Residual Cones Research Articles

Cones and cone pathways remain functional in advanced retinal degeneration

Most defects causing retinal degeneration in retinitis pigmentosa (RP) are rod-specific mutations, but the subsequent degeneration of cones, which produces loss of daylight vision and high-acuity perception, is the most debilitating feature of the disease. To understand better why cones degenerate and how cone vision might be restored, we have made the first single-cell recordings of light responses from degenerating conesand retinal interneurons after most rods have died and cones have lost their outer-segment disk membranes and synaptic pedicles. We show that degenerating cones have functional cyclic-nucleotide-gated channels and can continue to give light responses, apparently produced by opsin localized either to small areas of organized membrane near the ciliary axoneme or distributed throughout the inner segment. Light responses of second-order horizontal and bipolar cells are less sensitive but otherwise resemble those of normal retina. Furthermore, retinal output as reflected in responses of ganglion cells is less sensitive but maintains spatiotemporal receptive fields at cone-mediated light levels. Together, these findings show that cones and their retinal pathways can remain functional even as degeneration is progressing, an encouraging result for future research aimed at enhancing the light sensitivity of residual cones to restore vision in patients with genetically inherited retinal degeneration.

Optogenetic approaches to therapy for inherited retinal degenerations.

Inherited retinal degenerations such as retinitis pigmentosa (RP) affect around one in 4000 people and are the leading cause of blindness in working age adults in several countries. In these typically monogenic conditions, there is progressive degeneration of photoreceptors; however, inner retinal neurons such as bipolar cells and ganglion cells remain largely structurally intact, even in end-stage disease. Therapeutic approaches aiming to stimulate these residual cells, independent of the underlying genetic cause, could potentially restore visual function in patients with advanced vision loss, and benefit many more patients than therapies directed at the specific gene implicated in each disorder. One approach investigated for this purpose is that of optogenetics, a method of neuromodulation that utilises light to activate neurons engineered to ectopically express a light-sensitive protein. Using gene therapy via adeno-associated viral vectors, a range of photosensitive proteins have been expressed in remaining retinal cells in advanced retinal degeneration with in vivo studies demonstrating restoration of visual function. Developing an effective optogenetic strategy requires consideration of multiple factors, including the light-sensitive protein that is used, the vector and method for gene delivery, and the target cell for expression because these in turn may affect the quality of vision that can be restored. Currently, at least four clinical trials are ongoing to investigate optogenetic therapies in patients, with the ultimate aim of reversing visual loss in end-stage disease.

Deep Phenotyping of PDE6C-Associated Achromatopsia.

PurposeTo perform deep phenotyping of subjects with PDE6C achromatopsia and examine disease natural history.MethodsEight subjects with disease-causing variants in PDE6C were assessed in detail, including clinical phenotype, best-corrected visual acuity, fundus autofluorescence, and optical coherence tomography. Six subjects also had confocal and nonconfocal adaptive optics scanning light ophthalmoscopy, axial length, international standard pattern and full-field electroretinography (ERG), short-wavelength flash (S-cone) ERGs, and color vision testing.ResultsAll subjects presented with early-onset nystagmus, decreased best-corrected visual acuity, light sensitivity, and severe color vision loss, and five of them had high myopia. We identified three novel disease-causing variants and provide phenotype data associated with nine variants for the first time. No subjects had foveal hypoplasia or residual ellipsoid zone (EZ) at the foveal center; one had an absent EZ, three had a hyporeflective zone, and four had outer retinal atrophy. The mean width of the central EZ lesion on optical coherence tomography at baseline was 1923 μm. The mean annual increase in EZ lesion size was 48.3 μm. Fundus autofluorescence revealed a central hypoautofluorescence with a surrounding ring of increased signal (n = 5). The mean hypoautofluorescent area at baseline was 3.33 mm2 and increased in size by a mean of 0.13 mm2/year. Nonconfocal adaptive optics scanning light ophthalmoscopy revealed residual foveal cones in only one of two cases. Full-field ERGs were consistent with severe generalized cone system dysfunction but with relative preservation of S-cone sensitivity.ConclusionsPDE6C retinopathy is a severe cone dysfunction syndrome often presenting as typical achromatopsia but without foveal hypoplasia. Myopia and slowly progressive maculopathy are common features. There are few (if any) residual foveal cones for intervention in older adults.

