Diurnal variation of c-Fos expression in subdivisions of the dorsal raphe nucleus of the Mongolian gerbil (Meriones unguiculatus).

Recent studies suggest that the dorsal raphe nucleus (DRN) of the brainstem contains several subdivisions that differ both anatomically and neurochemically. The present study examined whether variation of c-Fos expression across the 24-hour light-dark cycle may also be different in these subdivisions. Animals were kept on a 12:12 light-dark cycle, were perfused at seven different time points, and brain sections were processed by using c-Fos immunocytochemistry. At all coronal levels of the DRN, c-Fos expression reached a peak 1 hour after the light-dark transition (lights-off) and reached its lowest levels in the middle of the light period. In contrast to the light-dark transition, c-Fos levels did not change significantly after the dark-light transition (lights-on). One-way analysis of variance (ANOVA) revealed that the diurnal variation of c-Fos expression was highly significant in the caudal ventral DRN. Similar variation in c-Fos expression also was observed in the other DRN subdivisions, but this variation appeared to gradually diminish in the caudal-to-rostral and ventromedial-to-dorsomedial directions. Double-label immunocytochemistry revealed that, 1 hour after lights-off, only 11% of c-Fos-positive neurons in the caudal ventral DRN were serotonin (5-HT)-immunoreactive. These results suggest that DRN subdivisions may differ functionally with regard to the diurnal cycle, and that these differences may be reflected in the activity of nonserotonergic cells in the DRN.

GABAergic visual pathways in the frog Rana pipiens.

Gamma-aminobutyric acid (GABA) is the most prevalent inhibitory neurotransmitter in the vertebrate brain. It can exert its influence either as GABAergic projection pathways or as local interneurons, which play an essential role in many visual functions. However, no GABAergic visual pathways have been studied in frogs so far. In the present study, GABAergic pathways in the central visual system of Rana pipiens were investigated with double-labeling techniques, combining immunocytochemistry for GABA with Rhodamine microspheres for retrograde tracing. Three GABAergic visual pathways were identified: (1) a retino-tectal projection, from retina to the contralateral optic tectum (OT); (2) an ipsilateral projection from the nucleus of the basal optic root (nBOR) to the pretectal nucleus lentiformis mesencephali (nLM); and (3) a second-order pathway from the nucleus isthmi (NI), bilaterally, to the optic tectum. These results indicate that GABA is involved in both first-order (retina to optic tectum) as well as second-order (nucleus isthmi to optic tectum) visual projections in Rana pipiens, and may play a major role in mediating visuomotor reflexs such as optokinetic nystagmus or other visually guided behaviors.

Retinal projection to the dorsal raphe nucleus in the Chilean degus (Octodon degus).

A substantial projection from the retina to the dorsal raphe nucleus (DRN) has been demonstrated in the Chilean degus, a diurnal/crepuscular hystricomorph rodent. Following intraocular injection of cholera toxin subunit B (CTB), immunocytochemically labeled CTB-positive axons and terminals were observed in all major retinorecipient nuclei as well as in the DRN and periaqueductal gray (PAG) of the mesencephalon. Two streams of optic axons to the DRN were observed: one descending from the optic tract at the level of the pretectum and anterior superior colliculus, the other emerging as a small fascicle at the anterior pole of the inferior colliculus and descending bilaterally through the PAG. Contralateral retinal afferents in the DRN appeared to terminate primarily in the dorsomedial and lateral subdivisions of the DRN, and a less extensive ipsilateral component also was observed. Axonal arborizations were characterized by short branches and multiple varicosities, both in the DRN and in the PAG. The extent and density of DRN retinal afferents were not as extensive as previously observed in Mongolian gerbils using identical techniques, but the retinal-DRN projection is considerably larger in degus than in rats. The functional significance of the retinal-DRN pathway remains to be determined, although a variety of evidence indicates that light may directly affect the activity of neurons and serotonin levels in the DRN.

