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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC Oct 19, 2008.
Published in final edited form as:
PMCID: PMC2570025
NIHMSID: NIHMS53990

Kalirin-7 Is an Essential Component of both Shaft and Spine Excitatory Synapses in Hippocampal Interneurons

Abstract

Kalirin, a multifunctional Rho GDP/GTP exchange factor, plays a vital role in cytoskeletal organization, affecting process initiation and outgrowth in neurons. Through alternative splicing, the Kalirin gene generates multiple functionally distinct proteins. Kalirin-7 (Kal7) is the most prevalent isoform in the adult rat hippocampus; it terminates with a PDZ binding motif, is localized to the post-synaptic density, interacts with PSD95 and causes the formation of dendritic spines when over-expressed in pyramidal neurons. Levels of Kal7 are low in the dendrites of hippocampal aspiny interneurons. In these interneurons, Kal7 is localized to the postsynaptic side of excitatory synapses onto dendritic shafts, overlapping clusters of PSD95 and NMDA receptor subunit NR1. Selectively decreasing levels of Kal7 decreases the density of PSD95 positive, bassoon positive clusters along the dendritic shaft of hippocampal interneurons. Over-expression of Kal7 increases dendritic branching, inducing formation of spine-like structures along the dendrites and on the soma of normally aspiny hippocampal interneurons. Essentially all of the spine-like structures formed in response to Kal7 are apposed to VGLUT1 positive, bassoon positive presynaptic endings; GAD positive, VGAT positive inhibitory endings are unaffected. Almost every Kal7 positive dendritic cluster contains PSD95 along with NMDA (NR1) and AMPA (GluR1 and GluR2) receptor subunits. Kal7-induced formation of spine-like structures requires its PDZ binding motif, and interruption of interactions between the PDZ binding motif and its interactors decreases Kal7-induced formation of spine-like structures. Kal7 thus joins Shank3 and GluR2 as molecules whose level of expression at excitatory synapses titrates the number of dendritic spines.

Keywords: Rho GEF, PSD95, GABA, dendritic growth, synaptogenesis

Introduction

A delicate balance between synaptic excitation and inhibition is critical for maintaining normal circuits in the CNS and interneurons play an essential role in regulating local circuit excitability (Maccaferri and Lacaille, 2003). Most hippocampal interneurons release γ-aminobutyric acid (GABA) as their primary neurotransmitter (Freund and Buzsaki, 1996). Disruption of GABAergic interneuron development may play a role in epilepsy, bipolar disorder, schizophrenia, autism and Alzheimer’s disease (Benes and Berretta, 2001;Powell et al., 2003;Ramos et al., 2005).

Based on their morphological, biochemical and electrophysiological properties, hippocampal interneurons are diverse, allowing them to play a wide range of roles in information processing (Freund and Buzsaki, 1996;Parra et al., 1998;McBain and Fisahn, 2001;Maccaferri and Lacaille, 2003). Neurochemical markers for interneurons include calcium binding proteins and neuropeptides (Sloviter and Nilaver, 1987;Gulyas et al., 1991;Baraban and Tallent, 2004). Hippocampal interneurons are generally free of spines, and both excitatory and inhibitory inputs synapse onto the dendritic shaft (Benson et al., 1994;Gulyas et al., 1999).

Hippocampal interneurons express several GABAA receptor subunits and their dendritic shafts receive inhibitory inputs from GABAergic endings (Mody and Pearce, 2004). Formation of spiny vs. aspiny excitatory synapses is cell-type specific and developmentally regulated (Rao et al., 1998;Anderson et al., 2004). Like excitatory synapses onto spines, aspiny excitatory glutamatergic post-synaptic endings contain PSD95 and glutamate receptors, GABAergic inhibitory post-synaptic endings do not (McBain et al., 1999;Zhang et al., 1999;Craig et al., 2006). Expression of the extracellular domain of the AMPA receptor subunit GluR2 causes spine formation in hippocampal interneurons (Passafaro et al., 2003). Synapse-specific calcium compartmentalization can be achieved at both spiny and aspiny excitatory synapses.

Small GTPases of the Rho subfamily and their GDP/GTP exchange factors (GEFs) play vital roles in the function of dendritic spines in hippocampal pyramidal neurons and cortical neurons (Nakayama et al., 2000;Tashiro et al., 2000;Ryan et al., 2005;Tolias et al., 2005). Kalirin, a multifunctional Rho GEF, is expressed in both hippocampal pyramidal neurons and interneurons (Ma et al., 2001). Antisense-mediated reductions in endogenous Kalirin expression (all isoforms) in hippocampal pyramidal neurons caused simplification of the dendritic tree and a reduction in spine density (Ma et al., 2003). Kal7, the major splice variant in the adult rat hippocampus, terminates with a PDZ (PSD95/DLG/ZO-1)-binding motif, interacts with PSD95 and localizes to the post-synaptic density (Penzes et al., 2001b;Ma et al., 2003) (Fig. 1A). Over-expression of Kal7 in cortical neurons causes an increase in spine density (Penzes et al., 2001b). Since hippocampal interneurons largely lack dendrite spines (Benson et al., 1994;Gulyas et al., 1999), but do express Kal7, our aim was to identify the roles of Kal7 in this neuronal cell type. We show that over-expression of Kal7 in hippocampal interneurons causes formation of spine synapses and that endogenous Kal7 is essential for maintenance of excitatory synapses onto dendritic shafts. The formation of spine synapses in interneurons induced by Kal7 requires its PDZ binding motif, presumably via its binding of interactors. A better understanding of the roles of Kalirin in interneurons will contribute to understanding learning and memory and to developing strategies for the treatment of neurological and psychiatric disorders (Wang et al., 2006).

Fig. 1
Kal7 is expressed in hippocampal interneurons

Experimental methods

Animals

Timed pregnant adult female Sprague Dawley rats from Charles River Laboratories were housed 1 per cage with a 14-10 h light-dark cycle with food and water available ad libitum. All experiments were conducted in accordance with the guidelines established by the UCHC Animal Care and Use Committee. For preparations of organotypic slices and dissociated cells, rat pups were sacrificed at postnatal day 9 (P9) or P1, respectively. For immunohistochemistry, adult male Sprague Dawley rats were anesthetized with ketamine and perfused transcardially with saline followed by 4% paraformaldehyde in PBS.

