This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreadingprocess.
This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.
Research Articles: Neurobiology of Disease
Optogenetic stimulation of the superior colliculus confers retinalneuroprotection in a mouse glaucoma model
E. Geeraerts1, M. Claes1, E. Dekeyster1, M. Salinas-Navarro1, L. De Groef1, C. Van den Haute1, I.
Scheyltjens3, V. Baekelandt2, L. Arckens3 and L. Moons1
1Laboratory of Neural Circuit Development and Regeneration, Department of Biology, KU Leuven, Leuven,Belgium2Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Leuven, Belgium.3Laboratory of Neuroplasticity and Neuroproteomics, Department of Biology; KU Leuven, Leuven, Belgium
https://doi.org/10.1523/JNEUROSCI.0872-18.2018
Received: 6 April 2018
Revised: 15 November 2018
Accepted: 29 December 2018
Published: 17 January 2019
Author contributions: E.G., E.D., L.D.G., V.B., L.A., and L.M. designed research; E.G., M.C., M.S.-N.,C.V.d.H., and I.S. performed research; E.G. and M.C. analyzed data; E.G. wrote the first draft of the paper; E.G.,M.C., E.D., M.S.-N., L.D.G., C.V.d.H., I.S., V.B., L.A., and L.M. edited the paper; E.G., L.A., and L.M. wrote thepaper; C.V.d.H. contributed unpublished reagents/analytic tools.
Conflict of Interest: The authors declare no competing financial interests.
Corresponding author: Prof. Dr. Lieve Moons, Neural Circuit Development and Regeneration Research Group,Animal Physiology and Neurobiology Section, Department of Biology, KU Leuven, Naamsestraat 61, Box 2464,B-3000 Leuven, Belgium, Tel: (32)-16-32.39.91, Fax: (32)-16-32.42.62, Email: [email protected]
Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.0872-18.2018
Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted versionof this article is published.
1
Optogenetic stimulation of the superior colliculus 1
confers retinal neuroprotection in a mouse 2
glaucoma model 3
Geeraerts, E. 1, Claes, M.1, Dekeyster, E.1, Salinas-Navarro, M.1, De Groef, L.1, Van den 4
1 Laboratory of Neural Circuit Development and Regeneration, Department of Biology, KU Leuven, 6 Leuven, Belgium 7 2 Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven, Leuven, 8 Belgium. 9 3 Laboratory of Neuroplasticity and Neuroproteomics, Department of Biology; KU Leuven, Leuven, 10 Belgium 11
12
13
14
Corresponding author: 15
Prof. Dr. Lieve Moons 16
Neural Circuit Development and Regeneration Research Group 17
12.41; stimulation: p = 0.37, F(1,248) = 0.80; interaction: p = 0.84, F(7, 248) = 0.4832) (Fig. 5B). There 392
was a significant IOP elevation for around 5 days, similar to previous reports using this model (Tukey 393
Post Hoc test, p < 0.05) (Valiente-Soriano et al., 2015; De Groef et al., 2016). Habituation due to 394
chronic stimulation could also be excluded for this LP + stimulation group. The effect on behavior of 395
the second-to-last stimulation was recorded and quantified in a subset of chronically stimulated mice 396
(N = 4), and indeed revealed that mobility and rotation patterns closely matched those of previous 397
experiments (Fig. 5B). Corroborating these findings, c-Fos expression patterns in the SC after the final 398
stimulation also showed strong resemblance to those observed in non-glaucomatous mice 399
stimulated one or three times, confirming that no habituation to repeated optogenetic stimulation 400
occurred (Fig. 5C). 401
17
Next, RGC degeneration was assessed by immunohistochemical staining on retinal flatmounts for the 402
RGC nuclear marker Brn3a and semi-automatic quantification of the surviving RGC cell bodies at 14 403
dpi was performed (Fig. 5D). The LP + sham group showed an average survival of 2596 ± 176 404
RGCs/mm2 in LP eyes contralateral to the stimulated SC (N = 20). This represented an average 405
survival rate of 74%, compared to contralateral eyes without LP. The LP + stimulation group had 406
significantly more surviving RGCs, 3181 ± 117 RGCs/mm2 corresponding to an average survival rate of 407
90% (N = 17, two-tailed unpaired t-test, p = 0.0114, t = 2.67, degrees of freedom = 35). As 929 408
RGCs/mm2 were lost in the LP model at 14 dpi, compared to only 344 RGCs/mm2 after repeated 409
optogenetic SC stimulation, RGC loss was reduced by 63%. Isodensity maps of RGC survival for LP + 410
sham eyes often showed the increased sectorial RGC loss, associated with the model, as blueish 411
sectors, along with a general diffuse loss shown as orange-red hues (Fig. 5F). This sectorial loss 412
occurred in 10/20 of the LP + sham eyes, while the other retinas just showed a diffuse loss. In 413
contrast, in the LP + stimulation group, this sectorial loss occurred significantly less frequent, 414
occurring in 3/17 eyes, with the rest of the retinas showing diffuse loss (one-sided Fisher’s exact test, 415
p = 0.04) (Fig. 5F). 416
Finally, as retrograde transduction of RGCs could allow direct activation of transduced RGC terminals 417
upon light stimulation in the SC, in contrast to the intended stimulation of the SC cells, possible 418
confounding effects of retrograde SSFO transduction of RGCs in the retina had to be excluded. After 419
immunostaining to enhance reporter visibility, we observed 49 to 371 transduced RGCs/retina with a 420
mean ± SEM of 144 ± 44, representing 0.4% of the total RGC population (N = 7, Fig. 4F). Therefore, it 421
seems unlikely that this limited population of SSFO-expressing RGCs can be responsible for the 422
