Takeki Kamiyama

Shigeko Shinohara

ROLE OF PROSODY IN SEGMENTAL PERCEPTION IN L2

Keywords: Prosody, L2 acquisition, Japanese, French

This paper focuses on the role of prosody in segmental perception of CCV cluster vs. CVCV in French by Japanese speakers learning French. We studied Japanese speakers' perception of minimal pair words in French of the type /blt/ vs. /bult/ embedded in different intonation contours, with higher or lower F0 on the cluster. Results indicate that tonal prominence facilitates the perception of segmental sequences in L2.

INTRODUCTION

In the very early stage of L1 acquisition, infants seem to have the predisposition to be sensitive to the prosodic cues to phrase boundary. Previous studies report a heavy reliance on the stress patterns for word segmentation in the early stage of lexical acquisition (newborn to 7.5month) ( [START_REF] Christophe | Do infants perceive word boundaries? An empirical study of the bootstrapping of lexical acquisition[END_REF] for French; [START_REF] Jusczyk | The beginnings of word segmentation in English-learning infants[END_REF] for English), which moves towards multiple strategies within a few months [START_REF] Jusczyk | The beginnings of word segmentation in English-learning infants[END_REF]. In the production of young children, consonant clusters are mostly resolved through consonant deletion [START_REF] Bernhardt | Handbook of Phonological Development (From the Perspective of Constrain-Based Nonlinear Phonology)[END_REF]: the result is that prosodic patterns remain more or less intact as compared to the adult target. In contrast, it is shown in recent loanword studies that consonant clusters are overwhelmingly resolved through vowel epenthesis rather than through consonant deletion [8][10]. This pattern seems to be also reflected in adult production of L2 for Japanese speakers [START_REF] Shinohara | Prononciation du français par des locutrices japonaises -le cas de l'insertion de[END_REF]. A consequence of vowel epenthesis is a distortion of prosody in the target language, the number of nuclei being increased. We wonder if this production pattern reflects adult speakers' insensitivity toward prosodic information in L2. In this study we would like to test if prosodic information is still exploited in access to segmental information in L2 speakers of French.

BACKGROUD

In French, the fall in fundamental frequency (F0) at the end of affirmative sentences tends to extend to the last three syllables. [START_REF] Vaissière | Cross-linguistic prosodic transcription: French versus English. Problems and methods in experimental phonetics[END_REF] and [START_REF] Vaissière | La Phonétique[END_REF] characterize the tonal patterns of affirmative and yes-no question sentences as follows. If the last lexical word contains less than three syllables and is preceded by a function word, which tends to be pronounced low, the beginning of that lexical word can have an optional local pitch peak. In the case of total questions, F0 stays relatively flat until the last syllable, where a sharp rise occurs. Following these observations, given that the first syllable of disyllabic words can be produced with a higher pitch than the following one in affirmatives and not in total questions, we hypothesize that the higher pitch, presumably more prominent prosodic patterns, in affirmatives facilitate the perception of segmental sequences.

We undertook a series of experiments. We chose minimal pair words in French differing only by the presence or absence of a vowel, and which are adapted into Japanese as words containing the same segments, but one requires a vowel epenthesis because Japanese does not allow complex onset (e.g. boulette /bult/ 'small ball' > /buretto/, and blette /blt/ 'beet' > /buretto/; the epenthetic vowel is marked by italics) [START_REF] Shinohara | Prononciation du français par des locutrices japonaises -le cas de l'insertion de[END_REF]. As to the perception of clusters, Japanese speakers are known to perceive an illusory epenthetic vowel (e.g. /ebzo/ perceived as /ebuzo/ [START_REF] Dupoux | Epenthetic vowels in Japanese: A perceptual illusion?[END_REF]). Therefore, we can suppose that they have difficulties differentiating these minimal pairs. However, this task might be facilitated by the prosodic prominence in affirmatives. In Experiment 1, we recorded the minimal pair words in the two types of intonation in order to confirm the reported patterns, and in Experiments 2 and 3, these recordings were used for auditory perception tests with native speakers of Japanese learning French as a second language.

EXPERIMENT 1: PRODUCTION BY NATIVE SPEAKERS OF FRENCH

Methods

The corpus consists of 28 words (14 pairs) of CCV-and CVCV-types embedded in 68 sentences. 2 female native speakers of French read the sentences 3 times, which were recorded on a Minidisc recorder in a semi sound-proof room and converted to WAVE files with a sampling rate of 22050 Hz, 16 bits.

Analysis

F0 was measured on Praat first to observe the general patterns, secondly for a more systematic comparison between the mono-and disyllabic words by normalizing F0 on the time axis.

Results

The 2 speakers showed similar F0 patterns in the interrogatives, but not in the affirmatives in particular with disyllabic words.