Antibodies

The following rabbit antibodies were used: affinity-purified Kal7 (JH2959, 1:200), GAD65/67 (1:2000, Sigma), GluR1 (1:500, Upstate), VGAT (1:1000, Synaptic Systems), GFP (for Western blot, Abcam), MAP2 (1:500, Chemicon), NPY (JH4, 1:1000), PAM (JH629, 1:500). Mouse monoclonal antibodies were: PSD95 (clone28/43, 1:300, gift from Dr. Matt Rasband, UCHC), Bassoon (1:200, Stressgen), NR1 (1:300, BD Pharmingen), GluR2 (1:500, Chemicon), GAD65 (GAD6, 1:200, Developmental Studies Hybridoma Bank, University of Iowa), parvalbumin (1:2000, Swant, Switzerland), MAP2 (1:500, Sigma), βIII tubulin (TUJ1, 1:500, Covance), GABAA receptor (β-chain, 1:200, Chemicon), GFP (1:200, Chemicon), Myc (9E10, 1:20), Kal7 (1:10, clone 20-D8). Guinea pig antibody to VGLUT1 (1:3000, Chemicon) and rat antibody to GFP (1:1000, Nacalai Tesque, Japan) were used where indicated.

DNA constructs

pEAK-Kal7GFP constructs

Enhanced green fluorescent protein (EGFP) and monomeric (A206K)EGFP were appended to the N-terminus or inserted into sites within spectrin repeats 5 and 9 of HisMycKal7 in the pEAK10 vector (Edge Biosystems, Gaithersburg, MD). Each of the EGFP-tagged variants of Kal7 produced massive lamellipodia and activated Rac when expressed in fibroblasts, but N-terminally EGFP-tagged Kal7 formed large fluorescent aggregates. Kal7 with EGFP or monomeric EGFP inserted into spectrin repeat 9 did not form large aggregates when expressed in hippocampal neurons. The GEF activity of GFP-tagged Kal7 was verified using cell based Rac activation assays as described (May et al., 2002;Schiller et al., 2005). pEAK vectors encoding HisMyc-Kal-GEF1 and HisMycKal7 were described previously (May et al., 2002). pCW-PSD95 was a generous gift of Dr. Morgan Sheng (Harvard University). pSCEP-Kalirin-7- CT was generated by replacement of the NsiI–NotI fragment of pSCEP-Kalirin-7 with a fragment truncated after nt 4728 of Kalirin-7, thus encoding a protein lacking the C-terminal 60 amino acids. pCMS-Kal7-antisense: The dual promoter pCMS vector (BD Biosciences Clontech) puts EGFP expression under control of the SV40 promoter and expression of the gene of interest under control of the CMV promoter. The 60 nt of coding region unique to Kal7 mRNA plus the following 845 nt (5′-GGC ACC CTT GTT to TCC AAT TTT TCA T-3′) of 3′-untranslated region were cloned into pCMS-EGFP in the antisense orientation. The cDNA was amplified by PCR from rat brain mRNA and the DNA sequence was verified against the rat genome (http://genome.ucsc.edu/). pDsRed-Monomer-N1 (Clontech) expresses DsRed under control of the CMV promoter. pSIREN-Kal shRNA constructs and pSIREN-PAM shRNA constructs: The RNAi-Ready-pSiren-DNR-DsRed-Express vector (Clontech) places shRNA expression under control of the human U6 promoter and DsRed expression under control of the CMV promoter. Sequences for shRNAs were selected using the rules at http://bioinfo.clontech.com/rnaidesigner/frontpage.jsp. pSIREN-Kal7 shRNA encodes a Kal7 shRNA located 394 nt after the stop codon in the unique 3′ UTR region of rat Kal7 mRNA (374 nt after the stop codon in mouse) (5′-AAAGTCTGCAACTCAAGTA-3′). pSIREN-PAM shRNA encodes a PAM shRNA within the PHM domain (5′-AACCTGTACAGATAAAGCC-3′). All DNA constructs were verified by sequencing.

Primary cultures of rat hippocampal neurons and transfection

Dissociated hippocampal cultures were prepared from P1 Sprague Dawley rats as described (Ma et al., 2003). Briefly, the hippocampi were digested with 0.25% trypsin for 25 min at 37 °C. Dissociated cells were plated in Neurobasal A medium containing 7% heat-inactivated horse serum, 1 × B27 supplement, 0.5 mM glutamine and maintained at 37 °C in 5% CO2. Three hours later, plating medium was replaced with fresh medium containing only 3% horse serum (heat inactivated). Three days after plating, the culture medium was exchanged with maintenance medium [Neurobasal A medium containing 2% B27 supplement, 0.5mM glutamine, 25 units/ml penicillin, 25 μg/ml streptomycin]. Thereafter, 50% of the medium was replaced twice a week for up to 3 weeks. Freshly dissociated P1 neurons were nucleofected (Amaxa Gmbh, Germany) using the rat neuron kit. Following nucelofection, neurons were plated and allowed to recover in DMEM for 3 hours; nucleofected neurons were kept in maintenance medium up to 3 weeks.

Treatment of hippocampal cultures with cell permeant peptide

To disrupt binding of the Kal7 PDZ binding motif to its interactors, we synthesized a peptide comprising the final eight residues of Kal7 (GDPFSTYV; Kal7CT) preceded by seven arginine residues (R7-Kal7CT). In the control peptide, the C-terminal Val was replaced with Asp (GDPFSTYD) (mutant R7 Kal7CT). To determine whether the peptides entered neurons, dissociated hippocampal neurons (DIV 18) were incubated with the R7-Kal7CT (1, 5, 10, 20 μM, peptides dissolved in medium) for 45 min; neurons were them washed with medium and returned to the incubator with half the original medium. Control neurons were treated with mutant R7-Kal7CT (1, 5, 10 and 20 μM) or vehicle at the same time under the same conditions. Neurons were fixed with 4% paraformaldehyde 1 h, 12 h, 24 h, or 3 days after removing peptide and double-stained with a polyclonal antibody to Kal7 (which detects both the peptide and endogenous Kal7) and a monoclonal antibody to Kal7 (which detects endogenous Kal7, but not R7-Kal7CT). Based on signal to noise ratio, the optimal concentration of peptide is 10 μM. To determine whether R7-Kal7CT can alter Kal7 induced spine formation, dissociated hippocampal neurons were transfected with vector encoding Kal7GFP at DIV1. The R7-Kal7CT peptide (10 μM) was added to the cultures at DIV 7 and DIV 13. Control neurons receive mutant R7-Kal7CT (10 μM) or vehicle at the same times. Cells were fixed with 4% paraformaldehyde and triple-stained at DIV18 with antibodies to GFP, MAP2 and GAD65.