observed difference in RGC survival between stimulated and non-stimulated animals (585 423
RGCs/mm2, corresponding to around 7605 RGCs/retina or 16% of the entire population). Importantly, 424
further detailed evaluation disclosed that a higher number of retrogradely transduced RGCs did not 425
result in an increased RGC survival; there was no correlation between the number of retrogradely 426
transduced RGCs and RGC survival for light stimulated animals (Spearman r = -0.57, p = 0.2, N = 7). 427
18
The observation that maximally 2% of the protected RGCs is retrogradely transduced, together with 428
the absence of a correlation between retrograde transduction and survival of RGCs, argues against a 429
substantial role for such retrogradely transduced RGCs in the observed neuroprotective effect. 430
In summary, our findings indicate that the repeated optogenetic stimulation of neuronal activity in 431
the SC confers retinal neuroprotection in a glaucomatous mouse model. 432
Discussion 433
In this study, we set out to unveil whether target neuron stimulation can increase survival of 434
projecting neurons. Following intracollicular injection of an AAV2/7-CaMKII-SSFO vector, SSFO was 435
expressed throughout the SC and could be repeatedly activated with consistent effects. Repeated 436
optogenetic stimulation that increased neuronal activity of the SC was then shown to reduce RGC 437
loss by 63% at 14 dpi in an LP mouse glaucoma model. Concomitantly, there was a significant 438
reduction in the occurrence of sectorial cell loss, an important neurodegenerative aspect of the 439
disease model (Valiente-Soriano et al., 2015). 440
Upon SSFO-mediated stimulation of the SC, a strong upregulation of c-Fos in the SSFO-transduced 441
region of the SC was observed specifically after light delivery (Fig. 2B, 4B). The immediate-early gene 442
c-Fos is a well-known transcription factor involved in coupling neuronal activity to translational 443
changes and widely used as a histological indicator of neuronal activation, also in the context of 444
optogenetic neurostimulation (Curran and Morgan, 1995; Kovacs, 1998; Yizhar et al., 2011). 445
Behavioral investigation provided complementary information, unveiling that optogenetic activation 446
recapitulated electrical and pharmacological stimulation of the SC by inducing a spectrum of running, 447
freezing-like immobility and turning responses (Kilpatrick et al., 1982; Schmitt et al., 1985; Sahibzada 448
et al., 1986; Coimbra and Brandao, 1993; Brandao et al., 1994). In Sahibzada et al. (1986), 449
directionality of turning was interpreted as orientation or aversive behavior, with orientation defined 450
as a turn towards the visual hemifield governed by the stimulated SC, while aversion was marked by 451
a turn in the opposite direction. In our study, the right SC was stimulated, so counterclockwise turns 452
19
towards the left visual field can be interpreted as orientation behavior. Optogenetic stimulation of 453
the right SC thus increases and biases orientation to the left hemifield. This is in partial agreement 454
with Stubblefield et al. (2013), who report a bias and no observation of direct movement by 455
optogenetic stimulation alone, but this could be attributed to technical factors like stimulation power 456
and opsin. Apart from orientation behavior, defensive behaviors such as running and increased 457
freezing-like immobility were also observed here, especially with higher stimulation powers. Our 458
stimulation series of increasing light power suggests an interesting interplay between these 459
defensive behaviors, with a stimulation of 2.2 mW eliciting bouts of running behavior intermixed 460
with periods of increased freezing-like immobility, while in sessions with a higher light power, this 461
running was superseded by a further increase in immobile freezing-like behavior. Although previous 462
reports have disclosed that optogenetic stimulation of various subpopulations of SC neurons can 463
mediate defensive behaviors, we cannot exclude that direct activation of the periaqueductal gray, a 464
known neuronal substrate of defensive behaviors, might have contributed to the observed responses 465
(Carrive, 1993; Shang et al., 2015; Wei et al., 2015; Tovote et al., 2016). Indeed, despite considerable 466
distance between the optic fiber positioned just above the SC and the periaqueductal gray under the 467
deep SC, the excellent light sensitivity of the SSFO might allow direct stimulation of the 468
periaqueductal gray with higher light power, as suggested by the augmented c-Fos expression seen in 469
this region after a 2.2 mW light pulse (Fig. 4B) (Yizhar et al., 2011). In conclusion, the behavioral 470
responses observed after SSFO-mediated stimulation of the SC recapitulate well-known midbrain-471
related orientation and defensive behavior, thus confirming specificity of the stimulation paradigm 472
employed here (Dean et al., 1989; Brandao et al., 1994). 473
Both immunohistochemical and behavioral read-outs showed highly consistent results after repeated 474
activation, suggesting a lack of habituation to the stimulation. The stimulation series with increasing 475
light power resulted in a mounting behavioral response, indicating that a 2 hour interstimulus 476
interval was already enough to allow the response to reflect the stimulus parameters (Fig. 3). 477