Topographic organization of serotonergic dorsal raphe neurons projecting to the superior colliculus in the Mongolian gerbil (Meriones unguiculatus).

Recent evidence suggests that the dorsal raphe nucleus (DRN) of the brainstem is a collection of neuronal clusters having different neurochemical characteristics and efferent projection patterns. To gain further insight into the neuroanatomic organization of the DRN, neuronal populations projecting to the superior colliculus (SC) were mapped in a highly visual rodent, the Mongolian gerbil (Meriones unguiculatus). Retrograde tracers Fluoro-Gold (FG) or cholera toxin subunit-B (CTB) were injected into the superficial layers of the SC, and serotonin (5-hydroxytryptamine, 5-HT) -positive cells were identified by using immunocytochemistry in the FG-injected animals. Based on its projections to the SC, the DRN was divided into five rostrocaudal levels. In the rostral and middle levels of the DRN, virtually all FG-filled cells occurred in the lateral DRN, and 36-55% of 5-HT-immunoreactive (5-HT-ir) cells were also double-labeled with FG. Caudally, FG-filled cells occurred in the lateral, ventromedial, and interfascicular DRN; and 44, 12, and 31% of 5-HT-ir cells, respectively, were also FG-filled. The dorsomedial DRN contained only a small proportion of FG-filled cells at its most caudal level and was completely devoid of FG-filled cells more rostrally. The CTB-injected animals showed a similar distribution of retrogradely labeled cells in the DRN. Topographically, the dorsal tegmental nucleus and the laterodorsal tegmental nucleus appeared to be closely associated with 5-HT-ir cells in the caudal DRN. These results suggest that the lateral DRN and the ventromedial/interfascicular DRN may be anatomically, morphologically, and neurochemically unique subdivisions of the gerbil DRN.

Retinal afferents to the dorsal raphe nucleus in rats and Mongolian gerbils.

A direct pathway from the retina to the dorsal raphe nucleus (DRN) has been demonstrated in both albino rats and Mongolian gerbils. Following intraocular injection of cholera toxin subunit B (CTB), a diffuse stream of CTB-positive, fine-caliber optic axons emerged from the optic tract at the level of the pretectum/anterior mesencephalon. In gerbils, CTB-positive axons descended ventromedially into the periaqueductal gray, moving caudally and arborizing extensively throughout the DRN. In rats, the retinal-DRN projection comprised fewer, but larger caliber, axons, which arborized in a relatively restricted region of the lateral and ventral DRN. Following injection of CTB into the lateral DRN, retrogradely labeled ganglion cells (GCs) were observed in whole-mount retinas of both species. In gerbils, CTB-positive GCs were distributed over the entire retina, and a nearest-neighbor analysis of CTB-positive GCs showed significant regularity (nonrandomness) in their distribution. The overall distribution of gerbil GC soma diameters ranged from 8 to 22 micrometer and was skewed slightly towards the larger soma diameters. Based on an adaptive mixtures model statistical analysis, two Gaussian distributions appeared to comprise the total GC distribution, with mean soma diameters of 13 (SEM +/-1.7) micrometer, and 17 (SEM +/-1.5) micrometer, respectively. In rats, many fewer CTB-positive GCs were labeled following CTB injections into the lateral DRN, and nearly all occurred in the inferior retina. The total distribution of rat GC soma diameters was similar to that in gerbils and also was skewed towards the larger soma diameters. Major differences observed in the extent and configuration of the retinal-DRN pathway may be related to the diurnal/crepuscular vs. nocturnal habits of these two species.

Distribution of GABA-like immunoreactive neurons and fibers in the central visual nuclei and retina of frog, Rana pipiens.