Preparation of rat hippocampal organotypic cultures

Hippocampal slices were prepared from P9 Sprague Dawley rats as described (Ma et al., 2003). Briefly, hippocampi were dissected into ice-cold, sterile Gey’s balanced salt solution (Sigma) containing 0.5% glucose. Medial hippocampi were sliced transversely (400 μm) using a slice chopper. Slices were kept in ice-cold Gey’s balanced salt solution and then placed onto 30 mm Millicell CM membrane inserts in Petri dishes filled with 1.1 ml of culture medium containing 0.5X basal Eagle’s medium, 0.25X Hank’s balanced salt solution, 0.25X horse serum (defined, heat-inactivated), 25 units/ml penicillin, 25 μg/ml streptomycin, and 1 mM L-glutamine (Life Technologies). Slices were kept under 5% CO2 at 37°C, with media changes at 1 DIV and every 3 days thereafter.

Immunohistochemistry (brain sections and slices)

Coronal sections (12 μm) were cut and mounted on gelatin-coated slides. Antibody specificity was evaluated by replacement of antibody with pre-immune serum and pre-incubation of antibody with its antigen (10 μg/μl) as described (Ma et al., 2003); staining was eliminated in both controls. Sections were blocked in PBS containing 1% BSA/10% normal goat serum/0.25% Triton X-100 (pH 7.4) for 1 h at room temperature. Sections were stained subsequently with polyclonal Kal7 and GAD65/67 antibodies at 4°C overnight. Sections were stained simultaneously with polyclonal Kal7 and monoclonal parvalbumin antibodies. Primary antibodies were visualized with Cy3-labeled donkey anti-rabbit IgG (Jackson Lab) and FITC-labeled goat anti-mouse IgG (Jackson Lab). Images were taken with a Zeiss LSM510 confocal microscope. Replacement of the Kal7 antibody with pre-immune serum or pre-incubation of the antibody with its antigen (10 μg/ml) completely eliminated staining (Ma et al., 2003).

Hippocampal slices were fixed for immunostaining in 4% paraformaldehyde in PBS as described (Ma et al., 2003). Slices were stained simultaneously with polyclonal Kal7 and monoclonal GFP antibodies and visualized as described above. For measurement of Kal7 staining intensity in Kal7GFP neurons and neighboring non-transfected neurons, images were taken under identical conditions and NIH image J was used to analyze fluorescence intensity (Ma et al., 2003). At least 20 neurons were analyzed for each group.

Immunocytochemistry (dissociated neurons)

Neurons were generally fixed for 20 min in 4% paraformaldehyde in PBS. After blocking in PBS containing 1% BSA/5% normal goat or donkey (or both) serum/0.20% Triton X-100 (pH 7.4) for 1 h at room temperature, cells were doubly or triply stained with appropriate primary antibodies overnight at 4°C. For visualization of NR1, VGLUT1, GluR1, GluR2, VGAT, GABAA receptor and Kal7, cells were fixed with methanol for 12 min at −20°C, except where indicated. Primary antibodies were visualized with appropriate secondary antibodies: Cy3-donkey anti-rabbit IgG (Jackson Lab), Cy3-donkey anti-mouse IgG (Jackson Lab), Cy3-donkey anti-guinea pig IgG (Jackson Lab), FITC-goat anti-mouse IgG (Jackson Lab), FITC-goat anti-rat IgG (Jackson Lab), Alexa Fluor 633-goat anti-rabbit IgG (Molecular Probes), Alexa Fluor 633-goat anti-mouse IgG (Molecular Probes). Coverslips were mounted with Prolong Gold antifade reagent (Molecular Probes).

Biolistic transfection

Plasmid DNA was precipitated onto 1.0 μm gold microcarrier particles at a concentration of 1 μg plasmid/mg gold as described (Ma et al., 2003). At DIV2, slices were transfected with plasmid coated gold particles using the Helios Gene Gun System (Bio-Rad). After transfection (48, 72 and 96 h), slices were fixed and processed for immunostaining as described above. High density cultures of dissociated hippocampal neurons were biolistically transfected with pCMS-EGFP, pCMS-Kal7-antisense, pDsRed-Monomer-N1, pSIREN-Kal7 shRNA or pSIREN-PAM shRNA at DIV10 as described (Ma et al., 2003). Cells were fixed and processed for immunostaining 6 days after transfection.

Image analysis and quantification

Images captured using a Zeiss LSM510 confocal microscope were analyzed as described (Ma et al., 2003). For organotypic slices, only GFP positive neurons in the CA1 area were analyzed; interneurons and pyramidal neurons were identified based on their locations and their morphology. For dissociated hippocampal neurons, GFP positive interneurons were identified by GAD staining (Esclapez et al., 1994). Z-stacks (Z step, 1 μm) were taken using a 20X objective (0.7 digital zoom factor); images of the entire neuron were generated with Zmaris 3.2 software (Bitplane AG, Zürich). Dendrites were traced, total dendritic length and dendritic branch segments were calculated, and Sholl profiles were analyzed using Neurolucida (Ma et al., 2003).

For quantification of spine density and synaptic clusters, a stack of images (Z step, 0.3 μm) was acquired using a 63X objective (2.5 digital zoom factor) and dendrites were visualized in 3-dimensions (Ma et al., 2003). For analysis of colocalization of Kal7GFP clusters and synaptic markers, single plane images through the brightest point were used. For each experiment, all images were taken with identical settings under the same conditions. Spine density and synaptic clusters were counted after images were calibrated and thresholds were set to ensure that all interesting structures were included in the analysis. Quantifications were performed using Metamorph (University of Imaging, West Chester, PA) and were limited to dendrites within 100 μm of the soma. The length of each traced spine was measured using Metamorph. Data are presented as average ± SEM. Statistical analyses were performed with JMP6 software (SAS Institute, Cary, NC) using one way analysis of variance (ANOVA) followed by Dunnett’s test to assess statistical significance between groups; *P < 0.05 or **P<0.01 was considered statistically significant.

Results

Kal7 is expressed in hippocampal interneurons and localized at excitatory synapses

Antisera specific for the unique C-terminus of Kal7 also detect ΔKal7, a splice variant generated from a different promoter (Fig. 1A). Since Kal7 protein is much more prevalent than ΔKal7 (McPherson et al., 2002), we simply refer to cross-reactive material as Kal7. Interneurons are very heterogeneous, and we focus here on interneurons in the CA1 region that can be identified in coronal sections of adult rat hippocampus based on their location and staining with antibody to GAD65/67 (Fig. 1B–D1), a marker for inhibitory interneurons, or parvalbumin (PV, Fig. 1E–G1) (Freund and Buzsaki, 1996). Two different antibodies to GAD were used to identify GABAergic neurons. The polyclonal antibody to GAD65/67 labels both presynaptic GABAergic endings and the soma of GABAergic neurons (Esclapez et al., 1994). Since the monoclonal antibody is specific for GAD65, it visualized presynaptic endings more readily than the cell soma (Chang and Gottlieb, 1988).