Furthermore, we demonstrated consistent c-Fos+ cell densities and behavioral responses to repeated 478
20
stimulations spread over five days (Fig. 4). When optogenetic stimulation was combined with the LP 479
model and animals were stimulated twice per day for two weeks, we still observed a behavioral and 480
histological response similar to animals stimulated for the first time (Fig. 5). Together, these 481
observations strongly argue against habituation, indicating that SSFO-mediated optogenetic 482
activation presents a reliable long-term stimulation strategy. 483
The most prominent finding of this work is that this repeated optogenetic stimulation of a major RGC 484
target site, the SC, has a neuroprotective effect in the retina. Since we excluded a major contribution 485
of the sparse RGCs that might have been directly activated due to SSFO expression in their axon 486
terminals within the superficial layers of the SC, the mechanism of action is likely retrograde 487
molecular signaling. Knowledge about retrograde signaling in RGCs is scarce and focused almost 488
exclusively on neurotrophins such as BDNF. Importantly, the signaling pathways induced in RGCs by 489
retrogradely derived BDNF have been shown to be distinct from those induced by BDNF supplied to 490
the RGC soma in the retina, highlighting a key role for area-specific trophic stimulation (van 491
Oterendorp et al., 2014; Dekeyster et al., 2015). Notably, during development, supplementation of 492
BDNF to the SC reduces developmental RGC loss (Raff et al., 1993; Frade et al., 1997; Ma et al., 1998). 493
Furthermore, retrograde neurotrophic factor deprivation is believed to be an important aspect of 494
glaucomatous neurodegeneration, with a failure of retrograde axonal transport and spatially 495
coincident increase of BDNF in collicular astrocytes (Nickells, 2007; Crish et al., 2013). Additionally, 496
transient blocking of axonal transport using retrobulbar lidocaine injections rapidly reduces pattern 497
electroretinogram amplitude, a measure of RGC function (Chou et al., 2013). These findings all 498
suggest an important role for the SC in RGC function and survival, and are now supplemented by an 499
innovative and controllable approach of collicular stimulation with demonstrated neuroprotective 500
effect. 501
A possible limitation of the current study is its reliance on loss of Brn3a expression to assess RGC 502
survival, as indeed glaucomatous neurodegeneration has a much broader range of pathological 503
21
features, including loss of functional antero- and retrograde axonal transport, axon degeneration and 504
dendritic remodeling (Calkins, 2012). Here, we refrained from investigating axonal transport because 505
the administration of fluorescent tracers entails a damaging manipulation of the retino-collicular axis, 506
which might have acted as a confounder. Of note, Valiente-Soriano et al. (2015) describe a near-507
perfect linear correlation between survival of Brn3a+ cells and preservation of retrograde axonal 508
transport in the LP model applied here. Furthermore, comparisons between axon counts in the optic 509
nerve and Brn3a+ cell numbers are lacking for the LP model, but these measurements are in 510
excellent correspondence in the microbead model, where elevated IOP is achieved by intracameral 511
injection of beads (Cone et al., 2010; Chen et al., 2011; Ito et al., 2016). Together, these results 512
demonstrate that preservation of somatic Brn3a immunoreactivity is linked with other aspects of 513
RGC integrity. There also has been some controversy on the validity of Brn3a as an RGC marker after 514
injury because of its downregulation before actual RGC death (Nuschke et al., 2015; Mead and 515
Tomarev, 2016). While the loss of Brn3a immunoreactivity precedes clearing of the RGC soma by 516
phagocytosing cells, discrepancies between both measurements are only temporary and progress 517
towards the same endpoint, indicating that Brn3a is a sensitive predictor of RGC integrity (Salinas-518
Navarro et al., 2010; Galindo-Romero et al., 2011). Furthermore, previous literature clearly reports 519
that loss of Brn3a staining coincides with an increase in activated caspase-3 expression after injury, 520
indicating that the disappearance of Brn3a immunoreactivity signals the RGC entering apoptotic 521
pathways (Sanchez-Migallon et al., 2016). Additionally, Brn3a expression is also maintained by other 522
known neuroprotective treatments, such as intraocular BDNF administration (Sanchez-Migallon et 523
al., 2011; Galindo-Romero et al., 2013). Thus, while this manuscript does not provide a full 524
quantification of all aspects of glaucomatous neurodegeneration, the use of Brn3a, an established 525
marker for RGC survival with demonstrated correlation with other degenerative features, strongly 526
argues in favor of an increased neuronal survival by target stimulation, with retrograde axonal 527
signaling as the only known intermediate. 528
22
The SSFO-mediated collicular stimulation developed here might serve as a tool to unveil molecular 529
signaling pathways by which target areas in the brain can support neuroprotection. Discovery of key 530
molecular players underlying this survival signaling could provide new targets for future 531
neuroprotective therapies. The eye and its connection to the central brain targets can potentially 532