Photoreceptor degeneration in a new Cacna1f mutant mouse model

The Cacna1f gene encodes the α1F subunit of an L-type voltage-gated calcium channel, Cav1.4. In photoreceptor synaptic terminals, Cav1.4 channels mediate glutamate release and postsynaptic responses associated with visual signal transmission. We have discovered a new Cacna1f mutation in nob9 mice, which display more severe phenotypes than do nob2 mice. To characterize the nob9 phenotype at different ages, we examined the murine fundus, applied retinal optical coherence tomography, measured flash electroretinograms (ERGs) in vivo, and analyzed the retinal histology in vitro. After identifying the X-linked recessive inheritance trait, we sequenced Cacna1f as the candidate gene. Mutations in this gene were detected by polymerase chain reaction (PCR) and confirmed by restriction fragment length polymorphism. Morphologically, an early-onset of retinal disorder was detected, and the degeneration of the outer plexiform layers progressed rapidly. Moreover, the mutant mice showed drastically reduced scotopic ERGs with increasing age. In 14-month-old nob9 retinas, immunostaining of cone opsins demonstrated a reduction in the number of short-wavelength opsins (S-opsins) to 54% of wild-type levels, and almost no middle-wavelength opsins (M-opsins) were observed. No cone ERGs could be detected from residual cones, in which S-opsins abnormally migrated to inner segments of the photoreceptors. The mutations of the Cacna1f gene in nob9 mice involved both a single nucleotide G to A transition and a 10-nucleotide insertion, the latter resulting in a frame-shift mutation in exon 14.

Retrograde Melanopsin Signaling Increases With Age in Retinal Degenerate Mice Lacking Rods and the Majority of Cones.

Following on from reports of retrograde retinal signaling in mice, we sought to investigate the influence of age and retinal location on this phenomenon using mice that lack rods and the majority of cones. We used functional anatomy for c-fos (Fos) and tyrosine hydroxylase (TH) to measure light-driven activation of dopamine neurons along a dorsal-ventral transect in C3H/He wild-type and rodless-coneless rd/rd cl (rdcl) mice aged 3, 5, and >14 months. A parallel series of retinae from 3-month-old mice was also stained for cone opsins and melanopsin. Analysis by confocal microscopy revealed light-driven Fos activation in TH cells residing in the middorsal retina of the youngest rdcl mice. This region was largely devoid of residual cones but contained a large number of intrinsically photosensitive retinal ganglion cells (ipRGCs) and the highest density of melanopsin neurites. With advancing age, there was a paradoxical increase in retrograde signaling from ∼3% Fos-positive (Fos+) TH cells at 3 months to ∼36% in rdcl mice >14 months. This increased activation occurred in more central and peripheral retinal regions. Our data provide new insights into the anatomy and plasticity of retrograde melanopsin signaling in mice with severe rod/cone dystrophy. The increased retrograde signaling we detect may result from either an increased potency of melanopsin signaling with advancing age and/or postsynaptic modification to dopaminergic neurons.

Genotype-dependent variability in residual cone structure in achromatopsia: toward developing metrics for assessing cone health.

Gene therapy trials for inherited photoreceptor disorders are planned. Anatomical metrics to select the best candidates and outcomes are needed. Adaptive optics (AO) imaging enables visualization of photoreceptor structure, although analytical tools are lacking. Here we present criteria to assess residual photoreceptor integrity in achromatopsia (ACHM). Two AOSLOs, at the Medical College of Wisconsin and Moorfields Eye Hospital, were used to image the photoreceptor mosaic of 11 subjects with ACHM and 7 age-matched controls. Images were obtained, processed, and montaged using previously described methods. Cone density and reflectivity were quantified to assess residual cone photoreceptor structure. All subjects with ACHM had reduced numbers of cone photoreceptors, albeit to a variable degree. In addition, the relative cone reflectivity varied greatly. Interestingly, subjects with GNAT2-associated ACHM had the greatest number of residual cones and the reflectivity of those cones was significantly greater than that of the cones in the subjects with CNGA3/CNGB3-associated ACHM. We present cone reflectivity as a metric that can be used to characterize cone structure in ACHM. This method may be applicable to subjects with other cone disorders. In ACHM, we hypothesize that cone numerosity (and/or density) combined with cone reflectivity could be used to gauge the therapeutic potential. As gene replacement would not be expected to add cones, reflectivity could be a more powerful AO-metric for monitoring the cellular response to treatment and could provide a more immediate indicator of efficacy than behavioral measures, which may take longer to change.

