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Neurobiology of Aging 30 (2009) 819–828
Age-dependent remodelling of retinal circuitry
E. Terzibasi a,1, M. Calamusa b, E. Novelli c, L. Domenici a,d,E. Strettoi a, A. Cellerino b,∗
a Istituto di Neuroscienze, Consiglio Nazionale delle Ricerche (CNR), Via G. Moruzzi 1, 56100 Pisa, Italyb Scuola Normale Superiore, P.zza Cavalieri 7, 56100 Pisa, Italy
c Fondazione G.B.Bietti per l’Oftalmologia, Rome, Italyd Dipartimento di Scienze e Tecnologie Biomediche, Universita di L’Aquila, 67010 L’Aquila, Italy
Received 27 April 2007; received in revised form 27 July 2007; accepted 14 August 2007Available online 24 October 2007
bstract
We have investigated morphological changes in second-order neurons of the mouse retina during aging by using immunohistochemistrynd electron microscopy. We observed sprouting of rod bipolar cells dendrites and horizontal cells arborizations: neuronal processes of botheuronal types showed irregular extensions beyond the outer plexiform layer, toward the outer limiting membrane, as well as into the outeruclear layer (ONL). These processes were first observed in animals of 12 months of age and increased in numbers steadily until 24 months,hich represent the last age examined. The ectopic processes are decorated by puncta immunoreactive for pre-synaptic markers typical ofhotoreceptor terminals juxtaposed to post-synaptic neurotransmitter receptors, demonstrating the presence of the entire molecular machineryf functional synapses. Electron microscopy confirmed that ectopic processes receive synapses from photoreceptor terminals.
We conclude that during the second year of life retinal rod bipolar and horizontal cells undergo sprouting and form ectopic synapses in theNL.2007 Elsevier Inc. All rights reserved.
eywords: Plasticity; Mouse retina; Aging; Ectopic synapses
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. Introduction
The central nervous system (CNS) undergoes remodellinguring many physiological processes, such as development,earning and also as consequence of aging.
Aging of the CNS is a natural phenomenon of progressivend irreversible functional decay characterized by region-pecific neuronal loss (Trillo and Gonzalo, 1992; Lossi etl., 2005), decrement of neurotransmitter release (Zsilla et
l., 1994; Hebert and Gerhardt, 1999) and deterioration ofognitive functions (Herzog and Rodgers, 1989; Rapp and∗ Corresponding author. Current address: Leibniz Institute for Agingesearch, Beutenberg Strasse 11, 07745 Jena, Germany.el.: +49 3641 656493; fax: +49 3641 656040.
E-mail address: [email protected] (A. Cellerino).1 Current address: Leibniz Institute for Aging Research, Beutenbergtrasse 11, 07745 Jena, Germany.
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197-4580/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.neurobiolaging.2007.08.017
maral, 1989; Milgram et al., 1994; Small, 2001; Keller,006). Some causative agents of these processes are oxida-ive stress (Gotz et al., 1994; Tritschler et al., 1994; Beal,995, 2005; Williams, 1995; Hogg, 1998; Urano et al., 1998;onzalez-Fraguela et al., 1999; Galli et al., 2005; Moreira et
l., 2005) and accumulation of aberrant proteins which escaperoteolytic cleavage (Bahr and Bendiske, 2002; Keller et al.,002; Abd El Mohsen et al., 2005).
The retina is part of the CNS whose neuronal types andonnections are known in great detail and large numbersf cell-specific markers have been described. A completeatalogue of all retinal subtypes is now available for someammalian retinas (LaVail and Lawson, 1986; Masland,
001). The retina is potentially a sensitive target of age-
ependent degeneration. In particular, photoreceptors areharacterized by a very high metabolic rate, are chronicallyxposed to light and are sensitive to light damage (LaVail etl., 1987).
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Partial degeneration and age-dependent functional impair-ent of rod photoreceptors have been described in the mouse
etina (Gresh et al., 2003). A reduction of ganglion cell num-ers as an effect of aging has also been described (Katz andobison, 1986; Weisse et al., 1990; Gao and Hollyfield, 1992;im et al., 1996; Harman et al., 2000; Danias et al., 2003;earson et al., 2006). Yet, despite extensive knowledge oformal retinal circuitry, little is known of putative struc-ural modifications of the inner retina as a consequence ofging. Retinal circuit is known to undergo plastic changes asconsequence to photoreceptor degeneration, which includeoth degeneration of neuronal processes and compensatoryormation of new connections. For example, rapid degen-ration of rod photoreceptors in rd1 mice is associated tooss of rod bipolar dendrites, re-distribution of glutamateeceptors and reduced complexity of horizontal cells networkStrettoi and Pignatelli, 2000) In other forms of retinal degen-ration, rod bipolar cells were shown to transiently contactones (Peng et al., 2000) and substantial sprouting of bothhotoreceptors and horizontal cells is observed in a mouseutant which lack functional photoreceptor synapses (Dick
t al., 2003). Similar changes are likely to take place alson the aging retina. Preliminary results from our laboratoryTerzibasi et al., 2003) and a recent publication from Lietst al. (2006) have indeed shown aberrant processes in rodipolar neurons as a consequence of aging. Here, we usedmmunohistochemistry, confocal and electron microscopyo investigate age-dependent morphological alterations inecond-order retinal neurons, namely bipolar and horizontalells.