spearhead this search because in mammals it lacks recurrent connections characterizing many other 533
central brain regions. For example, in a rodent stroke model affecting the striatum and 534
somatosensory cortex, optogenetic stimulation of the ipsilesional primary motor cortex was shown 535
to promote functional recovery (Cheng et al., 2014). The intricate network of anterograde and 536
retrograde connections between these brain structures, however, complicates interpretation of how 537
optogenetic stimulation could mediate these effects (Ebrahimi et al., 1992; Petrof et al., 2015; 538
Mohan et al., 2018). As such, the retino-collicular projection provides a unique opportunity to isolate 539
the effect of retrograde signaling, in addition to the many advantages of the eye as a model system 540
for neurodegenerative diseases (De Groef and Cordeiro, 2018). In summary, this work makes 541
headway for the exploration of retrograde signaling as a route to novel neuroprotective therapies in 542
the central nervous system. 543
Authors’ contributions 544
Study was conceived and designed by EG, ED, LDG, VB, LA and LM. Experiments were performed by 545
EG, MC and MSN, with the help of IS and CVDH. Data analysis was done by EG and MC. The 546
manuscript was written by EG, LA and LM. 547
All authors reviewed and approved the final manuscript. 548
Acknowledgements 549
This work was supported by the Hercules Foundation (Belgium, AKUL-09-038, AKUL1309), the 550
Research Foundation Flanders (FWO-Vlaanderen, Belgium, fellowships to MC, ED, MSN and LDG), the 551
Flemish government agency for Innovation by Science and Technology (IWT-Vlaanderen, Belgium, 552
fellowships to LDG, IS and EG, and SBO/110068 project OPTOBRAIN) and Funds for Research in 553
Ophthalmology (FRO, Belgium, prizes awarded to EG and ED). The authors declare no competing 554
23
financial interests. The authors would like to thank Samme Vreysen, Lut Noterdaeme, Marijke 555
Christiaens, Evelien Herinckx and Véronique Brouwers for their excellent technical assistance. Pieter 556
Vancamp and all the members of the OPTOBRAIN consortium are acknowledged for their helpful 557
comments regarding the manuscript and the experiments described therein. 558
References 559
Almasieh M, Levin LA (2017) Neuroprotection in Glaucoma: Animal Models and Clinical Trials. Annu 560 Rev Vis Sci 3:91-120. 561
Balkowiec A, Katz DM (2000) Activity-dependent release of endogenous brain-derived neurotrophic 562 factor from primary sensory neurons detected by ELISA in situ. Journal of Neuroscience 563 20:7417-7423. 564
Brandao ML, Cardoso SH, Melo LL, Motta V, Coimbra NC (1994) Neural substrate of defensive 565 behavior in the midbrain tectum. Neurosci Biobehav Rev 18:339-346. 566
Calkins DJ (2012) Critical pathogenic events underlying progression of neurodegeneration in 567 glaucoma. Prog Retin Eye Res 31:702-719. 568
Carrive P (1993) The periaqueductal gray and defensive behavior: functional representation and 569 neuronal organization. Behav Brain Res 58:27-47. 570
Chen HH, Wei X, Cho KS, Chen GC, Sappington R, Calkins DJ, Chen DF (2011) Optic Neuropathy Due to 571 Microbead-Induced Elevated Intraocular Pressure in the Mouse. Invest Ophth Vis Sci 52:36-572 44. 573
Cheng MY, Wang EH, Woodson WJ, Wang S, Sun G, Lee AG, Arac A, Fenno LE, Deisseroth K, Steinberg 574 GK (2014) Optogenetic neuronal stimulation promotes functional recovery after stroke. Proc 575 Natl Acad Sci U S A 111:12913-12918. 576
Chou TH, Park KK, Luo XT, Porciatti V (2013) Retrograde Signaling in the Optic Nerve Is Necessary for 577 Electrical Responsiveness of Retinal Ganglion Cells. Invest Ophth Vis Sci 54:1236-1243. 578
Cohen LP, Pasquale LR (2014) Clinical characteristics and current treatment of glaucoma. Cold Spring 579 Harb Perspect Med 4. 580
Coimbra NC, Brandao ML (1993) GABAergic nigro-collicular pathways modulate the defensive 581 behaviour elicited by midbrain tectum stimulation. Behav Brain Res 59:131-139. 582
Cone FE, Gelman SE, Son JL, Pease ME, Quigley HA (2010) Differential susceptibility to experimental 583 glaucoma among 3 mouse strains using bead and viscoelastic injection. Experimental Eye 584 Research 91:415-424. 585
Crish SD, Dapper JD, MacNamee SE, Balaram P, Sidorova TN, Lambert WS, Calkins DJ (2013) Failure of 586 axonal transport induces a spatially coincident increase in astrocyte BDNF prior to synapse 587 loss in a central target. Neuroscience 229:55-70. 588
Curran T, Morgan JI (1995) Fos - an Immediate-Early Transcription Factor in Neurons. J Neurobiol 589 26:403-412. 590
De Groef L, Cordeiro MF (2018) Is the Eye an Extension of the Brain in Central Nervous System 591 Disease? . Journal of Ocular Pharmacology and Therapeutics 34:129-133. 592
De Groef L, Dekeyster E, Geeraerts E, Lefevere E, Stalmans I, Salinas-Navarro M, Moons L (2016) 593 Differential visual system organization and susceptibility to experimental models of optic 594 neuropathies in three commonly used mouse strains. Experimental Eye Research 145:235-595 247. 596
Dean P, Redgrave P, Westby GWM (1989) Event or Emergency - 2 Response Systems in the 597 Mammalian Superior Colliculus. Trends Neurosci 12:137-147. 598
Dekeyster E, Geeraerts E, Buyens T, Van den Haute C, Baekelandt V, De Groef L, Salinas-Navarro M, 599 Moons L (2015) Tackling Glaucoma from within the Brain: An Unfortunate Interplay of BDNF 600 and TrkB. Plos One 10. 601
24