Kal7 positive interneurons are scattered throughout the stratum oriens, stratum pyramidal and stratum radiatum (Fig. 1B–D; arrows). Of the 128 GAD65/67 positive interneurons examined, all contained Kal7; levels were generally lower than in CA1 pyramidal neurons. Of the 98 parvalbumin positive interneurons examined, all contained Kal7; levels were lower than in the soma of most parvalbumin negative neurons. (Fig. 1E–G1). The level of Kal7 in the dendrites extending from the soma of parvalbumin positive interneurons is much lower than in the soma (Fig. 1E1–G1).

To evaluate Kal7 staining in the dendrites of mature interneurons, DIV21 cultures of dissociated hippocampal neurons were fixed with methanol and stained for Kal7 and GAD65/67. Kal7 staining was observed in the soma and in the dendrites of both interneurons and other neurons (Fig. 2A–2F). However, Kal7 levels in the dendrites of interneurons are lower than in other neurons (Fig. 2A, 2D). Kal7 staining in the dendrites of GAD65/67 positive interneurons appears in clusters (Fig. 2D1); the Kal7-positive clusters are not colocalized with GAD65/67, which is present in presynaptic inhibitory terminals (Fig. 2D1–F1).

Fig. 2
Kal7 is localized at excitatory synapses of hippocampal interneurons

To determine whether the Kal7 positive clusters along the dendrites of interneurons identified by GAD65/67 staining were synaptic structures, Kal7 and a variety of synaptic markers were visualized simultaneously: Bassoon, a presynaptic terminal marker; VGLUT1, a marker for glutamatergic presynaptic endings; PSD95, a post-synaptic density marker; NMDA receptor subunit NR1 and AMPA receptor subunit GluR2, markers for the postsynaptic side of excitatory synapses. Kal7 positive clusters were apposed to almost all VGLUT1 positive clusters (Fig. 2G–I) and to most Bassoon positive clusters (Fig. 2J–L) along interneuron dendrites. Bright Kal7 positive clusters were always positive for PSD95 (Fig. 2M–O) and NR1 (Fig. 2P–R). While a subset of the Kal7 positive clusters (13±1%) were positive for GluR2, most (95±4%) of the GluR2 positive clusters were positive for Kal7 (Fig. 2S–U). These data indicate that Kal7 in interneurons is localized to the postsynaptic side of excitatory synapses onto the dendritic shaft.

Reduced endogenous Kal7 expression causes a decrease in excitatory synapses onto interneurons

To determine whether Kal7 plays an essential role in the formation of excitatory synapses onto the dendritic shafts, we sought a means of selectively reducing expression of this isoform of Kalirin. Antisense-mediated reductions in the expression of all of the major isoforms of Kalirin in hippocampal pyramidal neurons revealed an essential role for Kalirin in maintenance of the dendritic arbor and dendritic spines (Ma et al., 2003). Kal7 is the major isoform in the adult brain (Johnson et al., 2000) and its appearance at the time of synapse formation suggests a specific role for this isoform in synaptogenesis. Kal7 immunoreactivity in CA1 interneurons and pyramidal cells was first detected at P7, with increasing levels of staining in both cell types from P7 to P30 (data not shown). A Kal7/ΔKal7-specific shRNA targeted to the unique 3′-untranslated region of Kal7/ΔKal7 was expressed in dissociated hippocampal neurons (Fig. 3A). Expression of Kal7 shRNA caused a decrease in endogenous Kal7 immunoreactivity in GAD65 positive interneurons in comparison to neighboring non-transfected interneurons (Fig. 3A, G); staining for Kal12 was not affected, demonstrating specificity. Expression of Kal7 antisense RNA (pCMS-Kal7-antisense) produced effects identical to those of Kal7 shRNA (data not shown). To address specificity, we created a shRNA vector targeted to PAM, a transcript highly expressed in hippocampal interneurons (Ma et al., 2002). Expression of PAM shRNA decreased PAM immunoreactivity by 56% in GAD65 positive interneurons, but did not alter the number of PSD95 clusters along their dendrites (data not shown).

Fig. 3
Expression of Kal7 shRNA causes a reduction in the number of excitatory synapses onto dendritic shafts of interneurons

Dissociated hippocampal neurons transfected with control vector (pSiren. DsRed) or pSiren-Kal7 shRNA were fixed after 6 days and simultaneously visualized with antibodies to GAD65/67 and either PSD95 (Fig. 3B, C) or bassoon (Fig. 3D–F). Expression of Kal7 shRNA caused a decrease in the density of PSD95 clusters along the dendrites of interneurons (Fig. 3B1, C1, H). Similarly, the density of presynaptic bassoon positive clusters along the dendrites of interneurons expressing the Kal7-shRNA is decreased when compared to control DsRed expressing interneurons (Fig. 3D, E, H) or neighboring non-transfected interneurons (Fig. 3F). The prevalence of GAD65/67 positive clusters along the dendrites of Kal7-shRNA expressing interneurons was not altered (Fig. 3H). Kal7 thus plays an essential role in the formation of excitatory synapses on spines and on dendritic shafts.