. Materials and methods
.1. Animals
Mice from the Jackson Laboratory (C57BL/6J strain) were
sed for the present study. Animals were maintained in anrtificial 12 h light/dark cycle, with food and water ad libitumnd illumination level below 60 photopic lux. All experimen-al procedures were done in compliance with the ARVOputF
able 1ynoptic table of the antibodies employed
ntibody Source
ouse anti-PKC� Sigma, St. Louis, MOabbit anti-PKC� Santa Cruz Biotechnologyouse anti-Go-� Chemicon, Temecula, CAabbit anti-Calbindin D Chemicon, Temecula, CAouse anti-Calbindin D Sigmaouse anti-neurofilament, 200 kDa Sigmaabbit anti-mGluR6 Neuromics antibodiesouse anti-tubulinIII Sigmaouse anti-kinesin II Chemicon, Temecula, CAabbit anti-GFAP Sigmaouse anti-synaptophysin Boehringer Mannheim Biochemicaassoon Stressgen
f Aging 30 (2009) 819–828
tatement for the use of animals in research and with thetalian regulations.
Animals were anesthetized by intra-peritoneal injection ofvertin (1.2% tribromoethanol and 2.4% amylene hydrate inistilled water, 0.02 ml/g body weight) and perfused transcar-ially with 4% paraformaldehyde/0.1 M phosphate bufferpH 7.4). All procedures were done in compliance with thetalian laws for the use of animals in research.
.2. Tissue preparation and immunocytochemistry
After perfusion, eyes were enucleated and opened alonghe ora serrata. The cornea, lens and vitreous body wereemoved and the posterior eyecups with the retinas attachedere post-fixed for 15 min by immersion in 4% paraformalde-yde/0.1 M phosphate buffer (pH 7.4). Subsequently, theyere infiltrated with 30% sucrose to ensure cryoprotection,
mbedded in Tissuetek (Reichart-Jung, Nubloch, Germany)rozen and serially sectioned at 14 �m on a cryostat.
Immunocytochemistry was performed following standardrocedures. A list of the primary antibodies used to labeletinal neurons, their concentration and specificity is pro-ided in Table 1. Binding of primary antibodies was detectedy goat anti-mouse and goat anti-rabbit IgG secondaryntibodies coupled respectively to Alexa Fluor 488 (greenuorescence) and Alexa Fluor 568 (red fluorescence) fromolecular Probes and a goat anti-mouse Alexa Fluor 568
rom Sigma–Aldrich, all used at 1:400 dilution. To label conehotoreceptors Arachis ipogea-derived lectin protein (PNA)onjugated with Alexa Fluor 488 were used (1:1000, fromolecular Probes).
.3. Light microscopy and sampling
Single- and double-labelled retinas were observed using auorescence microscope (Eclipse E600, Nikon). Fluorescent
ictures were acquired at resolution of 1024 × 1024 dot/in.sing a CCD camera (Roper Scientific Photometrics) pluggedo the fluorescence microscope and a 40×/0.75 objective.or statistical analysis, three mice for each age and threeDilution Localization of interest
1:1000 Rod bipolar cells1:1000 Rod bipolar cells1:500 Rod bipolar cells and ON-cone bipolar cells1:1000 Horizontal cells1:1000 Horizontal cells1:200 Axonal arborization of horizontal cells1:7000 On-bipolar cells dendritic tips1:1000 Ganglion cells1:1000 Synaptic ribbons1:1000 Astrocytes1:500 OPL and IPL1:1000 Synaptic ribbons
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etinal sections for each mouse were sampled. In each hemi-etina, three fields were acquired: one in the centre (C,pproximately 10◦ of eccentricity), one in mid-periphery (CP,pproximately 45◦ of eccentricity) and one in periphery (P,pproximately 80◦ of eccentricity).
For quantitative analysis of sprouts, four sets of three ani-als of following ages were used: 12, 16, 20, 24 months,
o obtain a complete representation of the temporal evolu-ion of the degenerative process through the second year ofife-span. For further double-labelling procedures mice of6–20-month-old mice were used (sets of three animal forach age).
.4. Quantitative analysis and statistics
To quantify the density of PKC immunopositive sprout-ng processes a field of 650 �m × 500 �m comprising thentire retinal thickness was selected in each image. Withinhis region, a sprout was defined as a PKC+ process at leasthree times longer than the outer plexiform layer thickness.n each animal, the number of sprouts for the six imagesorresponding to each of the sampling regions (C, CP or) was computed separately. To evaluate the inter-observereliability in the counting procedure, one set of 18 imagesas analyzed by two observers. Their scores were strongly
orrelated (Pearson’s r = 0.54, p < 0.01).To test for age- and region-dependent sprouting, a two-
ay ANOVA using age and region as factors was performed.ukey’s post hoc test was used to measure the significance ofultiple comparisons.
.5. Confocal microscopy
To analyze double-labelled sections, retinal preparationsere examined with a Leica TCS-NT confocal micro-
cope equipped with a krypton–argon laser. The followingarameters were matched: age; fixation and immunostain-ng protocols; magnification, pinhole size, gain and offsetf the confocal microscope; thickness of extended-focusonfocal images. From each double-labelled section, areaspanning the whole thickness of the inner plexiform layerIPL) were chosen and the corresponding images were col-ected as 1024 × 1024 TIFF files.
.6. Pre-embedding immuno-electron microscopy
To examine sprouts by electron microscopy, one8-month-old animal was perfused transcardially with% paraformaldehyde/0.1 M phosphate buffer (pH 7.4).he eyes were enucleated; each retina was separated from
he sclera and choroid. The right retina was separated
rom the pigmented epitelium and sectioned in four quad-ants: one quadrant was maintained intact, whereas thethers were chopped with a razor’s blade into severalragments. The left retina was frozen, sectioned with aasww
f Aging 30 (2009) 819–828 821
ryostat in 60–80 �m vertical sections, and then reacted free-oating.
