Supplementary Information Optial ontrolling reveals time-dependent roles for adult-orn dentate granule ells Yan Gu, Maithe Arruda-Carvalho 3,4, Jia Wang, Stephen Janoshka,, Sheena Josselyn 3-5, Paul Frankland 3-5,* and Shaoyu Ge,,* Department of Neuroiology and Behavior, SUNY at Stony Brook, Stony Brook, NY 794 Program in Neurosiene, SUNY at Stony Brook, Stony Brook, NY 794 3 Program in Neurosienes and Mental Health, Hospital for Sik Children, 555 University Ave, Toronto, Ontario, Canada, M5G X8 4 Institute of Medial Siene, University of Toronto, Toronto, Ontario, Canada, M5S A8 5 Department of Physiology, University of Toronto, Toronto, Ontario, Canada, M5S A8 Contents: Supplementary Figures -9 Supplementary Tales
wpi M P D A L V wpi Injetion oordinates (in mm to Bregma) A/P M/L D/V. ±.6.5 3. ±.6 3. wpi d d wpi e e 4 wpi d e Supplementary Figure. Retroviral infetion in the adult hippoampus. a, Shemati drawing in the upper panel shows the viral injetion sites in the dentate gyrus. The lower panel lists the injetion oordinates. A/P: anterior/posterior; M/L: medial/lateral; D/V: dorsal/ventral., Sample images showing the injetion traks at (upper) and (lower) wpi. Note that injetion traks (arrow) were hardly seen at wpi. Sale ar: µm. -e, Laeling speifiity of neworn Cs with high-titer retrovirus. Shown in the upper pannel in, d and e are sample images of neworn neurons at, and 4 wpi. Shown in the lower pannel in, d and e are enlarged view of these neworn neurons, respetively. Note that, morphologially, there are no mature C-like ells at and wpi. Sale ars: 5 µm.
Single episode: ursts at 5 Hz Ten 5 ms pulses at Hz TBS paradigm = 4 episodes repeated at. Hz 3 wpi 4 wpi 8 wpi V-Clamp I-Clamp d e V-Clamp I-Clamp Supplementary Figure. Response of ChIEF+ adult-orn Cs to optial TBS. a, Shemati diagram depiting theta urst stimulation (TBS). TBS is omposed of four stimulation episodes at. Hz. Eah episode is a urst of ten Hz, 5ms pulses delivered at the theta frequeny (5 Hz)., Wholeell reordings were made on ChIEF+ adult-orn Cs in aute slies. Shown is a sample image of a reorded adult-orn C filled with ioytin through the reording pipette. Sale ar: 5 µm., Optial TBS-indued urrents (upper traes) and ation potentials (lower traes) reorded from neworn neurons at 3, 4 and 8 wpi in response to optial TBS (lue ars). Sale ars: vertial 5 pa or mv; horizontal ms. In oxes are enlarged traes of one urst ( Hz, pulses). Sale ars: vertial 5 pa or mv; horizontal 5 ms. d, A representative image showing a mossy fier terminal of neworn neurons filled with ioytin through the reording pipette. Sale ar: µm. e, Optial-indued urrents (upper traes) and ation potentials (lower traes) reorded from neworn neurons at 3 wpi. Sale ars: vertial 5 pa or mv; horizontal ms. In oxes are enlarged traes of one urst. Sale ars: vertial 5 pa or mv; horizontal 5 ms.
Optial Stimulation Field Potential Reording 3 M P D A V L Supplementary Figure 3. Sites of stimulation and reording in the - fepsps examination in vivo. a, Shemati drawing shows the optial stimulation site in the and reording site in the. A/P: anterior/posterior; M/L: medial/lateral; D/V: dorsal/ventral., Sample DIC images showing the traks of optial stimulation site in the (left panel) and reording site in the (right panel). Sale ar: 5 µm., Two-photon images showing ChIEF-dTomato expressing adult-orn neurons in the (left panel) and (axonal terminals, right panel). Sale ar: 5 µm.
4 Young Old Normalized EPSCs 6 5 4 3 Young Old d Potentiation 3 * 3 4 5 Time (min) Young Old Supplementary Figure 4. Enhaned plastiity in output synapses of young adult-orn neurons using a tetani indution paradigm. a, Shemati drawing shows whole-ell reording from a pyramidal neuron while optial stimulation was delivered on ChIEF expressing adult-orn Cs. -d, Exitatory postsynapti responses were reorded from pyramidal neurons y optially stimulating young (~4 wpi) or old (~ 8 wpi) ChIEF expressing adult-orn Cs. LTP was indued y a tetani optial stimulation (5 Hz, s)., Shown are traes efore (lak traes) and after (red traes) LTP indution (lue arrow). Sale ars: vertial: pa; horizontal: 5 ms., LTP expression. d, Histogram showing the amplitudes of LTP measured etween 45 and 6 minutes. All values represent mean±sem (*: p<.5, t-test).