TULP1 mutations causing early-onset retinal degeneration: preserved but insensitive macular cones.

To investigate visual function and outer and inner retinal structure in the rare form of retinal degeneration (RD) caused by TULP1 (tubby-like protein 1) mutations. Retinal degeneration patients with TULP1 mutations (n = 5; age range, 5-36 years) were studied by kinetic and chromatic static perimetry, en face autofluorescence imaging, and spectral-domain optical coherence tomography (OCT) scans. Outer and inner retinal laminar thickness were measured and mapped across the central retina. Comparisons were made with results from patients with RD associated with four ciliopathy genotypes (MAK, RPGR, BBS1, and USH2A). The TULP1-RD patients were severely affected already in the first decade of life and there was rapidly progressive visual loss. No evidence of rod function was present at any age. Small central islands showed melanized retinal pigment epithelium by autofluorescence imaging and well-preserved photoreceptor laminar thickness by OCT imaging. There was extracentral loss of laminar architecture and increased inner retinal thickening. Structure-function relationships in residual foveal cone islands were made in TULP1-RD patients and in other retinopathies considered ciliopathies. Patients with TULP1-RD, unlike the others, had greater dysfunction for the degree of foveal structural preservation. Retinal degeneration with TULP1 mutations leads to a small central island of residual foveal cones at early ages. These cones are less sensitive than expected from the residual structure. The human phenotype is consistent with experimental evidence in the Tulp1 knockout mouse model that visual dysfunction could be complicated by abnormal processes proximal to cone outer segments.

Human Cone Visual Pigment Deletions Spare Sufficient Photoreceptors to Warrant Gene Therapy

Human X-linked blue-cone monochromacy (BCM), a disabling congenital visual disorder of cone photoreceptors, is a candidate disease for gene augmentation therapy. BCM is caused by either mutations in the red (OPN1LW) and green (OPN1MW) cone photoreceptor opsin gene array or large deletions encompassing portions of the gene array and upstream regulatory sequences that would predict a lack of red or green opsin expression. The fate of opsin-deficient cone cells is unknown. We know that rod opsin null mutant mice show rapid postnatal death of rod photoreceptors. Using in vivo histology with high-resolution retinal imaging, we studied a cohort of 20 BCM patients (age range 5-58) with large deletions in the red/green opsin gene array. Already in the first years of life, retinal structure was not normal: there was partial loss of photoreceptors across the central retina. Remaining cone cells had detectable outer segments that were abnormally shortened. Adaptive optics imaging confirmed the existence of inner segments at a spatial density greater than that expected for the residual blue cones. The evidence indicates that human cones in patients with deletions in the red/green opsin gene array can survive in reduced numbers with limited outer segment material, suggesting potential value of gene therapy for BCM.

Identification of a Novel Mutation in the CDHR1 Gene in a Family With Recessive Retinal Degeneration

To describe the clinical phenotype and identify the molecular basis of disease in a consanguineous family of Palestinian origin with autosomal recessive retinal degeneration. Eight family members were evaluated with visual acuity and perimetry tests, color fundus photographs, full-field electroretinography, and optical coherence tomography. Cone photoreceptors surrounding the fovea were imaged in 2 members, using adaptive optics scanning laser ophthalmoscopy. Exome was captured using probes and sequenced. Readings were mapped to reference hg19. Variant calls and annotations were performed, using published protocols. Confirmation of variants and segregation analysis was performed using dideoxy sequencing. Analysis detected 24 037 single-nucleotide variants in one affected family member, of which 3622 were rare and potentially damaging to encoded proteins. Further analysis revealed a novel homozygous nonsense change, c.1381 C>T, p.Gln461X in exon 13 of the CDHR1 gene, which segregated with retinal degeneration in this family. Affected members had night blindness beginning during adolescence with progressive visual acuity and field loss and unmeasurable electroretinographic responses, as well as macular outer retinal loss, although residual cones with increased cone spacing were observed in the youngest individual. Exome analysis revealed a novel CDHR1 nonsense mutation segregating with progressive retinal degeneration causing severe central vision loss by the fourth decade of life. High-resolution retinal imaging revealed outer retinal changes suggesting that CDHR1 is important for normal photoreceptor structure and survival. Exome sequencing is a powerful technique that may identify causative genetic variants in families with autosomal recessive retinal degeneration.