Over-expression of Kal7 increases dendritic branching and spine formation in hippocampal interneurons

Since endogenous Kal7 is essential for excitatory synapses on the dendritic shafts, we wonder whether overexpression of Kal7 would cause the formation of spine-like structures or increase the number of shaft synapses. To determine the effects of slightly increasing Kal7 levels in interneurons, hippocampal slices were biolistically transfected with vectors encoding GFP or bioactive Kal7GFP (Fig. S2, Fig. 4). The use of organotypic slices facilitates identification of CA1 interneurons and pyramidal neurons. Slices prepared from P9 pups were transfected at DIV2 and were analyzed 48, 72 and 96 h later. GFP was visualized using an antibody to intensify the signal and ensure visualization of all processes. The validity of this approach was established by quantifying dendritic spines in interneurons stimultaneously expressing GFP and MycKal7; identical results were obtained using GFP-staining (turning the red channel off and visualizing only the green channel) or Myc-staining (turning the green channel off and visualizing only the red channel) to identify dendritic spines (Fig. S3). Similarly, the large increase in spine density produced by MycKal7 (visualized with co-transfected GFP) was identical to the increase seen using Kal7GFP (Fig. S3). Based on neuronal morphology and the intensity of the Kal7GFP signal, 72h post-transfection was selected as the optimal time for analysis; all slices in this experiment were analyzed at this time (Fig. 4). Interneurons can be distinguished from pyramidal neurons according to their localization and their short, thick dendrites with long, thin, complex axons (Fig. 4, D, G, H). Compared with non-transfected, neighboring neurons (n=22), expression of Kal7GFP (n=20) results in a 2-fold increase in Kal7 immunoreactivity in processes and a 2.5-fold increase in the cell soma (Fig. 4). Kal7GFP is observed in interneurons and pyramidal neurons (Fig. 4A–H) as well as in glia (open arrows, Fig 4G–H) after biolistic transfection.

Fig. 4
Expression of Kal7GFP alters the number of dendritic branches and spines in CA1 interneurons

Interneurons in the CA1 region expressing GFP (Fig. 4G) or Kal7GFP (Fig. 4H) were subjected to Sholl analysis. Expression of Kal7GFP in interneurons increased the number of dendritic branches within 125 μm of the cell soma (Fig. 5A); total dendritic length was unaltered (Fig. 5B). The total number of dendrites was unaltered following expression of Kal7. Exogenous Kal7GFP is found in long, highly branched axons, which were not analyzed because of their complexity (Fig. 4H).

Fig. 5
Expression of Kal7GFP alters the number of dendritic branches and spines in CA1 interneurons

To evaluate the possibility that expression of Kal7GFP might induce formation of spine-like structures in hippocampal interneurons, biolistically transfected neurons were systematically analyzed at higher magnification (Fig. 4I–M). As expected, the dendrites of GFP-expressing interneurons (controls) were not decorated with spine-like structures and spine-like structures were never observed on their soma (Fig. 4I–J). Over-expression of Kal7GFP in interneurons results in the appearance of spine-like structures along their dendrites and around their soma (Fig. 4K–M). Some of these spine-like structures have the appearance of mushroom spines, suggesting they are mature, functional spines (Fig. 4L–M). In addition, Kal7GFP positive clusters are observed along the dendritic shaft (open arrows, Fig. 4L–M). Quantification revealed a greater than 10-fold increase in dendritic spine density following expression of Kal7GFP in hippocampal interneurons (GFP, 0.28±0.31/10μm;Kal7GFP, 3.10±0.40/10μm). The length of these spines is not longer than 2 μm and most of them are between 0.5 to 1.75 μm (Fig. 5C).

Over-expression of Kal7GFP alters dendritic morphology and induces spine formation in hippocampal interneurons in culture

To determine whether over-expression of Kal7 can alter dendritic morphology and induce spine formation in interneurons in culture, dissociated cells were transfected with vectors encoding GFP or Kal7GFP. In order to identify interneurons, cultures were stained simultaneously with antibodies to GAD65/67 (Esclapez et al., 1994) (Fig. 6C–H), MAP2 to distinguish dendrites from axons (not shown) and GFP (Fig. 6A–H). As observed in slice cultures, expression of Kal7GFP increases the number of dendritic branches within 50 μm of the cell soma and decreases the number of long dendrites (more than 100 μm) (Fig. 6I). Unlike the response observed in organotypic slices, total dendritic length is diminished in Kal7GFP expressing interneurons in dissociated culture (GFP, 3504±165 μm; GFPKal7, 2112±121 μm, P< 0.01, Student’s T test). To identify neuropeptide (NPY)-expressing interneurons, neurons were triple-stained simultaneously with antibody to GFP, NPY and GAD65 (Fig. 7A–H). NPY is expressed in hippocampal interneurons under basal conditions (Ma et al., 2002). To identify parvalbumin-expressing interneurons (Gulyas et al., 1999), neurons were double-stained with antibodies to parvalbumin and GFP (Fig. 7I–J). To quantify spine density, neurons stained with antibodies to GFP and GAD65/67 were stained simultaneously for MAP2, to highlight dendritic shafts (Fig. 7L–M, Fig. S4). Spine formation in dissociated cultures was examined using higher power images. As observed in organotypic slices, the dendrites of interneurons expressing GFP are almost free of spine-like structures (Fig. 7A, L; Fig. S-4A). Spine-like structures are never found on the somas of interneurons expressing GFP (Fig. 7A, Fig S-4A). Expression of Kal7GFP induces a marked increase in spine-like structures along the dendrites and on the somas of interneurons (Fig. 7E, J-J1, M, Fig. S-4H–I). Most of these spines are 0.5 to 2.5 μm long (Fig. 7N). In GFP-expressing interneurons, GFP is diffusely merged with staining for MAP2 (Fig. 7L, Fig. S-4D). In contrast, in Kal7GFP-expressing interneurons, the GFP occurs in clusters both within the dendritic shaft and at the tips of dendritic spines (Fig. 7M, Fig. S-4H). Based on immunostaining, Kal7GFP is again being expressed in dendrites at levels that are 1.9-fold higher than endogenous levels of Kal7. Quantification reveals a far higher number of dendritic spine-like structures formed in response to Kal7GFP in cell culture (15.0±0.7/10 μm) than in slice culture (3.1±0.4/10 μm). The Kal7-induced spines formed in culture tend to be longer than the spines formed in slice cultures (Fig. 7N). To ensure that Kal7GFP and MycKal7 have similar effects on spine formation, dissociated hippocampal neurons were transfected with vector encoding soluble GFP alone, co-transfected with vectors encoding soluble GFP and MycKal7, or transfected with vector encoding Kal7GFP alone. Co-expression of MycKal7 with GFP or expression of Kal7GFP alone caused a similar increase in spine–like structures in hippocampal interneurons (Fig. S3).