Immunocytochemistry was used to study the distribution of gamma-aminobutyric acid (GABA) throughout the central visual nuclei and retina in Rana pipiens. In the retina, GABA immunoreactivity (both somata and fibers) was observed in all layers except the outer nuclear layer (ONL). Contrary to earlier reports, about 30% of total neurons within ganglion cell layer (GCL) expressed GABA immunoreactivity. Double-labeling studies indicated that about half of the GABA-containing perikarya in the GCL were retinal ganglion cells (RGCs). In the diencephalon, intensely labeled GABA-immunoreactive neurons and nerve fibers were observed within the neuropil of Bellonci (nB) and corpus geniculatum (CG), while only immunoreactive puncta were found in the rostral visual nucleus (RVN). In the pretectal region, the posterior thalamic nucleus (nPT) contained the most intensely labeled GABA immunoreactive perikarya and nerve fibers in the entire brain. Lightly immunoreactive perikarya were also found in the large-celled nucleus lentiformis mesencephali (nLM), as well as in the pretectal gray, which contains neurons postsynaptic to the retinal terminal zones within nLM. In the optic tectum (OT), both immunoreactive perikarya and fibers were found within superficial layers 8 and 9, whereas only densely packed immunoreactive perikarya were evident in the deep tectal layers (i.e. 2, 4, 6). The nucleus of the basal optic root (nBOR) contained a small number of lightly labeled GABA-immunoreactive perikarya, mostly located in the dorsal half of the nucleus. A large number of perikarya within the nucleus isthmi (NI) were also immunostained.

Organization of retinal axons within the optic nerve, optic chiasm, and the innervation of multiple central nervous system targets Rana pipiens.

Light microscopic analysis of the optic nerve, chiasm, and optic tracts of Rana pipiens after the anterograde and retrograde transport of horseradish peroxidase has shown that retinal ganglion-cell axons reach the optic nerve head in chronotopically organized fascicles that form bands across the intraocular optic nerve. These bands of fascicles are divided along the midline in a "zone of reorganization" to create two full maps of the retinal surface; however, this map is discontinuous in that nasal and temporal quadrants are adjacent to one another. In the intracranial portion of the optic nerve, axons undergo another reorganization such that peripheral retinal axons shift position and become localized laterally and ventrally, whereas centrally placed axons become localized dorsally. Within this reorganization, the nerve is reconfigured into laminae of axons, and each lamina consists of age-related axons organized into two retinal maps. In the ipsilateral chiasm, axons diverge to form three central, optic tracts: the medial optic tract, the projection to the corpus geniculatum, and the basal optic root. Ipsilateral axons leave the chiasm at the same level of the chiasm as do their contralateral counterparts. The remaining axons converge in the lateral diencephalon to form a fourth fascicle, the marginal optic tract. Thus, within the optic chiasm, a sequence of positional transformations occur that result in the formation of multiple optic pathways. The various changes in axonal trajectory always coincide with changes in the orientation of cell groups that lie within the nerve and optic chiasm.

NMDAR1-like immunoreactive fibers appear in the ipsilateral optic tract during optic nerve regeneration in Rana pipiens.

N-Methyl-D-aspartate receptor subunit 1-like immunoreactivity (NMDAR1-LI) was investigated in the brain of Rana pipiens during optic nerve regeneration. Following unilateral optic-nerve crush, frogs were tested for prey-catching and optokinetic nystagmus responses to assess return of visual function. At 1, 2, 3 and 5 months after the surgery, NMDAR1-LI was assessed in central visual pathways. At 3 and 5 months, conspicuous ipsilateral NMDAR1-LI fibers were detected in the thalamic and pretectal nuclei, and the time of their appearance coincided with the onset of behavioral recovery. Also, only ipsilateral retinorecipient layers in the optic tectum showed increased NMDAR1-LI during optic nerve regeneration. These results suggest that NMDA receptors may be present on retinal ganglion cell axons and terminals that have been misrouted during regeneration.

Distribution of the GABAA receptor complex beta 2/3 subunits in the brain of the frog Rana pipiens.