Staining with rabbit anti-PKC� antibody (from Santaruz) was performed on the free-floating sections, thehopped fragments and the whole retinal quadrant. After a0 h blocking step, retinal tissue was incubated in the primaryntibody diluted 1:200 for 4 days at 4 ◦C. The retinal samplesere incubated in a biotin-labelled secondary antibody (fromector, used 1:100), followed by avidin–biotin-peroxidaseomplex and glucose–oxydase DAB-nickel (Vector Labora-ories, Burlingame, CA, USA). Sections and fragments werehen fixed (2.5% gluteraldheyde and 2% paraformaldeyhde in.1 M phosphate buffer), post-fixed with osmium tetroxide,loc stained with uranyl acetate, dehydrated in ethanol andmbedded in Epon-Araldite for electron microscopy. Ultra-hin vertical sections were examined with a Jeol 1200 EXIIlectron microscope (Jeol Italia, Milan, Italy).
. Results
.1. Neurons of the senescent inner retina show ectopicrocesses in the outer plexiform layer (OPL)
We first examined the morphology of rod bipolar cellsidentified by PKC� immunoreactivity, Fig. 1A) and hori-ontal cells (labelled with Calbindin D, Fig. 1B) in retinasf 24-month-old C57BL/6J mice. We observed sprouting ofoth bipolar cells dendrites and horizontal cells arborizations:rocesses of both neuronal types showed irregular extensionseyond the outer plexiform layer (OPL), toward the outerimiting membrane, as well as into the outer nuclear layerONL).
.2. Aberrant processes in the OPL follow a spatial andemporal gradient
Density of processes was quantified in central (C),id-peripheral (CP) and peripheral retina (P) of mice aged
2, 16, 20 and 24 months. The density of aberrant processeshows a clear centre-to-periphery gradient, with the numberf sprouts increasing from the central area to the middle andar periphery. Fig. 1C illustrates this spatial gradient in a6-month-old retina.
In addiction, the length and the number of sprouts increaseradually with age. Fig. 1E illustrates the progression ofhe sprouting process from 12 to 24 months for both rodipolar and horizontal cells. The first detectable sproutsre evident in 1-year-old mice: their number increasesteadily up to 2 years. The plot presented in Fig. 1Dummarizes the spatial–temporal gradient in the densityf ectopic processes throughout the second year of life.e performed a two-way ANOVA, with retinal area and
ge as co-factors. Both factors have shown a statistical-ignificant influence on the number of sprout (p < 0.001),hile no significant interaction between the two factorsas detected. Multiple comparison matrices (Tukey’s post
822 E. Terzibasi et al. / Neurobiology of Aging 30 (2009) 819–828
Fig. 1. Sprouting of second-order neurons in the outer nuclear layer (ONL) of senescent C57bl/6J mouse retina. (A) Confocal image of rod bipolar cellsvisualized by imunocytochemistry for PKC� in vertical sections of 20-month-old C57bl/6J mouse retina. Many immunopositive cells show sprouting processesup to the ONL. (B) Confocal image of horizontal cells visualized by imunocytochemistry for Calbindin D in a 20 months mouse retina: neuronal processes ofseveral cells show irregular extensions beyond the OPL into the ONL. (C) Rod bipolar cells visualized by imunocytochemistry for PKC� in a 20-month-oldC57bl/6J mouse retina show a clear centre-to-periphery spatial gradient of the sprouting process: the sprouts number increases from the central zone (C) to themiddle (CP) and extreme periphery (P). Scale bar is 20 �m. (D) Diagram summarizing the spatial-temporal gradient of sprouting events through the secondyear of life of C57black mice: The number of sprouts in 2 years-old mice is four times higher than that observed in 1-year-old mice. (E) Rod bipolar cells( PKC� and Calbindin D, respectively: temporal gradient of sprouting occurrence,i II–VI = 16 months, III–VII = 20 months and IV–VIII = 24 months). The number ofs the second year of life.
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Table 2Pair wise comparisons of sprout density in the retinas of different ages
12m 16m 20m 24m
12m * s s s16m – * s s
I–IV) and horizontal cells (V–VIII) visualized by imunocytochemistry forllustrated by images of 12, 16, 20, 24-month-old retinas (I–V = 12 months,prouts of rod bipolar and horizontal cells shows a significant growth during
oc test) of different ages and retinal areas are reported inables 2 and 3.
.3. Aberrant processes from rod bipolar cells
To identify univocally the types of second-order neuronsndergoing sprouting, we double-labelled retinal sections foro-�, a marker of all (both rod and cone driven) ON bipo-
ar cells (Fig. 2A, green) and PKC� (specific for rod bipolar
20m – – * s24m – – – *
s = p < 0.05; 12m = 12 months; 16m = 16 months; 20m = 20 months;24m = 24 months; N = 3 for each age.
E. Terzibasi et al. / Neurobiology o
Table 3Pair wise comparisons of sprout density in different retinal regions at allinvestigated ages
C CP P
C * n.s. sCP – * sP
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ells, Fig. 2A, red); therefore, the PKC� negative, Go-� pos-tive cells are ON-cone bipolar cells. All aberrant processesre double-labelled, demonstrating that the sprouts originaterom rod bipolar cells only (Fig. 2A). This particular typef double staining does not provide information about OFF-one bipolar cells, but the scarceness of cone displacementoward the outer retina (see below) strongly suggests that theulk of the sprouts originate from rod bipolar cells.
