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Science 338 (6106): 536-540

Copyright © 2012 by the American Association for the Advancement of Science

Elfn1 Regulates Target-Specific Release Probability at CA1-Interneuron Synapses

Emily L. Sylwestrak1,2,3, and Anirvan Ghosh1,2,*

1 Neurobiology Section, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093–0366, USA.
2 CNS Discovery, F. Hoffmann La Roche, 4070 Basel, Switzerland.
3 Biozentrum of the University of Basel, 4056 Basel, Switzerland.


Figure 1
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Fig. 1. Elfn1 is expressed in hippocampal O-LM interneurons. (A) CA1 axons contact PV cells and O-LM cells, which provide feedback inhibition. DG, dentate gyrus; Pyr, pyramidal cell. (B) ISH for Elfn1 and somatostatin at P14, both showing expression in the stratum oriens and hilus of the hippocampus. s.p., stratum pyramidale; s.o., stratum oriens. Scale bars indicate 25 (left) or 10 (right) μm. (C) ISH (blue) for Elfn1 and immunostaining (brown) for tdTomato in transgenic mouse lines in which tdTomato is expressed in Sst or PV interneurons. Elfn1 expression is highest near the alveus in somata (arrows) and proximal dendrites (arrowheads) of Sst-tdTomato neurons. Elfn1-expressing cells show little colocalization in tdTomato-PV mice (asterisks). Scale bars indicate 25 or 2.5 μm. (D) Quantification of the percent of horizontal cells in each mouse line that contain Elfn1, tdTomato, or both. Sst:tdTomato, n = 145 cells, PV::tdTomato, n = 157 cells. (E) Dendrites of neurons from dissociated hippocampal cultures stained for Elfn1 and PSD95 or gephyrin. Scale bar, 5 μm.

 

Figure 2
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Fig. 2. Elfn1 knockdown reduces short-term facilitation at CA1-O-LM synapses. (A) Western blot from human embryonic kidney (HEK) cells expressing Elfn1-GFP cotransfected with Elfn1 shRNA, blotted for GFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Knockdown is rescued by a point mutation in the target sequence of the Elfn1-GFP cDNA (E1-GFPres). (B) Recording configuration and epifluorescence images of tdTomato and GFP in stratum oriens. Scale bar, 20 μm. (C) (Top) Average postsynaptic response of control and shElfn1-expressing cells to five stimuli to the alveus at 20 Hz, normalized to the amplitude of the first EPSC. Black, GFP; red, shElfn1 at P6; orange, shElfn1 at P1. (Bottom) Population data for EPSC amplitude normalized to first EPSC. GFP, n = 13; P6 shElfn1, n = 20; P1 shElfn1, n = 7. (D) (Top) Example cells comparing first to fifth EPSC at different interstimulus intervals. (Bottom) Population data for facilitation ratio, calculated as the amplitude ratio of the fifth EPSC to the first EPSC. GFP, n = 12; shElfn1, n = 8. **P < 0.01, analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD). (E) (Left) Example recordings of inhibitory synaptic responses in control and shElfn1-expressing cells to a 20-Hz stimulus before and after picrotoxin application. (Right) Average inhibitory postsynaptic current (IPSC) amplitude, normalized to the first IPSC amplitude. GFP, n = 7; shElfn1, n = 5. Error bars in all figures indicate SEM.

 

Figure 3
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Fig. 3. Postsynaptic Elfn1 regulates release probability at CA1-O-LM synapses. (A) Evoked EPSC amplitude recorded from neighboring O-LM interneurons, one uninfected and one infected with a lentivirus expressing GFP or GFP plus shElfn1. Each point represents one pair of simultaneously recorded neurons. Solid circle, GFP control; open circle, shElfn1. (Inset) Example traces of evoked EPSC amplitude from uninfected (black) and infected (green) neurons. (B) Average EPSC amplitude of data in (A). n = 7 pairs. P < 0.05, paired t test. (C) Kinetics of NMDA block in control and shElfn1-expressing neurons. NMDA EPSC is recorded at a holding potential of +40 mV in DNQX, and the alveus is stimulated in the presence of MK801 (40 μM) at 0.2 Hz. Peak NMDA-mediated current is normalized to the initial peak NMDA EPSC amplitude. (D) Average time to half maximum NMDA EPSC amplitude. GFP, n = 8; shElfn1, n = 10. P < 0.05, t test. (E) Cell-attached, simultaneous recording from neighboring uninfected and shElfn1-infected O-LM cells. Ten stimuli are delivered to the alveus (arrows) at 20 Hz. (Left) Overlayed traces of subsequent sweeps to show spiking distribution. (Right) Spiking probability is plotted as a function of stimulus number. n = 5 pairs.

 

Figure 4
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Fig. 4. Elfn1 is sufficient to modulate CA1 outputs. (A) Lentivirus overexpressing Elfn1-GFP is injected into P5 PV::tdTomato mouse pups. Stratum pyramidale or stratum oriens PV neurons in the infected area are targeted for recording. (Left) Response of control and shElfn1-expressing PV neurons to five stimuli delivered to the alveus at 20 Hz. (Right) Quantification of short-term plasticity in Elfn1 overexpressing PV cells. *P < 0.05 by Mann-Whitney U test. (B) (Right) Example recording and quantification of evoked EPSC in GFP-infected Sst neurons before and after application of the GluR6-selective kainate receptor antagonist, NS102 (20 μM). (Left) Average postsynaptic response of GFP-infected Sst interneurons before and after NS102, n = 8. (Right) Average postsynaptic response of shElfn1-infected Sst interneurons before and after NS102, n = 14. *P < 0.05; **P < 0.01 by ANOVA with Tukey’s post-hoc test. (C) Model of Elfn1 function at the synapse. In CA1, Elfn1 is selectively localized to excitatory synapses onto O-LM interneurons. Elfn1 signals transsynaptically to contacting CA1 axons to reduce probability of release and create a facilitating synapse.

 


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