Concerning the affirmatives, Speaker 1 tended to create a peak during the first syllable of the disyllabic target words before a fall, which was not observed in monosyllabic words, while Speaker 2 did not realize this peak clearly. As for the questions, both speakers showed a flat F0 pattern followed by a sharp rise during the last syllable of the sentence.

Figure 1 shows the F0 patterns of 32 sentences produced by Speaker 1. F0 (mean) during the vowel preceding the target word does not vary much from mono-to di-syllabic words (190 and 199 Hz for the affirmatives, 205 and 217 Hz for the questions). Concerning the target word in the affirmatives, it rises to 227 Hz at the beginning of the first vowel, and then falls to 186 Hz at the beginning of the second one, which is quite similar to that of the first vowel in monosyllabic words (188 Hz). As for the interrogatives, F0 rises at the beginning of monosyllabic words (285 Hz) and at the beginning of the second vowel of disyllabic words (264 Hz), while it stays flat (slightly falling) during the first syllable of disyllabic words. To sum up, these findings seem to confirm, overall, the expected tendencies: 1) the questions show a flat F0 pattern followed by a sharp rise during the last syllable of the sentence; 2) for the affirmatives, only one of the two speakers had expected F0 rise on the first syllable. Thus, besides the expected patterns, speaker difference was also observed.

EXPERIMENT 2: PERCEPTION BY JAPANESE-SPEAKING LEARNERS I

In our second experiment, native speakers of Japanese learning French as a second language listened to the sentences and tried to identify the target word. The aim is to examine the Japanese learners' ability to distinguish the two types of words (CCV and CVCV) in relation to the prosodic pattern of the sentence.

Methods

We used 48 out of the 68 sentences read by the 2 speakers in Experiment 1. The task was to listen to a sentence and decide which of the minimal pair words they had heard. The words were presented in French orthography on an answer sheet.

9 Japanese speakers living in Paris participated. They had lived there for 2 to 8 months at the time of the experiment. They were instructed to circle the word and the confidence rating corresponding to their perception. The subjects listened to the list of stimulus sentences pronounced by Speaker 1 once, and to those by Speaker 2 once after a break.

Results

The results are shown in Figure 2. First, the overall error rate was higher for the stimuli pronounced by Speaker 2. Recall that she did not make a clear peak in the affirmatives with disyllabic words. Secondly, there was little difference between the interrogatives produced by the two speakers, while the affirmatives lead to a higher error rate for Speaker 2 (significant difference for CVCV: t 22 = 2.53, p = 0.02). Thirdly, the error rate is higher for CVCV words (significant effect for both speakers: F (1, 44) = 4.38, p = 0.04, F (1, 44) = 7.38, p = 0.01, respectively), and this tendency is particularly clear for Speaker 1's interrogatives. These results suggest the following: when the test sentences are better articulated, the difference between the affirmatives and the interrogatives is clearly obtained. However, it is not clear if the tendency is entirely due to the prosodic differences, or also to some other factors such as hypo/hyper articulation.

EXPERIMENT 3: PERCEPTION BY JAPANESE-SPEAKING LEARNERS II

For this reason, we conducted another experiment with the same listeners, using stimulus sentences differing solely in f0 patterns of the last word.

Methods

affirmative sentences (8 minimal pairs)

pronounced by Speaker 1 were re-synthesized with a simplified f0 curve on Praat. Then f0 was manipulated so that each sentence would have two different f0 patterns during the last word, namely, one with a peak higher than the preceding function word, the other with a lower peak resulting in a continuous fall (see Figure 3).

The procedures were similar to those of Experiment 2, except that the list of stimuli was presented twice in different orders.

Results

Figure 4 shows the error rate and a score calculated on the basis of errors and confidence rating (if all subjects answered correctly with a top confidence rating, the score will be 5 x 9 listeners = 45). Even though there is no significant difference, CVCV words tend to be difficult to identify if pronounced with a low f0 pattern. This tendency corresponds to the difference found between the stimuli of the two speakers in Experiment 2.

To sum up the results of these two perceptual experiments, the tonal salience in the affirmatives seem to aid, as expected, the Japanese learners to differentiate the two types of segment sequences.

DISCUSSION

The findings in Experiment 1 indicate that the first syllable of disyllabic words preceded by a function word at the end of affirmative sentences tends to be pronounced with a higher F0 than that of the beginning of monosyllabic words. However, the realization of this tendency seems to depend on the strategy and the style adopted by the speaker: it was observed clearly in the recordings of Speaker 1, but not in those of Speaker 2. Speaker 1, who is a professional speech therapist, tended to hyperarticulate, while Speaker 2 did not.

The results in Experiment 2 could be explained better by taking into account this inter-speaker difference reflected in the stimuli. Concerning the higher error rate for CVCV words in questions produced by Speaker 1, we could suppose that CVCV tends to be perceived as CCV in the questions more than in the affirmatives. This is probably because the first syllable in the CVCV words in the total questions, which tends to be pronounced with little f0 movement approximately at the same f0 range as the preceding function word, is less easily perceived as a distinct syllable than that in affirmatives. Thus, the F0 rise in affirmative sentences might have facilitated the perception of the first syllable of the disyllabic words.