Fig. 6
Expression of Kal7GFP alters dendritic morphology in cultured hippocampal interneurons
Fig. 7
Over-expression of Kal7 forces aspiny interneurons to produce spine-like structures

The Kal7GFP positive spines are sites of excitatory synapses in interneurons

Kal7GFP clusters are localized to spine tips and within the dendritic shaft of interneurons. To determine the characteristics of these Kal7GFP clusters, hippocampal neurons expressing GFP (Fig. 8A, a) or Kal7GFP (Fig. 8A, b) were visualized simultaneously with antibodies to GFP, PSD95 and GAD65/67. In interneurons expressing GFP, all of the PSD95 positive clusters are localized within the dendritic shaft (Fig. 8A, a1); no PSD95 positive spine-like structures are present. In contrast, in Kal7GFP-expressing interneurons, PSD95 positive clusters are observed both within the dendritic shaft and at the tips of spine-like structures (Fig. 8A, b1). Colocalization analysis indicates that 97% of the Kal7GFP clusters are also stained for PSD95. The number of PSD95 positive clusters within the dendrites or in spine-like structures was quantified. Compared with control interneurons expressing GFP, expression of Kal7GFP causes a 2-fold increase in the linear density of dendritic PSD95 positive clusters (Fig. 8C). Classifying total PSD95 positive clusters as within the dendritic shaft or in spine-like structures revealed that 96% were in the dendritic shaft in GFP expressing interneurons while only 55% were in the dendritic shaft in interneurons expressing Kal7GFP.

Fig. 8
Kal7GFP is localized to synapses in interneurons

PSD95 is localized to glutamatergic synapses on spines and shafts and is involved in the formation of excitatory synapses (Hunt et al., 1996; El Husseini et al., 2000). To evaluate the presence of presynaptic endings, cultures expressing GFP (Fig. 8B, c) or Kal7GFP (Fig. 8B, d) were stained simultaneously with antibodies to GFP, bassoon and GAD65/67. In interneurons expressing GFP, bassoon positive presynaptic terminals are directly apposed to the dendritic shaft (Fig. 8c1). In Kal7GFP-expressing interneurons, almost every Kal7GFP cluster is aligned with a bassoon-positive presynaptic terminal (Fig. 8d1). Presynaptic endings terminate on Kal7GFP positive clusters that occur within the dendritic shaft and at the tips of spine-like structures (Fig. 8d1, arrowheads and arrows, respectively). Expression of Kal7GFP causes a 2-fold increase in the total number of bassoon positive presynaptic terminals along the dendrites of interneurons (Fig. 8C), matching the increase of PSD95 clusters.

In addition to excitatory input, the dendrites of interneurons receive inhibitory inputs from other GABAergic interneurons (Cobb et al., 1997). Inhibitory synapses were identified using antibody to GAD65/67. The dendrites of control interneurons were lined with GAD65/67-positive structures, which correspond to inhibitory synapses (Fig. 8a1, c1). As expected, GAD65/67 positive clusters overlap with a subset of the Bassoon positive clusters; GAD65/67 positive inhibitory synapses are much less prevalent than PSD95 positive clusters (Fig. 8a1). Expression of Kal7GFP did not alter the number of GAD65/67 positive presynaptic endings along the dendrites of interneurons (Fig. 8b1, d1, C). Kal7GFP positive clusters rarely aligned with GAD65/67 positive presynaptic terminals (Fig. 8b1, d1).

Localization of Kal7GFP at excitatory synapses of mature interneurons

To further characterize the Kal7GFP clusters formed in interneurons (identified using antisera to GAD65 or GAD65/67), cultures transfected at DIV1 and maintained in culture for 3 weeks were visualized with antibodies to GFP and to the vesicular GABA transporter (VGAT), a marker for inhibitory GABAergic presynaptic endings, or VGLUT1 (Fig. 9). VGLUT1 positive excitatory presynaptic terminals are localized to the surface of the dendritic shaft in control interneurons expressing GFP (Fig. 9A). VGLUT1-positive excitatory presynaptic terminals are localized to the tips of the spine-like structures formed in response to expression of Kal7GFP and along the dendritic shaft (Fig. 9B). Quantitative analysis reveals that 96% of the Kal7GFP positive clusters are aligned with VGLUT1 positive clusters along the dendrites of interneurons expressing Kal7GFP (Fig. 9I). In contrast, little co-localization of Kal7GFP positive clusters and VGAT (Fig. 9C, I) or GAD65 (Fig. 9D, I) positive inhibitory presynaptic terminals was observed.

Fig. 9
Kal7GFP is localized to the postsynaptic side of excitatory synapses onto interneurons

We next asked whether the clusters of Kal7GFP in the dendrites of hippocampal interneurons co-localized with excitatory glutamate receptors (NR1, GluR1 or GluR2) or with inhibitory GABAA receptors (Fig. 9E–H). Almost all (96%) of the Kal7GFP positive clusters co-localized with NR1 positive clusters (Fig. 9E, J). A slightly smaller percentage (89%) co-localized with GluR1 positive clusters (Fig. 9F, J). Most (96%) co-localized with GluR2 positive clusters (Fig. 9G, J). In contrast, only 1% of the Kal7GFP positive clusters overlap with GABAA receptor positive clusters (Fig. 9H, J). PSD95, which is present at excitatory endings on spines and on dendritic shafts, is present at 98% of the Kal7GFP positive clusters (Fig. 9J).

PDZ binding motif is essential for Kal7-induced spine formation, and over-expression of PSD95 does not make aspiny interneurons produce spine-like structures

The C-terminal of Kal7 interacts with several PDZ domain proteins localized to spine-like structures (Penzes et al., 2001b). To determine whether the PDZ binding motif of Kal7 is required for its effect on spine formation in interneurons, neurons were co-transfected with DsRed plus Kal7ΔCT, which lacks the PDZ binding motif, at P1 and examined at P16. Expression of Kal7ΔCT did not cause spine formation in interneurons; myc staining was diffuse in the cell soma and dendritic shafts (Fig. 10A–D). Of the 20 myc positive interneurons examined, all showed smooth dendritic shafts. As expected, expression of Kal7 in parallel cultures caused spine formation in interneurons (data not shown).

Fig. 10
PDZ binding motif is required for Kal7-induced spine formation but expression of PSD95 does not mimic expression of Kal7

Since the PDZ binding motif of Kal7 is essential, we next asked whether over-expression of one its targets, PSD95, might reproduce the effect of Kal7. Hippocampal neurons in dissociated culture were transfected with vector encoding PSD95 at DIV1 and neurons were doubly stained at DIV18 with antibodies to PSD95 and GAD65/67 (Fig. 10). The levels of PSD95 in PSD95-transfected interneurons are about 11 times higher than in non-transfected interneurons. Exogenous PSD 95 did not cause spine formation in hippocampal interneurons (Fig. 10E, 10H). In the 18 PSD95 positive interneurons examined, dendritic PSD95 clusters were always localized on dendritic shafts. Interneurons expressing lower levels of exogenous PSD95 also failed to develop spine-like structures (not shown).