Di Polo A, Aigner LJ, Dunn RJ, Bray GM, Aguayo AJ (1998) Prolonged delivery of brain-derived 602 neurotrophic factor by adenovirus-infected Muller cells temporarily rescues injured retinal 603 ganglion cells. Proc Natl Acad Sci U S A 95:3978-3983. 604
Ebrahimi A, Pochet R, Roger M (1992) Topographical Organization of the Projections from 605 Physiologically Identified Areas of the Motor Cortex to the Striatum in the Rat. Neurosci Res 606 14:39-60. 607
Ellis EM, Gauvain G, Sivyer B, Murphy GJ (2016) Shared and distinct retinal input to the mouse 608 superior colliculus and dorsal lateral geniculate nucleus. J Neurophysiol 116:602-610. 609
Frade JM, Bovolenta P, Martinez-Morales JR, Arribas A, Barbas JA, Rodriguez-Tebar A (1997) Control 610 of early cell death by BDNF in the chick retina. Development 124:3313-3320. 611
Galindo-Romero C, Valiente-Soriano FJ, Jimenez-Lopez M, Garcia-Ayuso D, Villegas-Perez MP, Vidal-612 Sanz M, Agudo-Barriuso M (2013) Effect of Brain-Derived Neurotrophic Factor on Mouse 613 Axotomized Retinal Ganglion Cells and Phagocytic Microglia. Invest Ophth Vis Sci 54:974-985. 614
Galindo-Romero C, Aviles-Trigueros M, Jimenez-Lopez M, Valiente-Soriano FJ, Salinas-Navarro M, 615 Nadal-Nicolas F, Villegas-Perez MP, Vidal-Sanz M, Agudo-Barriuso M (2011) Axotomy-induced 616 retinal ganglion cell death in adult mice: Quantitative and topographic time course analyses. 617 Experimental Eye Research 92:377-387. 618
Geeraerts E, Dekeyster E, Gaublomme D, Salinas-Navarro M, De Groef L, Moons L (2016) A freely 619 available semi-automated method for quantifying retinal ganglion cells in entire retinal 620 flatmounts. Exp Eye Res 147:105-113. 621
Gerits A, Vancraeyenest P, Vreysen S, Laramee ME, Michiels A, Gijsbers R, Van den Haute C, Moons L, 622 Debyser Z, Baekelandt V, Arckens L, Vanduffel W (2015) Serotype-dependent transduction 623 efficiencies of recombinant adeno-associated viral vectors in monkey neocortex. 624 Neurophotonics 2. 625
Hall J, Thomas KL, Everitt BJ (2000) Rapid and selective induction of BDNF expression in the 626 hippocampus during contextual learning. Nat Neurosci 3:533-535. 627
Heijl A (2013) The times they are a-changin': time to change glaucoma management. Acta 628 Ophthalmol 91:92-99. 629
Heijl A, Leske MC, Bengtsson B, Hyman L, Bengtsson B, Hussein M, Early Manifest Glaucoma Trial G 630 (2002) Reduction of intraocular pressure and glaucoma progression: results from the Early 631 Manifest Glaucoma Trial. Arch Ophthalmol 120:1268-1279. 632
Ito YA, Belforte N, Vargas JLC, Di Polo A (2016) A Magnetic Microbead Occlusion Model to Induce 633 Ocular Hypertension-Dependent Glaucoma in Mice. Jove-J Vis Exp. 634
Johnson EC, Guo Y, Cepurna WO, Morrison JC (2009) Neurotrophin roles in retinal ganglion cell 635 survival: lessons from rat glaucoma models. Exp Eye Res 88:808-815. 636
Johnson TV, Bull ND, Hunt DP, Marina N, Tomarev SI, Martin KR (2010) Neuroprotective Effects of 637 Intravitreal Mesenchymal Stem Cell Transplantation in Experimental Glaucoma. Invest Ophth 638 Vis Sci 51:2051-2059. 639
Jonas JB, Aung T, Bourne RR, Bron AM, Ritch R, Panda-Jonas S (2017) Glaucoma. Lancet 390:2183-640 2193. 641
Kilpatrick IC, Collingridge GL, Starr MS (1982) Evidence for the participation of nigrotectal gamma-642 aminobutyrate-containing neurones in striatal and nigral-derived circling in the rat. 643 Neuroscience 7:207-222. 644
Kovacs KJ (1998) c-Fos as a transcription factor: a stressful (re)view from a functional map. 645 Neurochemistry International 33:287-297. 646
Li XC, Jarvis ED, Alvarez-Borda B, Lim DA, Nottebohm F (2000) A relationship between behavior, 647 neurotrophin expression, and new neuron survival. P Natl Acad Sci USA 97:8584-8589. 648
Ma YT, Hsieh T, Forbes ME, Johnson JE, Frost DO (1998) BDNF injected into the superior colliculus 649 reduces developmental retinal ganglion cell death. Journal of Neuroscience 18:2097-2107. 650
Martin KRG, Quigley HA, Zack DJ, Levkovitch-Verbin H, Kielczewski J, Valenta D, Baumrind L, Pease 651 ME, Klein RL, Hauswirth WW (2003) Gene therapy with brain-derived neurotrophic factor as 652
25
a protection: Retinal ganglion cells in a rat glaucoma model. Invest Ophth Vis Sci 44:4357-653 4365. 654
Matusica D, Coulson EJ (2014) Local versus long-range neurotrophin receptor signalling: Endosomes 655 are not just carriers for axonal transport. Semin Cell Dev Biol 31:57-63. 656
Mead B, Tomarev S (2016) Evaluating retinal ganglion cell loss and dysfunction. Experimental Eye 657 Research 151:96-106. 658
Mohan H, de Haan R, Mansvelder HD, de Kock CPJ (2018) The Posterior Parietal Cortex as Integrative 659 Hub for Whisker Sensorimotor Information. Neuroscience 368:240-245. 660
Nadal-Nicolas FM, Jimenez-Lopez M, Sobrado-Calvo P, Nieto-Lopez L, Canovas-Martinez I, Salinas-661 Navarro M, Vidal-Sanz M, Agudo M (2009) Brn3a as a Marker of Retinal Ganglion Cells: 662 Qualitative and Quantitative Time Course Studies in Naive and Optic Nerve-Injured Retinas. 663 Invest Ophth Vis Sci 50:3860-3868. 664
Nanda SA, Mack KJ (2000) Seizures and sensory stimulation result in different patterns of brain 665 derived neurotrophic factor protein expression in the barrel cortex and hippocampus. Mol 666 Brain Res 78:1-14. 667
Nickells RW (2007) From ocular hypertension to ganglion cell death: a theoretical sequence of events 668 leading to glaucoma. Can J Ophthalmol 42:278-287. 669