However, as for the sentences produced by Speaker 2, the error rate of CVCV syllables in the affirmatives is almost as high as the interrogatives, suggesting that the first syllable in CVCV words in affirmatives, which is prosodically less prominent for Speaker 2 than for Speaker 1, did not help the listeners perceive this as a distinct syllable.

What is curious about the results is that the overall error rate is higher for CVCV words than for CCV words, implying that the learners perceived a cluster where there was a vowel between the two consonants more often than they perceived an inserted vowel where there was a cluster. This might be due to the learner's perceptual hypercorrection, but it seems to contradict earlier findings in which Japanese listeners perceived an inserted vowel in clusters [START_REF] Dupoux | Epenthetic vowels in Japanese: A perceptual illusion?[END_REF]. Further investigation is needed to clarify this point.

Regarding the final curious result, another interpretation concerns the prominence related to duration. Segmental distinctiveness can be enhanced by prosodically prominent position; and there may be more than one type of prosodic prominence. Durational prominence can be independent of tonal prominence biasing the perception in another dimension. While accentuation in French is a controversial issue in French phonology [START_REF] Dell | L'accentuation dans des phrases en français[END_REF][3], it is generally agreed that the phrase final syllables have longer duration than preceding syllables [START_REF] Delattre | Accent de mot et accent de groupe[END_REF] [START_REF] Vaissière | Cross-linguistic prosodic transcription: French versus English. Problems and methods in experimental phonetics[END_REF]. It might be the case that even though the first syllable of the disyllabic words have distinctive roles, the salience of those syllables are inferior to the phrase final ones so that their existence is confused with zero (=CCV sequence).

CONCLUDING REMARKS

The findings in the present study seem to indicate that tonal prominence facilitates the perception of segmental sequences in L2 perception, suggesting the importance of prosody in L2 phonetics.

We have discussed the role of prosody in relation to segments in L1 and L2 acquisitions. Assuming that prosody has a very important role in the early stage of L1 acquisition and some role in L2 acquisition, the next questions to ask are: 1) How much impact does the prosody have in L2 segmental acquisition; 2) If it turns out that the weight of prosody in L1 is still greater than that in L2, when and how does it decrease; in other words, how does L1 acquisition grammar weighing on prosody change to adult grammar.

Figure 1 :

 1 Figure 1: Mean F0 of the affirmatives (left) and the interrogatives (right) with mono-(dashed lines) and disyllabic words (plain lines) pronounced by Speaker 1 (16 sentences each). The first value ("prec. vowel mean") indicates the mean F0 of the vowel preceding the target word. The time axis is normalized, i.e. 0% and 100% points correspond to the beginning and the end of the vowel. Error bar: 1 standard deviation (SD).

Figure 2 :

 2 Figure 2:Error rate for stimuli pronounced by Speakers 1 (left) and 2 (right). "affirm" stands for affirmatives, "interro" for interrogatives, "CCV" for CCV words, and CVCV for CVCV words (9 listeners x 48 stimuli for each speaker).

Figure 3 :

 3 Figure 3: Sentence "Il s'agit des boulettes." pronounced by Speaker 1 and resynthesized with a simplified F0 curve (left); with a lowered f0 on the syllable /bu/ in the word /bult/ (right).

Figure 4 :

 4 Figure 4: Error rate (left) and score calculated on the basis of errors and confidence rating (right: -45 to 45). 9 listeners x 32 stimuli x 2 repetitions.


M.D Sarit Y Lesnik Oberstein
email: s.y.lesnikoberstein@amc.uva.nl

Ph.D Geoffrey P Lewis

Ph.D Thomas Dutra

Ph.D Steven K Fisher

Sarit Y Lesnik Oberstein

Evidence that neurites in human epiretinal membranes express melanopsin, calretinin, rod opsin and neurofilament protein

Keywords: Neurite, epiretinal membrane, rod opsin, melanopsin, calretinin, Müller cell

Aims: Previously we have identified neurofilament protein containing neurites in human epiretinal membranes (ERMs). The goal of this study was to further characterise these neurites by examining the expression of additional specific proteins in human ERMs and correlate this expression with various retinal disease conditions. Methods: Epiretinal membranes originating from 43 patients with proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR), or with no known pathology (idiopathic epiretinal membrane; iERM), were removed during vitrectomy at varying durations after diagnosis and immediately placed in fixative. The membranes were labeled immunohistochemically with different combinations of antibodies to the proteins melanopsin, calretinin, and neurofilament (to identify subclasses of ganglion cells), rod opsin (to identify rod photoreceptors), synaptophysin and SV2 (identifies synaptic vesicles), and vimentin (identifies glial cells).