.4. Displaced cone pedicles
To visualize the cone distribution into the OPL of agedice retina, we labelled retinal sections with fluorescent
ectin peanut agglutinin (PNA) and with the specific markeror horizontal cells, Calbindin D. This lectin stains conenner segments as well as their synaptic terminals in the OPL.lbeit rare cone pedicles were found displaced in the ONL,
long horizontal cells sprouts (Fig. 2B, green, arrowheads),he vast majority of them were normally positioned in thePL.
.5. Aberrant processes from horizontal cells
We labelled horizontal cells with the specific marker Cal-indin D and observed aberrant processes originating fromorizontal cells and directed toward the outer retina. Theserocesses followed time course similar to that described foripolar cell processes (see again Fig. 1E). Immunohistochem-stry with antibodies against neurofilaments and Calbindin
were used to label axonal arbours of horizontal cellsnd whole horizontal cell processes, respectively. We foundccasional double-labelled processes with both antibodies,howing an irregular, branched structure enlarged at its baseFig. 2C, arrow). Thus, horizontal cell axonal arborizationsive rise to sprouts as a consequence of aging (see againig. 1E, panels V–VIII).
Normal photoreceptor synapses comprise a stereotypedomplex of horizontal and bipolar cell processes knowns the triad. This close association is retained in synap-ic arrangement involving the aberrant processes (Fig. 2D):ALB+/NF+ sprouts identify the axonal terminal arboriza-
ion of horizontal cells invaginated with bipolar cell dendritesn the rod spherule at the typical triad synapse.
Ectopic contacts of rod bipolar cells with cone pediclesere reported in models of retinal degeneration (Peng et al.,000). To test this possibility we performed a triple labellingor PKC� (Fig. 2E in red), Calbindin (in green) and lectin (in
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f Aging 30 (2009) 819–828 823
righter green). Although Calbindin and PNA are both shownn green, cone pedicles can be easily recognized due to their
orphology and brighter staining (arrowheads). The stainingf horizontal cells helps to visualize the normal distributionf cone pedicles, regularly located into the OPL. We did notnd any evidence of spatial relations between cone pedicleslabelled in green, arrowheads) and dendritic sprouts of rodipolar cells (labelled in red, arrows).
Finally, some Calbindin positive processes appear to comento direct contact with cone pedicles visualized with lectintaining in double-labelling experiments (Fig. 2B, in red).
.6. Aberrant processes show pre- and post-synapticarkers
We investigated by double immunohistochemistry that theresence of the molecular machinery necessary for synapticransmission in the aberrant processes.
.7. Post-synaptic receptor
Double labelling with anti-PKC� (Fig. 2F green, for rodipolar cells) and anti-mGluR6 (Fig. 2F, red, specific for thelutamate metabotropic receptor 6 associated to the dendritesf ON-bipolar cells) reveals the presence of immunoreactiveuncta through the total length of the sprouts into the ONL.his finding suggests that aberrant processes might receive
unctional synapses.
.8. Pre-synaptic components
Presence of structured pre-synaptic opposite bipolar andorizontal sprouts was demonstrated by double-labellingxperiments using antibodies against Bassoon (a marker ofhe arciform density of the ribbon complex, Fig. 2G, green),he vesicle protein synaptophysin (Fig. 2H, green), the ribbonrotein kinesin 2 (Fig. 2I, green) on one hand and antibodygainst the rod bipolar marker PKC� (Fig. 2G, H and I, ined) on the other hand. All three antibodies (Bassoon, synap-ophysin, kinesin 2) showed a regular pattern of distributionlosely associated to the sprouting processes of rod bipolarells, which was undistinguishable from the labelling patternf normally placed synapses.
The spatial correspondence of pre- and post-synapticarkers was demonstrated by double labelling of Basson
Fig. 2L, green) and mGluR6 (Fig. 2L, red) A spatial cor-espondence between pre-synaptic marker Basson (Fig. 2M,reen) and horizontal cells sprouts (Fig. 2M, red) was alsobserved.
.9. Electron microscopy
We performed pre-embedding immunocytochemistry
ith anti PKC� antibodies to examine rod bipolar cellendrites and sprouts at the EM level. Sprouts were visibles dark spots in the ONL in semithin (1–2 �m) sections,bserved at the light microscope (Fig. 3A); they reached
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24 E. Terzibasi et al. / Neurobi
he central part of the ONL, at the height of the six to eightow of nuclei. At the EM level, we identified a scattered
opulation of ectopic rod spherules in the ONL. Thesepherules were dispersed among photoreceptor nucleind displayed a complement of synaptic vesicles, typicalibbons and arciform densities (Fig. 3B–D). Some of them(eit
f Aging 30 (2009) 819–828
stablished ribbon contacts with dendrites of rod bipolarells, visible by the dense product of PKC immunostaining
Fig. 3E). Thus, electron microscopy confirmed that thectopic sprouts of rod bipolar cells reach spherules misplacedn the outer nuclear layer and receive ribbon synapses fromhem.
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. Discussion
In the present paper we report that second-order neuronsf the mouse inner retina undergo structural remodelling asconsequence of aging. In particular, we show that two
lasses of second-order neurons of the mouse retina, namelyod bipolar and horizontal cells, develop aberrant processesrowing into the outer nuclear layer (ONL); sprouting affectsreferentially rod bipolar cells. Elongation of rod bipolar andorizontal cell processes is progressive and develops duringhe second year of life. These aberrant processes are alsoharacterized by a clear spatial gradient increasing in numberrom the centre to the periphery of the retina. Spatial gradi-nts are well described in many types of retinal degenerationsFisher, 1968; Lai et al., 1978; Trachimowicz et al., 1981).he present finding indicates that the peripheral retina is moreensitive to the effects of aging. Finally, aberrant processesstablish normally structured synapses ectopically placed inhe ONL. Thus, elongation of rod bipolar and horizontal cellrocesses occurring during the second year of the mouse lifean be interpreted as plastic changes through which existingrocesses grow.