Preservation of Cone Photoreceptors after a Rapid yet Transient Degeneration and Remodeling in Cone-OnlyNrl−/−Mouse Retina

Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor neural retina leucine zipper (NRL). The loss of Nrl (Nrl(-/-)) in mice results in a retina with predominantly S-opsin-containing cones that exhibit molecular and functional characteristics of wild-type cones. Here, we report that Nrl(-/-) retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by 4 months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic electroretinogram. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl(-/-) mouse illustrates the long-term viability of cones in the absence of rods and retinal pigment epithelium defects in a rodless retina. We propose that Nrl(-/-) retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.

Restoration of Cone Vision in the CNGA3−/− Mouse Model of Congenital Complete Lack of Cone Photoreceptor Function

Congenital absence of cone photoreceptor function is associated with strongly impaired daylight vision and loss of color discrimination in human achromatopsia. Here, we introduce viral gene replacement therapy as a potential treatment for this disease in the CNGA3(-/-) mouse model. We show that such therapy can restore cone-specific visual processing in the central nervous system even if cone photoreceptors had been nonfunctional from birth. The restoration of cone vision was assessed at different stages along the visual pathway. Treated CNGA3(-/-) mice were able to generate cone photoreceptor responses and to transfer these signals to bipolar cells. In support, we found morphologically that treated cones expressed regular cyclic nucleotide-gated (CNG) channel complexes and opsins in outer segments, which previously they did not. Moreover, expression of CNGA3 normalized cyclic guanosine monophosphate (cGMP) levels in cones, delayed cone cell death and reduced the inflammatory response of Müller glia cells that is typical of retinal degenerations. Furthermore, ganglion cells from treated, but not from untreated, CNGA3(-/-) mice displayed cone-driven, light-evoked, spiking activity, indicating that signals generated in the outer retina are transmitted to the brain. Finally, we demonstrate that this newly acquired sensory information was translated into cone-mediated, vision-guided behavior.

AO imaging of acute macular diseases

Abstract Purpose To show clinical cases of acute macular diseases and their follow‐up by adaptive optics flood imaging. Methods Cases of acute retinal ischemia, of acute macular neuroretinopathy, of photic injury and of poppers‐related retinopathy have been observed by a prototypic adaptive optics flood imaging (ImagineEye corporation). Images from follow‐up examinations have been registered in order to obtain retinal monitoring at the single photoreceptor level. Iamges were compared to high resolution OCT scans. Results Precise extension and progression/regression of lesions could be documented in all cases. Acute macular neuroretinopathy showed residual cones persisting within an area devoid of any detectable cone. Minute progression and regression of lesions could be documented. Acute ischemia of the inner retina due to central retinal vein occlusion resulted in focal masking of the cone mosaic. The cone mosaic reappeared during follow‐up. Photic injury showed no changes over a 1 year follow‐up. Images of poppers‐related retinopathy showed partial improvement over time. Conclusion Adaptive optics flood imaging allows documentation of the extension and progression of acute maculopathies of various origins.