Results: Anti-melanopsin, -calretinin, -neurofilament, and -rod opsin labeled neurites were routinely observed in the epiretinal membranes. Their presence did not appear to correlate with a specific disease condition or duration of the membrane. Generally neurites were observed in regions of glial cells.

Conclusions:

Based on the expression of selected markers for neurites, we show neurite processes in human ERMs of various aetiologies originating from rod photoreceptors and different populations of retinal ganglion cells although there was no obvious correlation with specific disease condition. Also synaptophysin and SV2 labeling was observed associated with all types of neurites indicating the presence of at least one component necessary for synaptic transmission. Our data suggest that the adult human retina retains a

significant capacity for neuronal remodeling under various disease conditions.

Introduction

The adult retina has long been considered a relatively static neuronal system with little potential for plasticity. However, it is becoming progressively clear that it can undergo remodeling in various injuries and diseases. The classical response includes glial cell proliferation, hypertrophy and migration, as well as neuronal changes including neurite pruning and, surprisingly, neurite sprouting. These events have been shown to occur in retinal degenerations and dystrophies, [START_REF] Strettoi | Modifications of retinal neurons in a mouse model of retinitis pigmentosa[END_REF][START_REF] Jones | Retinal remodeling triggered by photoreceptor degenerations[END_REF][START_REF] Jones | Retinal remodeling during retinal degeneration[END_REF][START_REF] Beltran | A frameshift mutation in RPGR exon ORF15 causes photoreceptor degeneration and inner retina remodeling in a model of X-linked retinitis pigmentosa[END_REF][START_REF] Fei | Cone neurite sprouting: An early onset abnormality of the cone photoreceptors in the retinal degeneration mouse[END_REF][START_REF] Vugler | Survival and remodeling of melanopsin cells during retinal dystrophy[END_REF] diabetes, [START_REF] Meyer-Rüsenberg | Pathological changes in human retinal ganglion cells associated with diabetic and hypertensive retinopathy[END_REF][START_REF] Gastinger | Dendrite remodeling and other abnormalities in the retinal ganglion cells of Ins2Akita diabetic mice[END_REF] retinal detachment (RD), [START_REF] Lewis | Neurite outgrowth from bipolar and horizontal cells after experimental retinal detachment[END_REF][START_REF] Sethi | Glial remodeling and neural plasticity in human retinal detachment with PVR[END_REF][START_REF] Fisher | Müller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences for visual recovery: a review and reconsideration of recent data[END_REF][START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF][START_REF] Wickham | Glial and neural response in short term human retinal detachment[END_REF] macular pucker, [START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF] macular degeneration, [START_REF] Sullivan | Dendritic and synaptic plasticity of neurons in the human age related macular degeneration retina[END_REF] retinitis pigmentosa (RP), [START_REF] Li | Rod photoreceptor neurite sprouting in retinintis pigmentosa[END_REF][START_REF] Milam | Histopathology of the human retina in retinitis pigmentosa[END_REF][START_REF] Fariss | Abnormalities in rod photoreceptors, amacrine cells, and horizontal cells in human retinas with retinitis pigmentosa[END_REF] and in aged retina. [START_REF] Liets | Dendrites of Rod bipolar cells sprout in normal aging retina[END_REF][START_REF] Eliasieh | Cellular reorganisation in the human retina during normal aging[END_REF] The newly generated neurites appear to originate from every class of retinal neuron including rod and cone photoreceptors, bipolar, amacrine and ganglion cells and these neurites often grow throughout the retina. 21,22 (for review) Interestingly, neurite growth does not always stop at the boundaries of the retina.

Once subretinal or epiretinal membranes (ERM) form, regardless of the disease condition, neurites are invariably observed. [START_REF] Sethi | Glial remodeling and neural plasticity in human retinal detachment with PVR[END_REF][START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF][START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF][START_REF] Fisher | Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment[END_REF] ERMs have been shown to contain various cell types including glial cells (Müller cells and astrocytes), retinal pigment epithelial cells, fibroblasts and immune cells [START_REF] Hiscott | Natural history of fibrocellular epiretinal membranes: a quantative, audioradiographic and immunohistochemical study[END_REF][START_REF] Hui | Glial epiretinal membranes and contraction. Immunohistochemical and morphological studies[END_REF][START_REF] Guerin | Immunocytochemical identification of Müller's cell glia as a component of human epiretinal membranes[END_REF] However, neurites are always found associated with Müller cells, suggesting that these cells are permissive to the growth of neurite processes. [START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF][START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF] Neurites have been found in ERMs removed from patients with the retinal conditions proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy (PDR) and RD [START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF] in addition to iERMS removed from eyes with no known retinal injury or disease. [START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF] To date, only anti-neurofilament labeled neurites have been identified in human ERMs. [START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF][START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF] Since neurofilament protein can be expressed by various cell types the goal of this study was to determine more precisely the cells of origin of neurites present in ERMs and correlate their presence with disease condition, using markers to proteins for cell types that have been shown to sprout neurites within the retina. We also used markers to synaptic vesicle proteins to determine if the neurites contained the machinery for synaptic transmission.