We detected perfect spatial correspondence of specificre- and post-synaptic markers on the terminal ending ofhe sprouts: pre-synaptic structures contain Bassoon, kinesinI and synaptophysin; these were juxtaposed to clusterf puncta positive for post-synaptic receptor mGluR6, theetabotrophic glutamate receptor 6, specific of the ON-
athway (Nakajima et al., 1993; Vardi and Morigiwa, 1997).ince punctuate labelling for mGluR6 is activity-dependentNomura et al., 1994), all these data strongly suggest thatctopic synapses are functional. Electron microscopy anal-sis revealed a normal structure of ectopic synapses, thatere positioned within the invagination of rod spherules,islocated to the outer retina, within the outer nuclear layer.
Aberrant processes have been previously described in sev-ral animal models of retinal degeneration. For example,prouting of rod bipolar dendrites is found in experimental
dm
ig. 2. Antibody staining of outer retinal neurons and synapses at 18 months. (A) DKC� (red) for rod bipolar cells shows that virtually all the sprouting processes are dorizontal cells (red) and PNA, labelling cones: several cone photoreceptor endingshe ONL. (C) Double-labelling with Calbindin D antibodies, staining the whole plexrborization of horizontal cells, show that both these components are labelled in theorizontal cells (green) and PKC� (red), for rod bipolar cells, shows a strict assocE) Triple-labelling with Calbindin D antibodies (green), PNA (brighter green punone pedicles (arrowheads) and dendritic sprouts of rod bipolar cells (arrows). (F) Vuncta are ectopically expressed in the ONL (arrowheads). (G) The close associatiod bipolar cells (red) confirms the presence of ectopic photoreceptor synapses alon(green), and PKC� (red), shows a close association of the vesicle marker along theetween rod bipolar cell sprouts and pre-synaptic elements is observed in retinal segreen puncta). (L) Vertical section, double-labelled for mGluR6 (red puncta) andlear topographical association of pre- and post-synaptic elements (arrowheads), alertical section, double-labelled for Calbindin (red) and Bassoon (green), showing
rom horizontal cells into the ONL. Scale bar: 20 �m (A–F, H and I) and 10 �m (G,he reader is referred to the web version of the article.)
f Aging 30 (2009) 819–828 825
etina detachment, in animal models of retinitis pigmentosa,n RCS rats, in mutant mice for Bassoon and Caf1 (Li et al.,995; Dick et al., 2003; Wang et al., 2005). Several studiesescribed secondary events of retinal remodelling caused byhotoreceptor degenerations, such as Muller cells hypertro-hy, neuronal migration to ectopic sites and formation of newonnections throughout the retina (Jones et al., 2003, 2005;ang et al., 2005). Analyses of experimental-induced (deaad et al., 1996) and inherited (Fletcher and Kalloniatis,996) rodent retinal degenerations revealed subtle changesn cellular phenotypes and morphology of the neural retina.emodelling of rod pathways is described to emerge rapidly
n the rd1 mouse (Strettoi and Pignatelli, 2000; Strettoi et al.,002), in which a mutation in a rod-specific gene causes theeath of rods by postnatal day 10. Rod bipolar cells showendritic retraction and their axon terminals retain immatureynaptic structures. Later on, modifications involve also theone circuits: both cones (Fei, 2002) and horizontal cell den-rites (Strettoi et al., 2002) in the young rd1 mouse sprout neweurites, whereas cone bipolar cells retract dendrites progres-ively (Strettoi et al., 2003). During human rod degeneration,urviving rods, horizontal and amacrine cells similarly extendnomalous neurites throughout the retina (Li et al., 1995;ariss et al., 2000).
Progressive reorganization of rod relay pathway, as con-equence of photoreceptor loss, and changes in synapticonnectivity between photoreceptor and their target cellsave bee also described in two further models of photore-eptor degeneration, such as the P23H line1 homozygouslbino rat (Cuenca et al., 2004) and the royal college ofurgeon (RCS) rat (Cuenca et al., 2005). In these studieshe authors describe photoreceptor degeneration-dependent
odifications in the synaptic machinery connecting photore-eptors with second-order neurons: rod and cone bipolar cellsnd horizontal cells.
Aberrant processes involving neurons of the inner retinairectly connected with photoreceptors were described in autant mouse for the pre-synaptic cytomatrix protein Bas-
ouble-labelling with Go-� (green) for rod and ON-cone bipolar cells andouble-labelled. (B) Double-labelling with Calbindin D antibodies, staining
(arrowheads) make contact with processes of horizontal cells sprouting intous of horizontal cells (red), and neurofilaments antibody, staining the axonalsprouts (arrow). (D) Double-labelling with Calbindin D antibodies, stainingiation between sprouting processes of horizontal and bipolar cells (arrow).cta) and PKC� (red): there is no evidence of physical interaction betweenertical section double-labelled for mGluR6 (red) and PKC (green): mGluR6on of the pre-synaptic protein Bassoon (green) and dendritic sprouts fromg these processes. (H) Vertical section double-labelled with sinaptophysinrod bipolar cell dendrites sprouting into the ONL (arrows). (I) Associationctions, double-labelled with anti-PKC� (red) and anti-kinesin2 antibodiesfor the pre-synaptic protein Bassoon (green, horseshoe stain). There is a
ong the footprint of an unlabelled process, originating from the OPLl. (M)the close proximity of the pre-synaptic marker with sprouting processes
L and M). (For interpretation of the references to color in this figure legend,
826 E. Terzibasi et al. / Neurobiology of Aging 30 (2009) 819–828
Fig. 3. Electron microscopy. (A) Semitin section from a retinal sample treated for pre-embedding ICCH with antibodies against protein kinase C�. The age ofthe mouse is 18 months. Rod bipolar cells (RBC) are heavily stained. Besides a normal complement of dendrites in the OPL, few processes are labelled in theONL as well (arrows). (B–D) Examples of small rod spherules (s) ectopically located in the ONL and identified at the EM level, in unlabelled retinal samples.Synaptic ribbons are clearly visible (arrows). These spherules were found at the height of the sixth to eighth row of nuclei in the ONL. (E) Electron micrographof a spherule (s), positioned in the ONL, invaginated by the process of a rod bipolar cell, labelled with PKC antibodies (asterisk). Two ribbons are facing thedendrite. n: nucleus of a photoreceptor.