Interruption of Kal7 PDZ binding motif interactions blocks Kal7-induced formation of spine-like structures

Since the Kal7 PDZ binding motif is necessary for spine formation in interneurons, we sought a means of disrupting its interactions. Cell-permeant peptides offer a means of disrupting specific protein-protein interactions in a controlled manner (Aarts et al., 2002). Wild type and mutant cell-permeant Kal-7 C-terminal peptides were designed; R7-Kal7CT is detected by our polyclonal Kal7 antibody, but not by our monoclonal Kal7 antibody (Fig. S5A–C). Neurons incubated with R7-Kal7CT (10pM) exhibited readily detectable staining in the soma, dendrites and synaptic structures, indicating intracellular peptide uptake (Fig. S5A–E, H). Neurons treated with mutant R7-Kal7CT peptide showed staining in the cell soma (Fig. S5F), with less peptide binding in dendrites (Fig. S5F). Cultures treated with vehicle revealed endogenous Kal7 staining in the soma (Fig. S5G).

To determine the effect of R7-Kal7CT on Kal7-induced spine formation, dissociated hippocampal neurons transfected with vector encoding Kal7GFP at DIV1 were allowed to mature for 18 DIV. R7-Kal7CT, R7-Kal7CTmutant or vehicle were added to the cultures at DIV7 and DIV13 and cultures were examined at DIV18. Staining with antibodies to GFP, MAP2 and GAD65 revealed the expected spine-like structures along the dendrites of GFP-Kal7 expressing neurons (Fig. 11A–D, 11I). The R7-Kal7CT-treated neurons appeared to be healthy; MAP2 stained dendrites were smooth and uniformly stained. The R7-Kal7CT peptide blocked Kal7-induced spine formation compared with control neurons treated with the R7-Kal7CT mutant peptide (Fig. 11, spine density: 13.2±1.2/10 μm for mutant peptide; 1.8±0.9/10 μm for Kal7CT peptide), which differs at only one residue from the R7-Kal7CT peptide.

Fig. 11
Disruption of Kal7 PDZ binding motif interactions blocks Kal7-induced formation of spine-like structures

Discussion

Kal7 is localized to excitatory synapses on the dendritic shafts of hippocampal interneurons

Although most glutamatergic synapses in the adult brain occur onto dendritic spines, glutamatergic synapses onto dendritic shafts are common in developing pyramidal neurons and mature GABAergic interneurons (Rao et al., 1998;Gulyas et al., 1999). Both shaft and spine synapses can produce highly localized changes in intracellular calcium (Goldberg et al., 2003). Proteins like PSD95, along with NMDA and AMPA receptors, are localized to the post-synaptic density of excitatory synapses on the dendritic shafts of interneurons (Allison et al., 1998;Zhang et al., 1999). In mature, dissociated hippocampal GABAergic interneurons, staining for Kal7, PSD95 and the NR1 subunit of the NMDA receptor is coincident (Fig. 2). Consistent with this, Kal7 clusters align with presynaptic endings identified using antibodies to bassoon or VGLUT1. By contrast, Kal7 clusters are not aligned with GAD65 positive terminals.

Kal7 is necessary for maintenance of excitatory synapses onto hippocampal interneurons

To understand the functions of endogenous Kal7 in hippocampal interneurons, levels of Kal7 and ΔKal7 were reduced using a small hairpin RNA and antisense RNA targeted to the 3′-UTR unique to these isoforms. Reducing Kal7 decreased the number of PSD95 clusters and bassoon positive presynaptic terminals on the dendrites of interneurons. In this experimental paradigm, levels of Kal12 (and presumably Kal9) were unaltered, identifying Kal7 as the isoform essential for maintenance of excitatory synapses. Excitatory synapses contain several PDZ domain proteins known to interact with Kal7 in yeast two hybrid assays (PSD95, Chapsyn-100, SAP102, SAP97, neurabin, spinophilin, afadin, S-SCAM) (Penzes et al., 2001b). By forming an intercellular adhesion complex, postsynaptic neuroligin and presynaptic neurexin serve as a bi-directional trigger for synapse formation, allowing complexes involving PSD95 to communicate with the presynaptic terminal (Graf et al., 2004;Craig et al., 2006;Dean and Dresbach, 2006). Disruption of this interaction as a result of reductions in Kal7 expression could trigger trans-synaptic changes that result in elimination of pre-synaptic inputs.

Over-expression of Kal7 induces the formation of dendritic spines on aspiny interneurons

As pyramidal neurons develop, synapses shift from dendritic shafts to dendritic spines (Rao et al., 1998;Fiala et al., 1998). Kal7, PSD95 and spinophilin are known to increase spine formation in neurons that have spines (El Husseini et al., 2000;Feng et al., 2000;Penzes et al., 2001b). However, hippocampal interneurons are spine-free under basal conditions (Benson et al., 1994;Gulyas et al., 1999;Passafaro et al., 2003). Like Kal7, expression of Shank3, a PSD scaffold protein, or GluR2 induced the formation of spine synapses in aspiny neurons that express low levels of endogenous Shank3 or GluR2 in their dendrites (Passafaro et al., 2003;Roussignol et al., 2005). The majority of interneurons express low levels of GluR2 (He et al., 1998) and only 13% of the endogenous Kal7 clusters contain GluR2. Exogenous Kal7 recruited GluR2 to Kal7-induced spines; experiments using shRNAs specific for Kal7 and GluR2 will be needed to determine whether one gene drives spine formation or there are parallel pathways.

The effects of Kal7 over-expression on spine formation are more dramatic in dissociated neurons than in slice cultures. It is striking that a two-fold increase in levels of Kal7 in dissociated interneurons brings about a greater than 50-fold increase in spine density (to 15 spines/10 μm). In slices, a two-fold increase in Kal7 levels increases spine density 10-fold, to 3 spines/10 μm. Expression of endogenous Kal7 was not detected until P7 in cultured hippocampal neurons (not shown). Differences in time of transfection or lack of normal cell-cell interactions may explain the more dramatic response seen in dissociated cell culture.

