but also decreases.

It is, however, important to note that the scenarios in which these EndoN-induced changes in interneuronal spines are occurring may be different between the organotypic cultures and the adult hippocampus: although PSA-NCAM levels are already strongly downregulated after 11–15 days in vitro, they are higher than those in the adult hippocampus. Moreover, in perinatal animals, PSA-NCAM may still be expressed by some neuronal populations, which do not express it in adults. It also has to be noted that the spine density in the interneurons of organotypic cultures is almost twice as high than in those of the adult hippocampus.

The mechanisms by which PSA-NCAM may regulate the structure of interneurons are still unclear. However, there is previous evidence pointing to brain derived neurotrophic factor (BDNF) as a key player in these mechanisms: the removal of PSA induces neuronal differentiation (Seidenfaden et al. 2003; Petridis et al. 2004) and increases BDNF-binding capacity (Burgess and Aubert 2006). These results led to the enunciation of the “shielding” hypothesis for the action of PSA, which poses that the addition of PSA to NCAM may cover membrane receptors, such as TrkB or p75, limiting the binding to their ligands, and consequently their effects, on the portion of plasma membrane where PSA is expressed. Since BDNF increases dendritic spine density (Tyler and Pozzo-Miller 2003) and its diffusion in the nervous parenchyma is very restricted (Horch and Katz 2002), it is possible that PSA-NCAM expression may regulate dendritic spine density by limiting the binding of BDNF, and probably other neurotrophins, to their receptors. If we consider this possibility in the present study, the increased interaction between BDNF and its receptors after PSA removal may result in the increase of the apparition rate and the dendritic spine density that we observed 2 days after the injection of EndoN in vivo and in organotypic cultures. However, the long-term disruption of a mechanism regulating the action of local trophic factors may reduce the correct distribution of BDNF, resulting in a decreased synaptic transmission, which may explain the reduction in interneuronal spine density observed 7 days after the injection of EndoN. In fact, supporting the “shielding” hypothesis, a previous report from our laboratory has shown that hippocampal interneurons expressing PSA-NCAM have lower spine density and less perisomatic synaptic contacts than neighboring interneurons lacking this molecule (Gómez-Climent et al. 2010). However, these results also suggest another nonexcluding hypothesis: PSA may be blocking certain synaptic contacts on interneurons, and consequently, PSA depletion may activate many of these synapses (Di Cristo et al. 2007; Castillo-Gómez et al. 2011). Some of these newly activated synapses may make contact with recently generated spines, stabilizing a greater portion of these structures (Muller et al. 2010). This may explain the increase in spine density 2 days after the injection of EndoN in vivo and the increase in their appearance rate in organotypic cultures after the addition of the enzyme. However, the long-term effects of PSA removal, that is, 7 days after the injection in vivo, decreasing the density of dendritic spines, are more complex to interpret. These effects may be due to a compensatory response of the local hippocampal circuits to PSA depletion. If shortly after PSA depletion interneurons increase their spine density and, consequently, their synaptic input, this may result in an overall inhibition of local circuits, which may produce in the long term a negative feedback, leading the interneurons to decrease their spine density.

Another possible mechanism by which PSA-NCAM may regulate the structure and function of interneuronal dendritic spines is through its known interaction with α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) glutamate receptors (Senkov et al. 2012). Previous studies have shown that soluble PSA increases the single-channel open probability of AMPA receptors and facilitates their activity (Vaithianathan et al. 2004) while inhibiting extrasynaptic GluN2B-containing NMDA receptors (Hammond et al. 2006). Consequently, these studies raise the possibility that the effects of PSA-NCAM on dendritic spine structural plasticity may be, at least in part, mediated by the influence of PSA-NCAM on glutamate receptors, particularly AMPA. The expression of glutamate receptors in the dendritic spines of interneurons has not been studied in detail yet. However, if they are present on these spines, it is reasonable to think that alterations in their distribution, composition, or functionality may also lead to changes in the density and morphology of these postsynaptic structures, similar to those observed in the spines of pyramidal neurons. Synaptic glutamate influences spine structure in pyramidal neurons through its effects on different receptors. NMDA and kainate induce a transient loss of dendritic spines (Halpain et al. 1998; Hasbani et al. 2001). In contrast, glutamate exerts a trophic effect on spines by acting on AMPA receptors (McKinney et al. 1999; Passafaro et al. 2003), and the chronic blockade of these receptors or their removal induces dendritic spine loss (Hsieh et al. 2006; Mateos et al. 2007). In consonance with these results in pyramidal neurons, a recent study has found that the overexpression of certain splice variants of AMPA receptors dramatically increases spine density in cortical interneurons (Hamad et al. 2011). Consequently, PSA-NCAM removal may promote the activity of AMPA receptors, resulting in the increased density of dendritic spines, which we observe after our short-term EndoN experiment. The effects of long-term PSA depletion are more difficult to explain, but they may involve the inhibition of GluN2B-containing NMDA receptors.

In summary, this study indicates that the polysialylation of the NCAM is an important factor in the modulation of interneuronal structural plasticity. These findings may be particularly relevant to psychiatric disorders, such as schizophrenia or major depression, in which alterations in the structure of cortical inhibitory networks and in PSA-NCAM expression have been described, both in patients (Barbeau et al. 1995; Sullivan et al. 2007; Tao et al. 2007; Brennaman and Maness 2010; Varea et al. 2011) and in animal models of these disorders (Phillips et al. 2003; Mattson et al. 2004; Castrén 2005).

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxford journals.org/

Notes

This work was supported by Spanish Ministry of Science and Innovation (MICINN-FEDER) BFU2009-12284/BFI, MICINN-PIM2010ERN-00577/NEUCONNECT in the frame of ERA-NET NEURON, Fundación Alicia Koplowitz (http://www.fundacionaliciakoplowitz.org) and Generalitat Valenciana ACOMP/2012/229 to J.N. R.G. had a FPI predoctoral fellowship from the Spanish Ministry of Education and Science (BES 2007-15757). Conflict of Interest: None declared.

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