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E. Terzibasi et al. / Neurobi
oon, an essential component of the photoreceptor ribbon:ibbons lacking Bassoon are not anchored to the pre-synapticctive zone preventing the development of a proper synap-ic function. Rod bipolar cells and horizontal cells of theassoon mutant retina make sprouting into the ONL androject aberrant processes throughout the photoreceptorsayer showing ectopic synaptic sites (Dick et al., 2003; tomieck et al., 2005). These and our results are very similar.owever, in our case immunohistochemistry excluded theossibility that Basson is down regulated as consequence ofging.
The nature and mechanisms driving the observed age-ependent growth of aberrant processes in ONL is notnderstood at this stage. The hypothesis can be raisedhat elongation of aberrant processes follows some kind ofhotoreceptor dysfunction. Although no overt loss of pho-oreceptors is detected in the aged mouse retina (Gresh et al.,003), it is possible that the abnormal growth of second-ordereurons processes is a direct consequence of an initial age-ependent photoreceptors dysfunction which is not reflectedn a significant level of cell death. The functional decay ofhe mouse retina has been demonstrated in 17-month-old
ice, in which the ERG recordings showed a significantecrease of the “a” wave amplitude compared to these mea-ured in young animals (Gresh et al., 2003). Support for thisypothesis comes from structural modifications which wereound in the previously mentioned mutant mouse lackinghe functional Bassoon protein. Although this mutant doesot show any photoreceptor degeneration, synaptic struc-ures in the outer retina show major abnormalities, includingree-floating ribbons in the rod spherules. Ultimately, rodynapses are non-functional. This causes a compensatoryhange in rod bipolar cells, which are very much reminis-ent of what we described here. Bipolar and horizontal cellsf the Bassoon mutant show sprouts into the ONL which areecorated by perfectly structured synapses. The number ofctopic synapses (Dick et al., 2003; tom Dieck et al., 2005)nd sprouting processes gradually increases over time, in theassoon mutant as well as in the aged animals we describeere. The only distinguishing factor between the remodellingetected in the Bassoon mutant and senescent mouse remod-lling is the different temporal scale: in the Bassoon mutant,odifications occur during an early phase of the mouse life,hereas the wild type mouse exhibits a slow and progressive
emodelling during the second year of life. Therefore, it iseasonable to suppose a direct causative link between age-ependent impairment of photoreceptor functionality and aompensatory sprouting response in second-order neurons inhe inner retina.
When we were writing this manuscript, data were pub-ished showing the presence of aberrant processes in rodipolar cells of the aged mouse retina (Liets et al., 2006).
ur experiments produced similar results; in addition, wehowed that aberrant processes are also present in horizontalells during aging and that their sprouts are also characterizedy a normal complement of synaptic proteins.
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isclosure statement
We disclose that:
(a) All the authors do not have actual or potential conflictsof interest including any financial, personal or other rela-tionships with other people or organizations within 3years of beginning the work submitted that could inap-propriately influence (bias) their work.
b) All procedures involving animals were approved andconformed to the Italian law and the ARVO regulationfor animal experimentation.
cknowledgements
L.D. is recipient of FIRB Grant RBAUO15YHM; A.C.s recipient of FIRB Grant RBAVO1A7BT. We thank Marcotebel (University of Trieste, Italy) for providing us the agedice.
eferences
bd El Mohsen, M.M., Iravani, M.M., Spencer, J.P., Rose, S., Fahim, A.T.,Motawi, T.M., Ismail, N.A., Jenner, P., 2005. Age-associated changesin protein oxidation and proteasome activities in rat brain: modula-tion by antioxidants. Biochem. Biophys. Res. Commun. 336 (2), 386–391.
ahr, B.A., Bendiske, J., 2002. The neuropathogenic contributions of lyso-somal dysfunction. J. Neurochem. 83 (3), 481–489.
eal, M.F., 1995. Aging, energy and oxidative stress in neurodegenerativediseases. Ann. Neurol. 38 (3), 357–366.
eal, M.F., 2005. Mitochondria take center stage in aging and neurodegen-eration. Ann. Neurol. 58 (4), 495–505.
uenca, N., Pinilla, I., Sauve, Y., Lu, B., Wang, S., Lund, R.D., 2004. Regres-sive and reactive changes in the connectivity patterns of rod and conepathways of P23H transgenic rat retina. Neuroscience 127 (2), 301–317.
uenca, N., Pinilla, I., Sauve, Y., Lund, R., 2005. Early changes in synapticconnectivity following progressive photoreceptor degeneration in RCSrats. Eur. J. Neurosci. 22 (5), 1057–1072.