We show here that there are distinct neurite populations in the membranes as evidenced by staining with antibodies to the photopigments melanopsin and rod opsin, as well as the calcium binding protein calretinin. In addition, these neurites grow on a bed of glial cells and many contain the synaptic vesicle proteins synaptophysin or SV2.

Materials and Methods

Human ERMs were collected during 3 port vitrectomy surgery from 43 patients with different types of retinal pathologies including PVR (n=14), PDR (n=9), and iERM (n=17). Three ERMs were also from non-insulin dependent diabetics without neovascular proliferation (NIDDM). One of the PDR membranes was large enough to be divided into 4 pieces and was used for 4 antibody combinations, and one divided into 2 pieces for 2 antibody combinations. The membranes were removed from the eyes at varying durations after diagnosis. The average durations were: 5 weeks for PVR, 12 months for PDR, 8 months for iERM, and 6 months for NIDDM. After removal, the membranes were immediately placed in fixative (4% paraformaldehyde in 0.1M sodium cacodylate buffer, pH 7.4; Electron Microscopy Sciences, Fort Washington, PA) and stored at 4 o C until used. The surgery was conducted at the Academic Medical Center, Amsterdam, The Netherlands. All procedures had institutional research ethics committee approval and adhered to the tenets of the Declaration of Helsinki.

The ERMs were processed whole, without embedding or sectioning. Following fixation the tissue samples were rinsed in phosphate buffer (PBS) and incubated in normal donkey serum (1:20) in PBS, 0.5% BSA, 0.1% Triton X-100, and 0.1% azide (=PBTA) overnight at 4 o C on a rotator. The following day various combinations of primary antibodies, in PBTA, were added overnight at 4 o C on a rotator. The antibodies, their concentration, and sources are listed in Table 1. Anti-SV2 was chosen in cases where the combination of primary antibodies did not allow the use of anti-synaptophysin, although both antibodies label synaptic vesicles in the neurite. Following rinsing of the primary antibodies in PBTA, the secondary antibodies conjugated to Cy2, Cy3, or Cy5, were added together overnight at 4 o C on a rotator (1:200 in PBTA). In 2 membranes the primary antibodies were omitted and only the secondary antibodies were added as negative controls. On the final day, the samples were rinsed in PBTA and a Hoechst nuclear stain was added at 1:5000 for 10 min. Without rinsing, the samples were then mounted on glass slides using 5% n-propyl galate in glycerol and viewed on an Olympus FluoView 500 laser scanning confocal microscope (New York, NY). Final images were collected as projections of 5 to 10 images taken at depths of 0.5 micron intervals.

Results

All membranes showed the presence of neurites as identified by various combinations of antibodies to neurofilament protein, melanopsin, calretinin and rod opsin (Table 2). Many of these proteins were double labeled with antibodies to synaptophysin or SV2, which are both normally present in synaptic vesicles.

No obvious correlation was observed with the specific antibody labeling of neurites and the disease condition or duration of the ERM before removal.

Anti-neurofilament (NF) staining was observed in all ERMs and in all disease conditions as reported previously (Table 2). [START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF][START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF] The neurofilament labeling pattern appeared as long, thin processes probably single fibers and thicker structures probably representing clusters of fibers (Fig 1A, C-F), or as bulbous structures (Fig. 1A,C). Anti-synaptophysin labeling of vesicles was seen both in association with these neurofilament labeled processes, as well as in areas without neurofilament labeling (Fig. 1A-D). Anti-NF labeling was only observed in regions that contained anti-vimentin labeled glia cells (Fig. 1E). see also [START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF] Antimicrotuble associated protein 2 (MAP2), a marker for dendrites, [START_REF] Bernhardt | Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons[END_REF] labeled some neurites in all 6 ERMs where it was used including PDR, PVR and iERMs. Many of the MAP2labeled process also labeled with anti-NF (Fig. 1E-G).

Anti-calretinin labeling, typically observed in retinal ganglion cells, amacrine cells, and horizontal cells, [START_REF] Goebel | Calretinin in the cat retina: Colocalisations with other calcium binding proteins, GABA and glycine[END_REF][START_REF] Nag | Developmental expression of calretinin immunoreactivity in the human retina and a comparison with two other EF hand calcium binding proteins[END_REF][START_REF] Linberg | Immunocytochemical evidence that rod connected horizontal cell axon terminals remodel in response to experimental retinal detachment in the cat[END_REF] was not as common as anti-neurofilament labeling nor did it co-localize with neurofilament in the ERMs (data not shown). It was only observed in 3 out of 11 ERMs including one iERM, one NIDDM and one PDR membrane (Table 2; Fig. 2,green). The calretinin labeled processes were always observed on a bed of antivimentin labeled glia (Fig. 2, blue). Anti-SV2 labeling (Fig. 2, red) often co-localised with the anti-calretinin labeled processes. In some cases, the vesicle labeling was only observed in specific regions of the calretinin labeled processes (Fig. 2A,B). In other cases, it was observed throughout the processes appearing much more discrete than in the neurofilament-labeled neurites and often assuming a pattern that appeared vesicular or in association with "beads" along the neurites (Fig. 2C-F).