This report describes the distribution of labeling of the monoclonal antibody bd-17 against the beta 2/3 subunits of the mammalian GABAA receptor complex throughout the brain of the frog Rana pipiens. The distribution matches quite closely those in homologous brain regions as previously described for this antibody in fishes, birds, and mammals, indicating that this antibody also labels beta 2/3 subunits of frog. A semiquantitative analysis of the distribution of labeling throughout the brain is based upon relative optical densities with respect to the structure showing maximal optical density in each brain, using standard illumination conditions. Comparison with distributions in birds and mammals suggests that these GABAA receptor complex subunits are strongly conserved in vertebrate evolution and play an important role in the visual, auditory, olfactory and motor systems.

Single-unit responses to whole-field visual stimulation in the pretectum of Rana pipiens.

Visually responsive single units were recorded from the pretectal region that includes the large-celled nucleus lentiformis mesencephali (nLM) in the leopard frog, Rana pipiens. During monocular stimulation of the contralateral eye, 60 single units responding to movement of a large-field, random-dot pattern were quantitatively analyzed using horizontal and vertical directions at each of four pattern velocities (0.4-40 degrees/s). All units were spontaneously active, motion sensitive, and the majority showed 'on'-'off' responses. Several different response profiles were observed, including velocity-sensitive units with peak response at 10 degrees/s, most of which showed directional selectivity, and speed-sensitive units that showed increasing spike frequencies as pattern velocity increased, but little or no directional selectivity. About one-third of all unit analyzed were direction-selective, and 55% of those responded optimally to the temporal-to-nasal (T-N) direction of motion. T-N units were recorded primarily from an area that lies dorsolaterally between nLM and the optic tectum, in the 'peri-nLM' region. The pronounced monocular optokinetic nystagmus (OKN) response asymmetry that occurs in anurans appears to be reflected in the response profiles of the T-N direction selective units.

Differences in hippocampal volume among food storing corvids.

The hippocampal complex (hippocampus and parahippocampalis) is known to play a role in spatial memory in birds and is known to be larger in food-storing versus non-storing birds. In the present study, we investigated the relative volume of the hippocampal complex in four food-storing corvids: gray-breasted jays (Aphelocoma ultramarina), scrub jays (Aphelocoma coerulescens), pinyon jays (Gymnorhinus cyanocephalus), and Clark's nutcrackers (Nucifraga columbiana). The results show that Clark's nutcrackers have a larger hippocampal complex, relative to both body and total brain size, than the other three species. Clark's nutcrackers rely more extensively on stored food in the wild than the other three species. Clark's nutcrackers also perform better during cache recovery and operant tests of spatial memory than scrub jays. Thus, greater hippocampal volume is associated with better performance in laboratory tests of spatial memory and with stronger dependence on food stores in the wild.

Segregation of optic axons based on central target: the medial optic tract in Rana pipiens.

The retinofugal projection to the nucleus of Bellonci (nB) was examined in Rana pipiens using both anterograde and retrograde transport of horseradish peroxidase (HRP). Following HRP injection into the nB, retrogradely labeled optic axons formed a discrete fascicle that crossed the lateral margin of the anterior diencephalon. We have designated this branch of the retinofugal pathway as the "medial optic tract." HRP-positive, medial optic tract axons projecting to nB occupied the rostral and most dorsal portion of the optic chiasm. The present findings indicate that within the optic chiasm, retinal axons are sorted according to their final destinations.

Distribution of GAD-like immunoreactivity in the retina and central visual system of Rana pipiens.