Nuschke AC, Farrell SR, Levesque JM, Chauhan BC (2015) Assessment of retinal ganglion cell damage 670 in glaucomatous optic neuropathy: Axon transport, injury and soma loss. Experimental Eye 671 Research 141:111-124. 672
Petrof I, Viaene AN, Sherman SM (2015) Properties of the primary somatosensory cortex projection 673 to the primary motor cortex in the mouse. Journal of Neurophysiology 113:2400-2407. 674
Quigley HA (2016) Understanding Glaucomatous Optic Neuropathy: The Synergy Between Clinical 675 Observation and Investigation. Annual Review of Vision Science, Vol 2 2:235-254. 676
Raff MC, Barres BA, Burne JF, Coles HS, Ishizaki Y, Jacobson MD (1993) Programmed Cell-Death and 677 the Control of Cell-Survival - Lessons from the Nervous-System. Science 262:695-700. 678
Rattiner LM, Davis M, French CT, Ressler KJ (2004) Brain-derived neurotrophic factor and tyrosine 679 kinase receptor B involvement in amygdala-dependent fear conditioning. Journal of 680 Neuroscience 24:4796-4806. 681
Reichardt LF (2006) Neurotrophin-regulated signalling pathways. Philos Trans R Soc Lond B Biol Sci 682 361:1545-1564. 683
Rueden CT, Schindelin J, Hiner MC, DeZonia BE, Walter AE, Arena ET, Eliceiri KW (2017) ImageJ2: 684 ImageJ for the next generation of scientific image data. BMC Bioinformatics 18:529. 685
Sahibzada N, Dean P, Redgrave P (1986) Movements resembling orientation or avoidance elicited by 686 electrical stimulation of the superior colliculus in rats. J Neurosci 6:723-733. 687
Salinas-Navarro M, Alarcon-Martinez L, Valiente-Soriano FJ, Jimenez-Lopez M, Mayor-Torroglosa S, 688 Aviles-Trigueros M, Villegas-Perez MP, Vidal-Sanz M (2010) Ocular hypertension impairs optic 689 nerve axonal transport leading to progressive retinal ganglion cell degeneration. 690 Experimental Eye Research 90:168-183. 691
Sanchez-Migallon MC, Valiente-Soriano FJ, Nadal-Nicolas FM, Vidal-Sanz M, Agudo-Barriuso M (2016) 692 Apoptotic Retinal Ganglion Cell Death After Optic Nerve Transection or Crush in Mice: 693 Delayed RGC Loss With BDNF or a Caspase 3 Inhibitor. Invest Ophth Vis Sci 57:81-93. 694
Sanchez-Migallon MC, Nadal-Nicolas FM, Jimenez-Lopez M, Sobrado-Calvo P, Vidal-Sanz M, Agudo-695 Barriuso M (2011) Brain derived neurotrophic factor maintains Brn3a expression in 696 axotomized rat retinal ganglion cells. Experimental Eye Research 92:260-267. 697
Scheyltjens I, Vreysen S, Van den Haute C, Sabanov V, Balschun D, Baekelandt V, Arckens L (2018) 698 Transient and localized optogenetic activation of somatostatin-interneurons in mouse visual 699 cortex abolishes long-term cortical plasticity due to vision loss. Brain Struct Funct. 700
Scheyltjens I, Laramee ME, Van den Haute C, Gijsbers R, Debyser Z, Baekelandt V, Vreysen S, Arckens 701 L (2015) Evaluation of the Expression Pattern of rAAV2/1, 2/5, 2/7, 2/8, and 2/9 Serotypes 702 With Different Promoters in the Mouse Visual Cortex. Journal of Comparative Neurology 703 523:2019-2042. 704
26
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, 705 Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A 706 (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676-682. 707
Schmitt P, Di Scala G, Brandao ML, Karli P (1985) Behavioral effects of microinjections of SR 95103, a 708 new GABA-A antagonist, into the medial hypothalamus or the mesencephalic central gray. 709 Eur J Pharmacol 117:149-158. 710
Shang C, Liu Z, Chen Z, Shi Y, Wang Q, Liu S, Li D, Cao P (2015) BRAIN CIRCUITS. A parvalbumin-711 positive excitatory visual pathway to trigger fear responses in mice. Science 348:1472-1477. 712
Stubblefield EA, Costabile JD, Felsen G (2013) Optogenetic investigation of the role of the superior 713 colliculus in orienting movements. Behav Brain Res 255:55-63. 714
Valiente-Soriano FJ, Salinas-Navarro M, Jimenez-Lopez M, Alarcon-Martinez L, Ortin-Martinez A, 718 Bernal-Garro JM, Aviles-Trigueros M, Agudo-Barriuso M, Villegas-Perez MP, Vidal-Sanz M 719 (2015) Effects of ocular hypertension in the visual system of pigmented mice. PLoS One 720 10:e0121134. 721
Van Der Gucht E, Vandenbussche E, Orban GA, Vandesande F, Arckens L (2000) A new cat Fos 722 antibody to localize the immediate early gene c-fos in mammalian visual cortex after sensory 723 stimulation. J Histochem Cytochem 48:671-684. 724
Van der Perren A, Toelen J, Carlon M, Van den Haute C, Coun F, Heeman B, Reumers V, 725 Vandenberghe LH, Wilson JM, Debyser Z, Baekelandt V (2011) Efficient and stable 726 transduction of dopaminergic neurons in rat substantia nigra by rAAV 2/1, 2/2, 2/5, 2/6.2, 727 2/7, 2/8 and 2/9. Gene Ther 18:517-527. 728
van Oterendorp C, Sgouris S, Schallner N, Biermann J, Lagreze WA (2014) Retrograde Neurotrophic 729 Signaling in Rat Retinal Ganglion Cells Is Transmitted via the ERK5 but Not the ERK1/2 730 Pathway. Invest Ophth Vis Sci 55:658-665. 731
Vidal-Sanz M, Salinas-Navarro M, Nadal-Nicolas FM, Alarcon-Martinez L, Valiente-Soriano FJ, de 732 Imperial JM, Aviles-Trigueros M, Agudo-Barriuso M, Villegas-Perez MP (2012) Understanding 733 glaucomatous damage: Anatomical and functional data from ocular hypertensive rodent 734 retinas. Prog Retin Eye Res 31:1-27. 735
Weber AJ, Viswanathan S, Ramanathan C, Harman CD (2010) Combined Application of BDNF to the 736 Eye and Brain Enhances Ganglion Cell Survival and Function in the Cat after Optic Nerve 737 Injury. Invest Ophth Vis Sci 51:327-334. 738