Anti-melanopsin labeled neurites were observed in 6 out of 8 ERMs including iERM, PDR and PVR membranes (Table 2; Fig. 3, green). Generally, these processes appeared distinct from the anti-neurofilament labeled processes (Fig. 3A). In a few cases, however, there was some faint labeling of melanopsin positive neurites with antineurofilament (Fig. 3B,C; arrows). While anti-SV2 labeling was often observed in the vicinity of melanopsin labeled processes, the two typically did not co-localize (Fig. 3D).

Anti-rod opsin labeled neurites were observed in 3 out of 7 ERMs including iERM, PVR and PDR membranes (Table 2; Fig. 4). These rod neurites were distinct from those neurites labeled with anti-neurofilament (Fig. 4A,B).The rod-opsin positive neurites generally co-localised with anti-synaptophysin labeling which often appeared as small uniform vesicles or in densely labeled areas which could be resolved as sites of heavy accumulation of smaller vesicles (Fig. 4C-E). The anti-rod opsin labeled neurites were often, but not always associated with anti-vimentin labeled glia (e.g. Fig. 4B).

Several of the membranes were double labeled with both anti-vimentin and anti-GFAP and we observed co-localization of the two proteins, indicating that the vimentin labeling represents glial cell labeling and not dedifferentiated RPE cells (data not shown).

Due to the restrictions of antibody combinations only vimentin was used to label the membranes in combination with the neuronal markers. Anti-vimentin labeling of glia was observed in all membranes where the antibody was used. As retinal astrocytes in general do not label with vimentin antibodies, and Müller cells label with both vimentin and GFAP antibodies, [START_REF] Lewis | Changes in the organization and expression of cytoskeletal proteins during retinal degeneration induced by retinal detachment[END_REF][START_REF] Lewis | Up-regulation of glial fibrillary acidic protein in response to retinal injury: its potential role in glial remodeling and a comparison to vimentin expression[END_REF] it is assumed that the glia labeled in the membranes are primarily Müller cells.

Discussion

ERMs can form in eyes with no known pathology, as well as in eyes with severe retinal disruption such as RD or diabetes. Our results show that regardless of the disease condition or length of time the membrane is present in the eye, neurites are invariably observed in all membranes. Indeed, the interval between diagnosis and removal ranged from 5 weeks (PVR),to 8-12 months (iERM and PDR), and there was no apparent difference in the type of neurites or the extent of their growth. This suggests that neurite growth begins almost immediately upon formation of any ERM, following Müller cell processes, the preferred substrate for neurite growth, as they grow onto the retinal surface. The early appearance of neurites in these ERMs agrees with our studies of RD in animal models and in human ERMs where neurite sprouting occurs rapidly. [START_REF] Lewis | Neurite outgrowth from bipolar and horizontal cells after experimental retinal detachment[END_REF][START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF][START_REF] Wickham | Glial and neural response in short term human retinal detachment[END_REF][START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF][START_REF] Coblentz | Evidence that ganglion cells react to retinal detachment[END_REF] In addition to neurofilament labeled neurites, we show neurites that are positive for melanopsin, calretinin and rod opsin. Many of these neurites also show labeling for the synaptic vesicle markers, synaptophysin or SV2.

Neurofilament protein is a cytoskeletal protein that is expressed in the dendrites and axons of most ganglion cells in the human retina. The expression of this protein is upregulated rapidly in a subset of ganglion cells within a few days after experimental RD of the feline retina. [START_REF] Fisher | Müller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences for visual recovery: a review and reconsideration of recent data[END_REF][START_REF] Coblentz | Evidence that ganglion cells react to retinal detachment[END_REF] Within a week of RD in animal models, fine processes can be seen extending from the cell body and later growing out of the retina into ERMs. This appears to be a common phenomenon in human retina too, since neurofilament labeled neurites were observed in 100% of the ERMs examined both in this and a previous study. [START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF] While it is not clear if these most closely resemble axons or dendrites, we did observe co-labeling with MAP2 in some neurites suggesting that these may form a heterogeneous mixture of processes that are dendritic and axon-like.