anias, J., Lee, K.C., Zamora, M.F., Chen, B., Shen, F., Filippopoulos, T., Su,Y., Goldblum, D., Podos, S.M., Mittag, T., 2003. Quantitative analysisof retinal ganglion cell (RGC) loss in aging DBA/2NNia glaucoma-tous mice: comparison with RGC loss in aging C57/BL6 mice. Invest.Ophthalmol. Vis. Sci. 44 (12), 5151–5162.
e Raad, S., Szczesny, P.J., Munz, K., Reme, C.E., 1996. Light damage inthe rat retina: glial fibrillary acidic protein accumulates in Muller cells incorrelation with photoreceptor damage. Ophthalmic Res. 28 (2), 99–107.
ick, O., tom Dieck, S., Altrock, W.D., Ammermuller, J., Weiler, R., Gar-ner, C.C., Gundelfinger, E.D., Brandstatter, J.H., 2003. The pre-synapticactive zone protein bassoon is essential for photoreceptor ribbon synapseformation in the retina. Neuron 37 (5), 775–786.
ariss, R.N., Li, Z.Y., Milam, A.H., 2000. Abnormalities in rod photorecep-tors, amacrine cells, and horizontal cells in human retinas with retinitispigmentosa. Am. J. Ophthalmol. 129 (2), 215–223.
ei, Y., 2002. Cone neurite sprouting: an early onset abnormality of the conephotoreceptors in the retinal degeneration mouse. Mol. Vis. 8, 306–314.
isher, R.F., 1968. The variations of the peripheral visual fields with age.Doc. Ophthalmol. 24 (1), 41–67.
letcher, E.L., Kalloniatis, M., 1996. Neurochemical architecture of thenormal and degenerating rat retina. J. Comp. Neurol. 376 (3), 343–360.
8 ology o
G
G
G
G
G
H
H
H
H
J
J
K
K
K
K
L
L
L
L
L
L
M
M
M
N
N
P
P
R
S
S
S
S
T
t
T
T
T
U
V
W
W
W
7 (1), 55–73.
28 E. Terzibasi et al. / Neurobi
alli, F., Piroddi, M., Annetti, C., Aisa, C., Floridi, E., Floridi, A., 2005.Oxidative stress and reactive oxygen species. Contrib. Nephrol. 149,240–260.
ao, H., Hollyfield, J.G., 1992. Aging of the human retina Differential lossof neurons and retinal pigment epithelial cells. Invest. Ophthalmol. Vis.Sci. 33 (1), 1–17.
onzalez-Fraguela, M.E., Castellano-Benitez, O., Gonzalez-Hoyuela, M.,1999. Oxidative stress in neurodegeneration. Rev. Neurol. 28 (5),504–511.
otz, M.E., Kunig, G., Riederer, P., Youdim, M.B., 1994. Oxidative stress:free radical production in neural degeneration. Pharmacol. Ther. 63 (1),37–122.
resh, J., Goletz, P.W., Crouch, R.K., Rohrer, B., 2003. Structure-functionanalysis of rods and cones in juvenile, adult, and aged C57bl/6 and Balb/cmice. Vis. Neurosci. 20 (2), 211–220.
arman, A., Abrahams, B., Moore, S., Hoskins, R., 2000. Neuronal densityin the human retinal ganglion cell layer from 16 to 77 years. Anat. Rec.260 (2), 124–131.
ebert, M.A., Gerhardt, G.A., 1999. Age-related changes in the capacity,rate, and modulation of dopamine uptake within the striatum and nucleusaccumbens of Fischer 344 rats: an in vivo electrochemical study. J.Pharmacol. Exp. Ther. 288 (2), 879–887.
erzog, A.R., Rodgers, W.L., 1989. Age differences in memory performanceand memory ratings as measured in a sample survey. Psychol. Aging 4(2), 173–182.
ogg, N., 1998. Free radicals in disease. Semin. Reprod. Endocrinol. 16 (4),241–248.
ones, B.W., Watt, C.B., Frederick, J.M., Baehr, W., Chen, C.K., Levine,E.M., Milam, A.H., Lavail, M.M., Marc, R.E., 2003. Retinal remodelingtriggered by photoreceptor degenerations. J. Comp. Neurol. 464 (1),1–16.
ones, B.W., Watt, C.B., Marc, R.E., 2005. Retinal remodelling. Clin. Exp.Optom. 88 (5), 282–291.
atz, M.L., Robison Jr., W.G., 1986. Evidence of cell loss from the rat retinaduring senescence. Exp. Eye Res. 42 (4), 293–304.
eller, J.N., 2006. Age-related neuropathology, cognitive decline, andAlzheimer’s disease. Ageing Res. Rev. 5 (1), 1–13.
eller, J.N., Gee, J., Ding, Q., 2002. The proteasome in brain aging. AgeingRes. Rev. 1 (2), 279–293.
im, C.B., Tom, B.W., Spear, P.D., 1996. Effects of aging on the densities,numbers, and sizes of retinal ganglion cells in rhesus monkey. Neurobiol.Aging 17 (3), 431–438.
ai, Y.L., Jacoby, R.O., Jonas, A.M., 1978. Age-related and light-associatedretinal changes in Fischer rats. Invest. Phthalmol. Vis. Sci. 17 (7),634–638.
aVail, M.M., Lawson, N.R., 1986. Development of a congenic strain ofpigmented and albino rats for light damage studies. Exp. Eye Res. 43(5), 867–869.
aVail, M.M., Gorrin, G.M., Repaci, M.A., Thomas, L.A., Ginsberg, H.M.,1987. Genetic regulation of light damage to photoreceptors. Invest. Oph-thalmol. Vis. Sci. 28 (7), 1043–1048.