Over-expression of Kal7 decreases dendritic branching in interneurons

Dendritic morphology is an important determinant of neuronal information processing. Dendritic growth and branching depend on the coordinated action of a number of different extracellular factors (Whitford et al., 2002;Dijkhuizen and Ghosh, 2005). The Rho GTPases are key integrators of factors regulating the dendritic cytoskeleton (Negishi and Katoh, 2005). In slice and dissociated cultures, over-expression of Kal7 caused an increase in the number of dendritic branches within 100 μm of the cell soma; in dissociated cells, a decrease in total dendritic length was also observed. These results suggest that Kal7 plays a role in limiting outgrowth of long dendrites in interneurons. Over-expression of the isolated GEF1 domain of Kalirin causes a more dramatic retraction of dendrites in cultured hippocampal interneurons than intact Kal7 (Ma et al., unpublished). Expression of the isolated GEF1 domain also caused dendritic retraction in cortical neurons (Penzes et al., 2001a), indicating that it is a key factor in control of dendritic morphology. Over-expression of PSD95 caused dendritic retraction in hippocampal neurons (Charych et al., 2006) and the effects of Kal7 on dendritic morphology may involve PSD95.

Interneurons in the CA1 region of the hippocampus are highly diverse, and different subpopulations have important differences in dendritic morphology (Freund and Buzsaki, 1996;Parra et al., 1998;Maccaferri and Lacaille, 2003). All parvalbumin-expressing CA1 interneurons examined stained for Kal7, with comparable levels in their soma and low levels in their dendrites. CA1 interneurons stained for calbindin or calretinin also contain Kal7 (not shown). Our quantification focused on the dendrites of interneurons in the CA1 region. Despite the variability inherent in these different subpopulations, the effects of Kal7 on dendritic branching were apparent. As for spine formation, expression of Kal7 in DIV1 dissociated interneurons, which do not yet express endogenous Kal7, had a more pronounced effect on branching than expression of Kal7 in P9 interneurons in organotypic slices. Coordinate control of the actin cytoskeleton and tubulin is required for normal dendritic growth and maturation, and GEFs that activate Rho GTPases may play a special role in this process (Negishi and Katoh, 2005). As for PSD95 (Charych et al., 2006;El Husseini et al., 2000), the Kal7 effectors essential to spine formation may differ from the effectors involved in dendritic branching.

Over-expression of Kal7 in hippocampal interneurons induces formation of excitatory synapses on dendritic spines

The ability of exogenous Kal7 to affect synaptic development was explored in dissociated interneurons. Kal7 interacts directly with PSD95 and the Kal7 clusters in dendrites overlap completely with PSD95 clusters; Kal7/PSD95 co-clusters increase and decrease as the level of Kal7 is manipulated. Functional synapses require NMDA and AMPA receptors (Petralia et al., 1999;Liao et al., 2001) and extensive overlap of exogenous Kal7 with endogenous NR1, GluR1 or GluR2 suggests that Kal7-induced synapses are functional. In contrast, exogenous Kal7 is not colocalized with endogenous GABAA receptors.

Bassoon is found in both excitatory and inhibitory endings (tom et al., 1998). Expression of exogenous Kal7 produces an increase in the number of bassoon positive structures apposed to the dendrites of interneurons that matches the increase in postsynaptic PSD95 positive clusters (Fig. 7C, D). The number of inhibitory presynaptic endings onto the dendrites of interneurons is smaller and is not altered by expression of exogenous Kal7. Neither GAD65 nor VGAT positive endings are apposed to Kal7-positive clusters. Almost all Kal7-positive clusters are apposed to VGLUT1 positive endings. VGLUT1 and 2 localize to distinct excitatory synapses, so the remaining Kal7-positive clusters may be apposed to VGLUT2 positive endings (Fremeau, Jr. et al., 2001;Fremeau, Jr. et al., 2004). We conclude that expression of Kal7 causes formation of dendritic spines that receive glutamatergic inputs. The degree of Kal7-induced spine formation in interneurons is dependent on the level of transfected Kal7 (not shown).

Kal7-induced spine formation is through interactions between the Kal7 PDZ binding domain and its interactors

Synaptogenesis is a complex process involving many proteins and two-way communication between presynaptic and postsynaptic elements (Craig et al., 2006). The PDZ domain proteins known to influence spine morphogenesis and synapse development include PSD95, Shank, spinophilin, NR2, GluR2, Drebin, neuroligin, synCAP and afadin, and many PDZ proteins localized to excitatory synapses organize glutamate receptors and their associated proteins (Tomita et al., 2001;Kim and Sheng, 2004;Craig et al., 2006). In addition to binding Kal7, PSD95 binds NMDA receptors and plays a role in their clustering (Kornau et al., 1995;Niethammer et al., 1996). Kal7 lacking a PDZ binding motif does not localize to the postsynaptic density and does not cause spine formation. While the PDZ binding motif of Kal7 is essential to this response, it is not clear whether known interactors such as PSD95, spinophilin, neurabin or afadin (Penzes et al., 2001b) or novel interactors play an essential role in synapse formation.

Disruption of interactions between the Kal7 PDZ binding domain and its interactors by a cell permeant peptide blocks Kal7-induced dendritic spine formation. A similar peptide was successfully used to block interaction between PSD95 and the NMDA receptor, protecting cultured neurons from excitotoxicity (Aarts et al., 2002) and decreasing the number of clusters of PSD95 and NMDA receptors and their colocalization in the dendrites of dissociated hippocampal neurons (Lim et al., 2003). PDZ proteins localized to the PSD of excitatory synapses control synaptic protein composition and structure (Kim and Sheng, 2004). Many of the PDZ proteins identified in spine synapses are also found at excitatory synapses onto the dendritic shafts of GABAergic interneurons (McBain et al., 1999;Zhang et al., 1999;Mi et al., 2002). Over-expression of PSD95 in hippocampal interneurons increases the number of GluR1 clusters in dendritic shafts and the frequency of miniature excitatory postsynaptic currents (El Husseini et al., 2000). While overexpression of PSD95 increased the levels of PSD95 in interneuron dendritic shafts, it did not cause spine formation. Coexpression of Kal7GFP plus PSD95 in interneurons did not increase spine density compared to Kal7GFP alone (not shown), indicating that PSD95 is not a limiting factor in Kal7-induced spine formation. Over-expression of the GluR2 subunit also induces spine formation in hippocampal interneurons (Passafaro et al., 2003;He et al., 1998). Interneurons clearly have the ability to form spines when instructed to do so and Kal7 and GluR2 can trigger this response. A crucial next step will be determining whether these factors form part of the same spine-formation pathway in hippocampal interneurons and, if so, identifying the order in which these factors act.

Supplementary Material

Acknowledgments

We thank Darlene D’Amato for her laboratory support and Drs. Zhao-wen Wang and Eric Levine for their comments on the manuscript. This work was supported by NIH grant DA-015464 to BAE.

Footnotes

Senior Editor: Dr. Marie Filbin

Revision of JN-RM-3649-07

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