Calretinin is a calcium binding protein found in the cytosol of amacrine and ganglion cells in the ganglion cell layer, and amacrine and horizontal cells in the inner nuclear layer in the human retina. [START_REF] Goebel | Calretinin in the cat retina: Colocalisations with other calcium binding proteins, GABA and glycine[END_REF][START_REF] Nag | Developmental expression of calretinin immunoreactivity in the human retina and a comparison with two other EF hand calcium binding proteins[END_REF][START_REF] Linberg | Immunocytochemical evidence that rod connected horizontal cell axon terminals remodel in response to experimental retinal detachment in the cat[END_REF] Importantly, the neurofilament protein antibody used in our study does not label horizontal or amacrine cells in the human retina. [START_REF] Sethi | Glial remodeling and neural plasticity in human retinal detachment with PVR[END_REF] In addition, calretinin did not label the same processes as the neurofilament antibody in the ERMs, again suggesting that the neurites arise from a heterogeneous collection of neurons within the retina. Calretinin labeled processes were observed less frequently than neurofilament labeled processes and since there is no evidence for neurites growing into ERMs from amacrine cells in other studies, these data, along with that detailed above, suggest that the origin of the anti-calretinin labeled processes is most likely from a different sub-class of ganglion cell. Interestingly, the calretinin labeled neurites were consistently co-labeled with the synaptic vesicle protein, SV2.

Melanopsin is a photopigment expressed by the intrinsically photosensitive retinal ganglion cell (ipRGC) as first described by Provencio et al. [START_REF] Provencio | Melanopsin: an opsin in melanophores, brain and eye[END_REF][START_REF] Provencio | A novel human opsin in the inner retina[END_REF] The ipRGCs form part of the retino-hypothalamic tract [START_REF] Gooley | Melanopsin in cells of origin of the retinohypothalamic tract[END_REF] and are involved in regulation of the circadian rhythm [START_REF] Berson | Phototransduction by retinal ganglion cells that set the circadian clock[END_REF][START_REF] Hannibal | The photopigment melanopsin is exclusively present in pituitary adenylate cyclise-activating polypeptide containing retinal ganglion cells of the retinohypothalamic tract[END_REF] and photoentrainment. [START_REF] Rollag | Melanopsin, ganglion cell photoreceptors, and mammalian photoentrainment[END_REF] The ipRGC expresses melanopsin in both dendrites and axons. [START_REF] Hattar | Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity[END_REF] In our study the frequency of ipRGC neurites in the ERMs was relatively high despite the fact that these ganglion cells form a small minority of all the ganglion cell population. [START_REF] Berson | Phototransduction by retinal ganglion cells that set the circadian clock[END_REF][START_REF] Hattar | Melanopsin-containing retinal ganglion cells: architecture, projections, and intrinsic photosensitivity[END_REF] These neurites are clearly distinct from the neurofilament and calretinin labeled neurites.

Although some of the melanopsin labeled neurites did show faint co-localisation with neurofilament, these processes were obviously different from those labeled brightly with the neurofilament antibody. It is possible that these double labeled neurites may be another subset of ipRGCs since two subpopulations of ipRGCs have been identified in mice. [START_REF] Baver | Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus[END_REF][START_REF] Schmidt | Functional and morphological differences among intrinsically photosensitive retinal ganglion cells[END_REF] Our study confirms that of Vugler et al., [START_REF] Vugler | Survival and remodeling of melanopsin cells during retinal dystrophy[END_REF] using the RCS rat with retinal dystrophy, demonstrating that ipRGCs remodel during degeneration. Finally, while the ipRGCs have been shown to have synaptic vesicles at the junctions with amacrine and bipolar cells in the mouse retina, [START_REF] Belenky | Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses[END_REF] they do not appear to label with anti-SV2 in the ERMs.

Rod neurite sprouting was first described in vivo in the disease RP in human donor retinas where long intra retinal rod opsin positive neurites were found. [START_REF] Li | Rod photoreceptor neurite sprouting in retinintis pigmentosa[END_REF] Since then rod neurite sprouting has been described within the retinal layers in different forms of retinal dystrophy in animals models and humans. [START_REF] Jones | Retinal remodeling triggered by photoreceptor degenerations[END_REF][START_REF] Jones | Retinal remodeling during retinal degeneration[END_REF][START_REF] Beltran | A frameshift mutation in RPGR exon ORF15 causes photoreceptor degeneration and inner retina remodeling in a model of X-linked retinitis pigmentosa[END_REF][START_REF] Wickham | Glial and neural response in short term human retinal detachment[END_REF][START_REF] Fariss | Abnormalities in rod photoreceptors, amacrine cells, and horizontal cells in human retinas with retinitis pigmentosa[END_REF][START_REF] Marc | Neural remodeling in retinal degeneration[END_REF][START_REF] Fisher | Cellular remodeling in mammalian retina: results from studies of experimental retinal detachment[END_REF][START_REF] Lewis | Experimental retinal reattachment: a new perspective[END_REF] These studies suggest that rod axons terminate within the inner retina as they do during development. [START_REF] Johnson | Rods and cones project to the inner plexiform layer during development[END_REF] We show here that these neurites can continue their growth outside the retina into an ERM. The fact that rod neurites grow the entire thickness of the retina and into iERMs suggests that there need not be significant damage to photoreceptors to stimulate growth. Moreover, since vision can often return to normal after iERM removal it would appear that the extraordinary growth of rod axons has minimal effect on vision. This may not be surprising since changes in small populations of rod photoreceptors probably would not affect typical scotopic visual function. Rod opsin labeling was not observed co-localized with neurofilament since these proteins do not overlap in the retina. However, we did observe synaptophysin to be co-localised to rod neurites, an observation congruent with the presence of synaptophysin labeling in elongated rod neurites within the retina in a feline model of RD, [START_REF] Lewis | Experimental retinal reattachment: a new perspective[END_REF] the canine RP model and in human RP. [START_REF] Beltran | A frameshift mutation in RPGR exon ORF15 causes photoreceptor degeneration and inner retina remodeling in a model of X-linked retinitis pigmentosa[END_REF] , [START_REF] Li | Rod photoreceptor neurite sprouting in retinintis pigmentosa[END_REF][START_REF] Milam | Histopathology of the human retina in retinitis pigmentosa[END_REF][START_REF] Fariss | Abnormalities in rod photoreceptors, amacrine cells, and horizontal cells in human retinas with retinitis pigmentosa[END_REF] There is no evidence that these neurites form functional synaptic connections.