Wei P, Liu N, Zhang Z, Liu X, Tang Y, He X, Wu B, Zhou Z, Liu Y, Li J, Zhang Y, Zhou X, Xu L, Chen L, Bi G, 739 Hu X, Xu F, Wang L (2015) Processing of visually evoked innate fear by a non-canonical 740 thalamic pathway. Nat Commun 6:6756. 741
West AE, Pruunsild P, Timmusk T (2014) Neurotrophins: Transcription and Translation. In: 742 Neurotrophic Factors (Lewin GR, Carter BD, eds), pp 67-100. Berlin, Heidelberg: Springer 743 Berlin Heidelberg. 744
Yizhar O, Fenno LE, Prigge M, Schneider F, Davidson TJ, O'Shea DJ, Sohal VS, Goshen I, Finkelstein J, 745 Paz JT, Stehfest K, Fudim R, Ramakrishnan C, Huguenard JR, Hegemann P, Deisseroth K (2011) 746 Neocortical excitation/inhibition balance in information processing and social dysfunction. 747 Nature 477:171-178. 748
Zafra F, Hengerer B, Leibrock J, Thoenen H, Lindholm D (1990) Activity Dependent Regulation of Bdnf 749 and Ngf Messenger-Rnas in the Rat Hippocampus Is Mediated by Non-Nmda Glutamate 750 Receptors. Embo Journal 9:3545-3550. 751
752
Figures & descriptions 753
27
754
Figure 1. Comparison of the AAV2/1-CMV-SSFO and AAV2/7-CaMKII-SSFO vector for transduction and activation of the SC. 755 A) Transduction spread of viral vectors, visualized by immunohistochemical staining for the SSFO-coupled RFP on vibratome 756 sections throughout the SC, spaced 300 μm apart. The AAV2/7-CaMKII-SSFO vector shows a more widespread transduction 757 than the AAV2/1-CMV-SSFO vector, especially in the superficial SC. An outline of the superficial SC and deep SC is sketched in 758 white. Scale bar: 500 μm. B) Theoretical example of semi-quantitative analysis method to investigate SSFO spread after viral 759 vector injection. For each SC, 6 sections immunostained for the SSFO-fused RFP reporter were sampled at 300 μm intervals, 760 spanning most of the colliculus (similar to those shown in the first panel). In each section, transduction was assessed in 4 761 indicated SC quadrants (superficial, medial, lateral and deep SC) and graded on a scale from 0 to 5, with 0 representing no 762 and 5 representing complete transduction of the quadrant area. Results are plotted along top-view illustrations of the 763 superficial and deep SC. These were averaged across animals to obtain an overview of SSFO transduction spread for a 764 vector. The numbers depicted in this panel are only meant to illustrate the analysis, the results are shown in panels C and D. 765 C, D) Top view illustration of SC transduction by both vectors, created as illustrated panel B. The resulting top view-like 766 images are displayed, with average transduction ± SEM for each position (N = 8-12). Both vectors showed good anterior-767 posterior and lateral medial spread in the superficial SC, with a more limited expression in the deep SC. The AAV2/7-CaMKII-768
28
SSFO generally transduced more of the SC than the AAV2/1-CMV-SSFO vector. Key: SC: superior colliculus; RFP: red 769 fluorescent protein; s: superficial SC; d: deep SC; P: periaqueductal gray; SSFO: stable step function opsin. 770
771 Figure 2. Optogenetic stimulation of the SSFO-transduced SC results in neuronal activation. A) Immunostaining for c-Fos on 772 coronal vibratome sections reveals that sham stimulation does not result in c-Fos upregulation. B) Light stimulation after 773 injection of a mCherry control vector also fails to induce increased c-Fos expression, in contrast to light stimulation of a 774 SSFO-transduced SC, where c-Fos is strongly upregulated in the SSFO-expressing region. This indicates that both AAV2/1-775 CMV-SSFO and AAV2/7-CaMKII-SSFO are able to mediate optogenetic neuronal activation. N = 4 for all conditions, scale bar: 776 500 μm, 50 μm for zoomed panels. Key: SC: superior colliculus; s: superficial SC; d: deep SC; P: periaqueductal gray; SSFO: 777 stable step function opsin. 778
29
779 Figure 3. Behavioral tracking of AAV2/7-CaMKII-SSFO transduced animals (N=3) before, during and after optogenetic 780 stimulation with various light intensities. Animal behavior was recorded for 60 minutes and light stimulation (2 s, 473 nm) of 781 specified light intensity was given at 30 minutes, as marked on the graphs with a lightning bolt and vertical grey line. A) 782 Example image of tracking software. The software tracked head, center and tail base of the animal (in cyan, red and purple 783 respectively). B) Schematic overview of behavioral quantification parameters. Movement rate was stratified into three rates: 784 ‘immobile’, ‘mobile’ and ‘highly mobile’ based on the body displacement over a four frame interval. For each movement 785 rate, a typical behavior is given. A turn was defined as a 90 degree deviation of the body-head vector, with a minimum 786 threshold of 45 degrees. C) Graph of mean time per minute in ‘immobile’ state for each stimulation intensity. Before 787 stimulation, animals spent 10-20 seconds per minute ‘immobile’ due to resting or grooming. Right after stimulation, time in 788 ‘immobile’ state increased for the stronger light powers for about 10 minutes (grey arrows), during which the animals were 789 highly alert and showed a high body tension (data not shown). D) Graph depicting mean time per minute in ‘mobile’ state 790 for each stimulation intensity. Before stimulation, animals spent around 40 seconds per minute exploring the arena, moving 791 at a normal, exploratory speed. After stimulation, this was reduced for the two highest stimulation powers for about 10 792 minutes (grey arrows). E) Graph depicting mean time per minute as ‘highly mobile’ per stimulation intensity. Light powers of 793 220 μW and more triggered a running response upon stimulation. F, G) Plots of the number of clockwise and 794 counterclockwise turns, respectively. Before stimulation, animals had no preference for clockwise or counterclockwise 795 movement. Stimulation induced a prominent increase in counterclockwise turning for up to 30 minutes with a concomitant 796 decrease in clockwise turning. For stimulation powers up to 220 μW, the number of counterclockwise turns steadily 797