The influence of physico-chemical material properties on erosion processes in the badlands of Basilicata, Southern Italy

Piping (tunnelling) appears to be one of the more significant methods of erosion in the badlands in the Plio-Pleistocene marine-sourced clays of the Basilicata area in Southern Italy. A detailed field investigation of the pedological, textural, mineralogical and geochemical properties of the clay-rich terrains widely outcropping in two selected badland field sites (the Fossa Bradanica area and the Bacino di Sant'Arcangelo) was undertaken to further elucidate process variability across the complex site. Paralleling findings from other piped badlands, certain physico-chemical properties of the clays were found to influence the different erosional processes in fundamental ways. The very dispersive nature of the materials enhances pipe enlargement so that subsurface flow rapidly becomes the dominant process, causing pipe enlargement by a process of positive feedback. The paper speculates on the progressive role of surface and subsurface processes in the genesis of all evolutional badland forms (calanchi, calanchi mammellonari and biancane). In an early stage, the calanchi mammellonari are produced, as slope mounds become increasingly separated by a dense network of small inclined pipes in the intermediate part of the slopes, a site which is presumed to favour elevated subsurface flow. When the overburden collapses, many cones are isolated in these portions of the slope. A second stage follows in which overland flow dominates, with the water canalizing into small gullies generated by the collapsed crust. At the base of many slopes, this same mechanism produces biancane as residual cones. Because of the structural weakness of piped materials, mass movements cannot be ruled out. In a few locations, landsliding may be involved, especially in the formation of calanchi mammellonari. In this case the intersection of the vertical pipes with the impermeable substratum focuses subsurface flow, and collapse of the surface along this failure plane. Subsequent remodelling occurs by surface processes, since the ‘catchment volume’ for large pipes no longer exists. This interpretation differs from that of others who have argued that in other locations the biancane formation is linked to the development of large vertical pipes along tectonic joints.

Impaired Opsin Targeting and Cone Photoreceptor Migration in the Retina of Mice Lacking the Cyclic Nucleotide-Gated Channel CNGA3

To characterize molecular and cellular changes in the mouse retina caused by the genetic deletion of the cone cyclic nucleotide-gated channel (CNG) subunit CNGA3. Retinas of wild-type and CNGA3-deficient (CNGA3(-/-)) mice from 9 days up to 22 months of age were analyzed by immunohistochemistry, electron microscopy, and molecular biological methods. CNGA3(-/-) cones failed to transport opsins into outer segments, downregulated various proteins of the phototransduction cascade, and induced apoptotic death. Loss of CNGA3 did not affect the transcription of cone-specific genes. Cone degeneration was evident from the second postnatal week on, and it proceeded significantly faster in the ventral than in the dorsal part of the retina. Ventral cones were completely missing after the third postnatal month, whereas residual dorsal cones were present, even in 22-month-old knockout mice. CNGA3(-/-) cone somata exhibited profoundly delayed migration during postnatal development. At the time of eye opening, most CNGA3(-/-) cones had displaced somata localized close to or in the outer plexiform layer. These cones lacked the characteristic synaptic pedicle, but revealed synapselike contacts to second-order neurons at their somata. At later stages, most of the surviving CNGA3(-/-) cones had correctly located somata and morphologically normal synapses. The loss of CNGA3 impairs the targeting of cone opsins and the expression of other visual cascade proteins. In addition, CNGA3 appears to be essential for normal postnatal migration of cone somata. After loss of cone outer segment proteins, CNGA3(-/-) cones induce apoptotic cell death.

Selective transplantation of rods delays cone loss in a retinitis pigmentosa model.

Rod-cone retinal degenerations (retinitis pigmentosa) are typified by initial rod loss followed by secondary cone death. Rod death, predominantly caused by gene mutations expressed specifically in these cells, induces scotopic vision loss. Cone death, the overriding cause of blindness, has no current explanation. Disease progression and preliminary data suggest that cone survival depends on rods. To establish whether rod transplantation into mutant rodless retinas could halt cone loss. We transplanted pure sheets of rods isolated from normal-sighted mice into the subretinal space of recipient retinal degeneration mice lacking rods but possessing approximately 30% residual cones. Control animals were unoperated on or grafted with inner retinal cells from young normal donors, entire retinas from aged retinal degeneration mice, or gelatin. Two weeks after surgery, we quantified by an unbiased method the numbers of host retinal cones after immunolabeling with specific markers. Only mice receiving rod-rich transplants demonstrated statistically significant greater cone numbers, with rescue of 40% of host cones normally destined to die during this period. Cone survival depends specifically on rods. Such findings indicate that transplantation of rods could limit loss of cones, thus preserving useful vision in human retinitis pigmentosa. Arch Ophthalmol. 2000;118:807-811