The presence of neurites is universal in ERMs, reinforcing the concept that significant disturbance of the retina is not necessary to induce the remodeling of both rod photoreceptors and ganglion cells. While it is not known what initiates neurite growth, it appears that molecules in the membranes, and in particular associated with Müller cells, may act as an attractant or stimulus for their outgrowth. Indeed, neurites in various pathologies have been shown to grow preferentially on a substrate of Müller cell glia,one of the main cellular components of ERMs. [START_REF] Fisher | Müller cell and neuronal remodeling in retinal detachment and reattachment and their potential consequences for visual recovery: a review and reconsideration of recent data[END_REF][START_REF] Lewis | Identification of ganglion cell neurites in human subretinal and epiretinal membranes[END_REF][START_REF] Oberstein | Ganglion cell neurites in human idiopathic epiretinal membranes[END_REF][START_REF] Jerdan | Proliferative vitreoretinopathy membranes. An immunohistochemical study[END_REF][START_REF] Morino | Variation in epiretinal membrane components with clinical duration of proliferative tissue[END_REF][START_REF] Vinores | Ultrastructural and electronimmunocytochemical characterization of cells in epiretinal membranes[END_REF][START_REF] Heidenkummer | Proliferative activity and immunohistochemical cell differentiation in human epiretinal membranes[END_REF][START_REF] Heidenkummer | Proliferative activity in epiretinal membranes. The use of the monoclonal antibody Ki-67 in proliferative vitreoretinal diseases[END_REF] Although the specific protein(s) that instigates neural growth and creates the permissive environment is unknown, it has been shown that human idiopathic and diabetic ERMs express neural growth factors and receptors, such as nerve growth factor (NGF), basic fibroblast growth factor (bFGF) and glial cell line-derived growth factor (GDNF), and the receptors p75 neurotrophin receptor and tyrosine kinase receptors trkA, trkB and trkC [START_REF] Harada | Neurotrophic factor receptors in epiretinal membranes after human diabetic retinopathy[END_REF][START_REF] Minchiotti | Human idiopathic epiretinal membranes express NGF and NGF receptors[END_REF] which might be involved in neurite growth.

Conclusion

It is clear from this and other studies that adult retinal neurons retain the ability to remodel given the appropriate cues. Here we demonstrate the presence of neurites derived from rod photoreceptors as well as three sub-types of retinal ganglion cell in ERMs. This growth appears unrelated to the particular disease condition since neurites were observed in all conditions examined, irrespective of the disease being severely disruptive to the retina, as in PVR and PDR, or minimally disruptive as in iERM. Future studies are aimed at identifying factors that may be involved in initiating and guiding this neurite growth.

Licence for Publication "The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in BJO editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence(http://group.bmj.com/products/journals/instructions-for-authors/licenceforms/)." Table 1. Antibodies used for the study along with their source and concentrations. Table 2. The antibody combinations used on different membrane types and whether the labeling was present (+) or absent (-). ("+/-" = positive but light staining.) (green), anti-SV2 (red), and anti-vimentin (blue). Anti-calretinin labeled processes can appear tortuous (A,B) or beaded (C-F) but invariably contain at least some anti-SV2 labeling. Figures "B" and "D" are the same as "A" and "C" with the green channel turned off. Figure "F" is the same as "E" with the red channel turned off. A&B are from PDR;

Figure 1 .

 1 Figure 1. Confocal micrographs of epiretinal membranes labeled with anti-neurofilament

Figure 2 .

 2 Figure 2. Confocal micrographs of epiretinal membranes labeled with anti-calretinin