Control Miefradil Normalized fepsps n.s...5 Control Miefradil Control SNX48 Miefradil 5 d Normalized fepsps 3 Control SNX48 Miefradil 3 4 5 Time (min) e Potentiation Supplementary Figure 5. Miefradil aolished LTP at the axon terminals of young Cs. a-, Miefradil does not alter asal MF- synapti transmission in vivo. a, Traes of fepsps efore and after miefradil appliation. Sale ar: vertial.5 mv; horizontal ms., Histogram showing normalized fepsps slopes efore and after appliation of miefradil (n=6, n.s.:p>.5, t-test). -f, Field EPSPs were reorded in in vitro y stimulating young adult-orn Cs (~4 weeks) in ontrol ACSF (n=6), or in the presene of R-type alium hannel loker SNX48 (n=6) or T-type alium hannel loker mieradil (n=5). Optial TBS were used for LTP indution., Shown are traes efore (lak traes) and after (red traes) LTP indution. Sale ars: vertial:. mv; horizontal: 5 ms. d, LTP expression in ontrol ACSF, SNX48 (5 nm) and miefradil ( µm). Gray ar indiates the applation of SNX48 or miefradil y perfusion, and lue arrow indiates the optial TBS. e, Histogram showing the amplitudes of LTP measured etween 45 and 6 minutes. LTP were aolished y miefradil ut not SNX48. f, Neither SNX48 nor Miefradil hanged synapti transmission. All values represent mean±sem (*: p<.5; n.s.: p>.5; t-test). * Control SNX48 Miefradil f Normalized fepsps..5 Control SNX48 Miefradil
Implantation Orange laser eam 6 Hippoampus Dorsal Arh- Orange laser eam dorsal Ventral ventral Supplementary Figure 6. Site for optrode implantation in the dorsal hippoampus. a, Sample image of a sagittal setion from mouse rain showing the loation of optrode implantation. Arrowhead: trak of optrode implantation. Sale ar: mm., A sample two-photon image showing Arh- expressing adult-orn neurons after ehavioral tests. Sale ar: µm., Shown is a gloal view of infeted neurons in the hippoampus after viral injetion. Diagrammati drawing roughly illustrates illumination area with the implanted optrode. Sale ar: mm.
Arh Hidden WM training 7, 4 or 8 weeks Lateny (s) 4 3 wpi Lateney (s) 4 3 4 wpi Lateny (s) 4 3 8 wpi 3 4 5 6 Training Sessions 3 4 5 6 Training Sessions 3 4 5 6 Training Sessions Supplementary Figure 7. Optial silening of neworn Cs does not influene aquisition of spatial memory. a, Shemati drawing shows, 4,or 8 weeks after injetion of Arh- retrovirus. Mie were trained in the hidden platform version of the water maze. Half of the animals were trained with light on in the dentate gyrus during training ( light group), while the other half were trained with no light ( no light group)., During training, lateny to find the platform delined normally oth in the no light and light groups at (Two-way ANOVA, signifiant effet of Training session only, F5,5=.8, p<.; n=6,6), 4 (Two-way ANOVA, signifiant effet of Training session only, F5,5=44.6, p<.; n=6,6) and 8 wpi (Two-way ANOVA, signifiant effet of Training session only, F5,5=34.54, p<.; n=6,6). All values represent mean±sem (n=6 in eah group at eah time point).
8 a 4 Hidden 5 * * Laterny (s) 3 Visile P3 P4 Perent searh 4 3 P P 3 4 5 6 Training sessions NE NW SW SE NE NW SW SE Supplementary Figure 8. illumination of -laeled neworn ells does not interfere with hippoampal memory retrieval. a-, illumination of a ohort of 4 week-old adult orn neurons laeled with showed no effet on hippoampal memory retrieval. a, During training (no light), lateny to find the platform delined normally in oth groups., During proe tests in oth ontrol ( no light ) and light onditions, the animals searhed seletively (One-way repeated measures ANOVA : F3,=8.438, p=.7; : F3,=4.83, p<.; NE>NW, SW, SE y paired t-test planned omparison in oth groups; n=8) spending more time in the target quadrant (NE) ompared to the other quadrants, displaying roust spatial memory. Visile platform tests were also performed, showing no differene of lateny in finding the platform (data not shown). All values represent mean±sem (*: p<.5).