i, Z.Y., Kljavin, I.J., Milam, A.H., 1995. Rod photoreceptor neurite sprout-ing in retinitis pigmentosa. J. Neurosci. 15 (8), 5429–5438.
iets, L.C., Eliasieh, K., van der List, D.A., Chalupa, L.M., 2006. Dendritesof rod bipolar cells sprout in normal aging retina. Proc. Natl. Acad. Sci.U.S.A. 103 (32), 12156–12160.
ossi, L., Cantile, C., Tamagno, I., Merighi, A., 2005. Apoptosis in themammalian CNS: lessons from animal models. Vet. J. 170 (1), 52–66.
asland, R.H., 2001. Neuronal diversity in the retina. Curr. Opin. Neurobiol.11 (4), 431–436.
ilgram, N.W., Head, E., Weiner, E., Thomas, E., 1994. Cognitive func-
tions and aging in the dog: acquisition of nonspatial visual tasks. Behav.Neurosci. 108 (1), 57–68.oreira, P.I., Smith, M.A., Zhu, X., Nunomura, A., Castellani, R.J., Perry,G., 2005. Oxidative stress and neurodegeneration. Ann. N.Y. Acad. Sci.1043, 545–552.
Z
f Aging 30 (2009) 819–828
akajima, Y., Iwakabe, H., Akazawa, C., Nawa, H., Shigemoto, R., Mizuno,N., Nakanishi, S., 1993. Molecular characterization of a novel retinalmetabotropic glutamate receptor mGluR6 with a high agonist selec-tivity for l-2-amino-4-phosphonobutyrate. J. Biol. Chem. 268 (16),11868–11873.
omura, A., Shigemoto, R., Nakamura, Y., Okamoto, N., Mizuno, N.,Nakanishi, S., 1994. Developmentally regulated post-synaptic localiza-tion of a metabotropic glutamate receptor in rat rod bipolar cells. Cell77 (3), 361–369.
earson, P.M., Schmidt, L.A., Ly-Schroeder, E., Swanson, W.H., 2006. Gan-glion cell loss and age-related visual loss: a cortical pooling analysis.Optom. Vis. Sci. 83 (7), 444–454.
eng, Y.W., Hao, Y., Petters, R.M., Wong, F., 2000. Ectopic synaptogenesisin the mammalian retina caused by rod photoreceptor-specific mutations.Nat. Neurosci. 3 (11), 1121–1127.
app, P.R., Amaral, D.G., 1989. Evidence for task-dependent mem-ory dysfunction in the aged monkey. J. Neurosci. 9 (10), 3568–3576.
mall, S.A., 2001. Age-related memory decline: current concepts and futuredirections. Arch. Neurol. 58 (3), 360–364.
trettoi, E., Pignatelli, V., 2000. Modifications of retinal neurons in a mousemodel of retinitis pigmentosa. Proc. Natl. Acad. Sci. U.S.A. 97 (20),11020–11025.
trettoi, E., Porciatti, V., Falsini, B., Pignatelli, V., Rossi, C., 2002. Mor-phological and functional abnormalities in the inner retina of the rd/rdmouse. J. Neurosci. 22 (13), 5492–5504.
trettoi, E., Pignatelli, V., Rossi, C., Porciatti, V., Falsini, B., 2003. Remod-eling of second-order neurons in the retina of rd/rd mutant mice. Vis.Res. 43 (8), 867–877.
erzibasi, et al., 2003. Proceedings of the Fourth FENS Forum, Lisbon,Portugal, July 10–14.
om Dieck, S., Altrock, W.D., Kessels, M.M., Qualmann, B., Regus,H., Brauner, D., Fejtova, A., Bracko, O., Gundelfinger, E.D., Brand-statter, J.H., 2005. Molecular dissection of the photoreceptor ribbonsynapse: physical interaction of Bassoon and RIBEYE is essentialfor the assembly of the ribbon complex. J. Cell Biol. 168 (5), 825–836.
rachimowicz, R.A., Fisher, L.J., Hinds, J.W., 1981. Preservation of reti-nal structure in aged pigmented mice. Neurobiol. Aging 2 (2), 133–141.
rillo, L., Gonzalo, L.M., 1992. Ageing of the human entorhinal cortex andsubicular complex. Histol. Histopathol. 7 (1), 17–22.
ritschler, H.J., Packer, L., Medori, R., 1994. Oxidative stress and mitochon-drial dysfunction in neurodegeneration. Biochem. Mol. Biol. Int. 34 (1),169–181.
rano, S., Sato, Y., Otonari, T., Makabe, S., Suzuki, S., Ogata, M., Endo,T., 1998. Aging and oxidative stress in neurodegeneration. Biofactors 7(1–2), 103–112.
ardi, N., Morigiwa, K., 1997. ON-cone bipolar cells in rat expressthe metabotropic receptor mGluR6. Vis. Neurosci. 14 (4), 789–794.
ang, S., Lu, B., Lund, R.D., 2005. Morphological changes in theroyal college of surgeons rat retina during photoreceptor degenera-tion and after cell-based therapy. J. Comp. Neurol. 491 (4), 400–417.
eisse, I., Loosen, H., Peil, H., 1990. Age-related retinalchanges—comparison between albino and pigmented rats. LensEye Toxic Res. 7 (3–4), 717–739.
illiams, L.R., 1995. Oxidative stress, age-related neurodegeneration, andthe potential for neurotrophic treatment. Cerebrovasc. Brain Metab. Rev.
silla, G., Zelles, T., Mike, A., Kekes-Szabo, A., Milusheva, E., Vizi,E.S., 1994. Differential changes in pre-synaptic modulation of trans-mitter release during aging. Int. J. Dev. Neurosci. 12 (2), 107–115.