30
decreased to baseline. For stimulations with 2.2 mW, and to a lesser extent 22 mW, the number of counterclockwise turns 798 peaked around 15 minutes after stimulation (grey arrows), when mobile behavior returned to baseline levels. All graphs 799 show only the mean for each time point because error bars would compromise graph readability. Arrowheads point to peaks 800 of their respective colors where the traces overlap. 801
802 Figure 4. Repeated optogenetic stimulation of the SC results in a consistent immunohistochemical and behavioral response. 803 Stimulation consisted of a 2 s 2.2 mW 473 nm light pulse and is indicated on graphs with a blue lightning bolt. A) Overview 804 of experimental setup. All animals were injected with AAV2/7-CaMKII-SSFO vector and light stimulated on day 1 and 3 with 805 recording of the behavioral response. On day 5, the animals either again received a light stimulation (N=4) or were sham 806 stimulated (N=4) and the SC was then processed for evaluation of c-Fos expression. B) The third stimulation on day 5 still 807 elicits c-Fos activation in the transduced region, which is not the case if a sham stimulation is given on this final day (N=4 for 808 each group). All scale bars are 500 μm. C) Quantitative comparison of the density of c-Fos+ cells in an SC section per mm2 809 transduced tissue after single (e.g. Fig. 2B) or triple stimulation (e.g. Fig 3B). There was no significant difference (N=4 for 810 both groups, one tailed unpaired t-test, p = 0.26, degrees of freedom = 6). D) SSFO-mediated optogenetic stimulation of the 811 SC results in a prolonged behavioral response. Animals were tracked for 60 min and light stimulated at the 30 minute mark 812 as indicated by gray dotted line with blue lightning bolt. Their response to the first optogenetic stimulation (day 1) is 813 characterized by brief running upon light pulse, followed by increased immobility for 10 minutes and a preference for 814
31
counterclockwise turning for up to 30 minutes. This response was highly similar in animals that were stimulated again 48 815 hours later on day 3. Key: SSFO: stable step function opsin; s: superficial SC; d: deep SC; P: periaqueductal gray. 816
817 Figure 5. Repeated optogenetic stimulation of the SC is neuroprotective in a mouse glaucoma model. A) Experimental 818 overview. Animals were injected with the AAV2/7-CaMKII-SSFO vector and implanted with an optic fiber three weeks later. 819 At least one week after implantation, LP was performed. Starting one day before LP, animals were optogenetically 820 stimulated twice a day for 14 days (LP + stimulation group). As a control group, animals were sham stimulated every time 821 with the laser off (LP + sham group). B) Ocular hypertension profiles after LP for stimulation and sham group show a similar 822
32
and significant elevation from baseline, which returns to normal levels from 6 dpi on (two-way ANOVA with Dunnett’s 823 multiple comparisons test versus LP + sham 0 dpi, letters indicate significance levels for each time point, with shading 824 corresponding to the respective group). C, D) As an additional confirmation that repeated optogenetic stimulation does not 825 lead to desensitization, behavior in response to stimulation was assessed at 13 days dpi. The behavioral response is highly 826 similar to animals stimulated for the first time (e.g. Fig. 4D). E) The final stimulation at 14 dpi still results in upregulated c-827 Fos expression in the SSFO transduced region, indicating that repeated stimulation produces consistent neuronal activation. 828 Scale bar: 500 μm. F) Repeated optogenetic stimulation of the SC leads to an increased RGC survival in the LP glaucoma 829 model at 14 dpi. Relative to contralateral right eyes, the LP + stimulation group shows significantly more survival (90%, N = 830 17) compared to the LP + sham group (74%, N = 20) (two-tailed unpaired t-test, P = 0.0114, t = 2.67, degrees of freedom = 831 35). G) Representative isodensity maps of retinal flatmounts. The No LP contralateral eye shows normal RGC density. LP + 832 sham animals often have increased sectorial loss (arrowhead), although occasionally only diffuse loss is observed (arrow). 833 For the LP + stimulation eyes, mostly diffuse loss is found. Scale bar: 2 mm. H) Limited retrograde transduction of the retina 834 is observed after collicular vector injection. Panels in H show a representative part of the retina after double-staining for the 835 RGC-marker Brn3a (green) and vector reporter RFP (black, artificial color for clarity). Retrogradely transduced RGCs are 836 indicated with arrowheads and they represent less than 1% of the total RGC population. Scale bar: 50 μm. Key: LP: laser 837 photocoagulation, glaucoma model; dpi: days post injury, days after LP. s: superficial SC; d: deep SC; P: periaqueductal gray. 838 839