9 a /Arh- Training Proe 4 weeks 9 min Fixation -fos staining and analysis DI -fos Merge DI -fos Merge + + + -fos / (%) 5 4 3 n.s. n.s. * Arh DI DI Arh -fos Merge -fos Merge Supplementary Figure 9. -fos staining showing the involvement of neworn Cs in spatial memory retrieval during the water maze test. a, Shemati drawing: 4 weeks after Arh- or -retrovirus injetion and optrode implantation, mie were trained in the water maze. After training, half of the mie were tested with light illumination, while the other half with no light. 9 minutes after the proe tests, mie were transardially perfused with PFA and rains were fixed. -fos expression was then stained, imaged and analyzed., Shown is -fos expression in the + or Arh+ 4-week-old neworn neurons. Arrows: -fos++ or -fos+arh+ neurons. Sale ar: µm., Histogram showing ativity-indued expression of -fos in +/Arh+ neworn neurons during water maze test. Optial illumination signifiantly redued the numer of -fos+arh+ neworn neurons. All values represent mean±sem (*: p<.5 and n.s.: p>.5, t-test).
wpi a. Intrinsi properties of adult-orn neurons expressing or optogenes at wpi. Memrane Capaitane (pf) Memrane Resistane (MΩ) Resting Potential Threshold Peak Amplitude Halfwidth (ms) 6.3±3.5 87.±78. -54.7±. -38.3±. 39.4±5. 76.9±7..6±.4 ChR 6.±3.4 99.4±47.9-54.±.6-3.9±. 33.±7.4 77.8±3.7.9±.6 ChIEF 5.±.3 78.±36.5-6.±5. -36.9±3. 4.±4.4 9.±.9.3±.5 Arh 6.4±3. 64.5±94.4-57.3±5.9-36.5±3.4 3.9±6. 76.±5.3 3.±.7. Intrinsi properties of adult-orn neurons expressing or optogenes at 3 wpi. Memrane Memrane Resting 3 wpi Capaitane Resistane Potential Threshold Peak Amplitude Halfwidth (pf) (MΩ) (ms) 3.8±. 885.5±57.4-6.±4.4-3.8±.7 6.±4.6.7±6.6.5±. ChR 8.5±4. 98.±386.4-6.7±3.7-3.4±.4 55.±4.3.8±4.9.7±. ChIEF 35.3±4.9 897.±3. -64.±5. -33.7±.6 55.6±6.3.9±.8.7±. Arh 6.9±.8 95.4±7.6-67.±.3-3.3±.9 64.8±3.9 8.8±6.5.6±.. Intrinsi properties of adult-orn neurons expressing or optogenes at 4 wpi. Memrane Memrane Resting 4 wpi Capaitane Resistane Potential Threshold Peak Amplitude Halfwidth (pf) (MΩ) (ms) 4.4±5.5 56.±89.9-73.6±3.3-38.4±.4 73.±3.9 36.9±5..5±. ChR 38.±5.7 498.8±33. -7.8±. -33.5±.3 74.±4.7 37.3±9.9.5±. ChIEF 35.5±5.3 4.±38.9-69.7±3.7-36.7±.7 7.±4.6 36.±9.8.5±. Arh 4.6±4.5 497.3±4.3-67.9±4. -36.±.8 73.6±.8 39.±7.6.5±. d. Intrinsi properties of adult-orn neurons expressing or optogenes at 8 wpi. 8 wpi Memrane Capaitane (pf) Memrane Resistane (MΩ) Resting Potential Threshold Peak Amplitude Halfwidth (ms) 5.8±5. 67.3±6. -77.4±.3-37.9±.7 74.±.4 38.±3.9.±. ChR 48.8±5. 98.8±33. -76.3±.9-4.8±3.3 77.8±3.9 34.3±5.3.±. ChIEF 46.3±3.5 3.±38.9-78.7±3. -4.3±3. 7.3±3.5 4.5±6..±. Arh 53.5±4.6 97.3±4.3-8.±3.5-36.9±3.8 7.9±3.6 37.±4..±. Supplementary Tale. Expression of optogenes does not hange intrinsi properties of neworn neurons along time. Mie were injeted with retrovirus expressing, Channelrhodopsin (ChR), Channelrhodopsin variant ChIEF, or Arheorhodopsin (Arh), and aute rain slies were ut at, 3, 4 and 8 week post viral injetion (wpi). Fluoresent adult-orn neurons were whole-ell pathed and intrinsi properties were reorded. Shown are the Memrane Capaitane, Memrane Resistane, Resting Memrane Potential, Ation Potential () Threshold, Peak, Amplitude and Halfwidth of adult orn ells at wpi (a), 3 wpi (), 4 wpi () and 8 wpi (d). All values represent mean±sem, and there is no statistial differene etween groups at eah time stage (n=-5, p>.5 y t-test).