Sensitivity losses in a long wavelength sensitive mechanism of patients with retinitis pigmentosa

The foveal vision of certain patients afflicted with retinitis pigmentosa may be mediated solely by a long wavelength sensitivity (1ws) mechanism, because (1) increment threshold data could always be fitted to a single template curve with unity slope, (2) test and field sensitivities differ only by a proportionality constant over the visible spectrum, (3) intense background fields have additive (quantum catch) effects on thresholds, and (4) wavelength differences of bipartite foveal stimuli could not be distinguished. Both test and field sensitivities were appreciably lower than the lws sensitivity values for unaffected deuteranopes. The action spectrum was similar (but not identical) to estimates of the erythrolabe absorption spectrum. The results provide a better understanding of the photopic vision of patients with retinitis pigmentosa and further evidence that their effective optical density of residual cones is reduced.

The Powers-Yonkee Bison Trap

Site 24 PR5 lies on the Fred Yonkee ranch in Powder River County, Montana, and was reported by Mrs, Margaret Powers, president of Sheridan Chapter of the Wyoming Archaeological Society. This site has a history of being pot-hunted by artifact collectors for at least 35 years. The Sheridan Chapter, with the kind per mission of the ranch owners, decided to conduct an investigation of this site while there was yet undisturbed soil. The bulk of the site had already been destroyed and hundreds of projectile points taken away. In July and August of 1961 a concrete datum post was placed; a survey made of the immediate and surrounding area; an examination made by two competent geolo gists (Dr. Frank Koucky, Professor of Geology at the University of Cincinnati, and Dr. Wilton Melhorn, Professor of Geology at Purdue University); and a camp set up at the site. Fourteen members worked a total of 39 man-days in exca vating 3 different locations on the site, and approximately 25 cubic yards of material was moved, all by hand labor and a wheelbarrow. Geologically, the site lies on a high terrace (elevation about 3600 feet above sea level) of pleistocene origin and con sisting of residual ridges and cones sup ported by sandstone or clinker beds, (Fig. 1). The present change rate is slow. The last period of heavy precipi tation (Little Ice Age) was probably about 3500 years ago, with heavy gully cutting. Since then there had been increasing dry ness and stabilization of gullies. The terrain consists of Fort Union or Wasatch beds, 40 to 60 million years old, with a contmuing sequence sandstone to shale to coal to limey-bed with fossils, follow ed by a repetition of this same sequence . The present surface is predominately heavy gumbo soil with outcroppings of red shale. Prairie grasses and sagebrush are the predominant vegetation, with Ponderosa pine and Colorado juniper scattered along ridges and arroyos . Mule deer and antelope are plentiful in the area, with considerable numbers of sage grouse, jackrabbits, cottontails, badgers, porcupines, and some raccoons. The country is quite arid, and the tem perature was extremely hot with the sun beating down mercilessly during the ex cavation period. The cultural area lies mostly along the north bank of a small arroyo which drains from east to west, emptying into a larger arroyo which drains to the north into a broad, rather flat valley. It appears that ancient man probably drove the bison from the broad valley or collecting basin into the large, north-south arroyo; then, with the aid of a barricade, shunted them into the smaller, east-west arroyo, which at that time probably had more or less verti cal walls. In the east or upper end of this arroyo, the bison were helplessly trapped, and it was an easy matter for the hunters to slay them at practically point-blank range with their stone-tipped projectiles. The rib cage was apparently the favorite target area on the bison, because the majority of the points were found in asso ciation with rib bones. It was decided to excavate at three differ ent locations on the site where surface evidence gave assurance of the presence of a cultural zone with undisturbed soil. The first location, which is called Site #1, was a small, loaf-shaped ridge lying at the head of the arroyo with a steep gully on either side. Some pot-hunting had been done here and much bone was evident in the disturbed dirt. A vertical profile was cut at right angles through the end of the ridge and in undisturbed soil, (Fig. 2). This revealed a cultural layer averaging