Immunohistochemistry was used to survey the distribution of glutamic acid decarboxylase (GAD), the synthesizing enzyme for gamma-aminobutyric acid (GABA), throughout the visual system of the frog Rana pipiens. GAD-like immunoreactivity (GAD-LI) was assessed in the retina, in primary retinorecipient targets, and in thalamic nuclear groups postsynaptic to primary retinal terminal zones. Within the retina, the inner plexiform layer displayed intense GAD-LI, but immunoreactivity was absent in the ganglion cell layer (GCL). Putative amacrine, bipolar, and horizontal cell somata were also labeled. Centrally, GAD-LI was observed in all primary visual nuclei. Dense, GAD-like immunoreactive punctate structures (puncta), presumably terminals, were observed in the pretectal nucleus lentiformis mesencephali, posterior thalamic neuropil, and uncinate neuropil. GAD-like immunoreactive puncta were noted in several laminae of the optic tectum, with the highest concentrations located within the 9th and 8th laminae. Moderate numbers of GAD-like immunoreactive puncta were found in the mesencephalic nucleus of the basal optic root, and two thalamic neuropils--corpus geniculatum and neuropil of Bellonci. The ventrolateral area, posterocentral nucleus, and posterolateral nucleus all contained sparse amounts of GAD-LI. These observations suggest that GABA plays an important physiological role in all central visual areas of Rana pipiens.

Drusen-like deposits in the outer retina of Japanese quail.

Histological examination of the outer retina of Japanese quail (Coturnix coturnix Japonica) ranging in age from 4-48 months revealed the presence of drusen-like deposits which closely resemble those described in primate and human retinas. At both light and electron microscopic levels, these deposits were characterized by randomly distributed, granular and heterogenous materials. Larger deposits with pleomorphic inclusions, often globular in shape, occurred more frequently in older quail, particularly in males. An increase in the incidence and size of drusenoid deposits occurred with increasing age, with a greater rate of accumulation in males than in females. Previously, we have shown that retinal pigment epithelial lipofuscin increases more rapidly with age in female quail and that cumulative light damage is associated with increased lipofuscin in females but not in males. In the present study, no increase in drusen-like deposits was observed in single light-damaged or double light-damaged retinas of either sex. Thus, lipofuscin and the frequency and size of drusenoid deposits do not appear to be directly linked, although both increase with age in both sexes.

Experimental light damage increases lipofuscin in the retinal pigment epithelium of Japanese quail (Coturnix coturnix japonica).

Lipofuscin accumulation in the retinal pigment epithelium (RPE) of Japanese quail was investigated in normal and light-damaged animals at 4 months and 12 months of age following a single, 18-hr light-damaging exposure (3000-3200 lx) evaluated 6 weeks postexposure. Quail in a second experimental group light-damaged at 4 months of age were exposed to a second, identical light-damaging exposure at 12 months of age. RPE lipofuscin was quantitatively assessed in three ways: (1) total lipofuscin, expressed as a percentage of the basal RPE cell area, (2) mean number of lipofuscin granules per RPE cell, and (3) mean area of lipofuscin granules. At 12 months of age, control female RPE cells showed substantially more lipofuscin than did males. Animals that received a single damaging light-exposure showed no significant differences in lipofuscin at either age when compared with controls, and none were observed between males and females. However, in the double light-exposure condition, both total lipofuscin and the number of lipofuscin granules were significantly higher in females compared to controls and also, when compared with animals in the single light-exposure condition. Mean lipofuscin granule size decreased in females in the double light-exposure condition when compared with control values. Histopathological evaluation of photoreceptor outer segments and outer nuclear layer indicated that double-exposed, 12-month-old females showed the most severe effects, including a substantial decrease (70%) in rod photoreceptor densities. However, only a small change was observed in cone densities for either sex (12-15%) in this condition. Overall, a strong, negative correlation (r = -0.78) was obtained between total lipofuscin and average rod density across age, sex, control and experimental conditions. A somewhat greater correlation (r = -0.82) was obtained for females, alone. These results suggest that rods, when severely damaged by light exposure, contribute to increased RPE lipofuscin. Age, sex and light-exposure history appear to be critical variables that can influence both the amount of lipofuscin present in RPE cells as well as the relative vulnerability of rod and cone photoreceptors to cumulative light-exposure damage. These results have implications for the development of a variety of age-related changes and diseases of the outer retina, including age-related macular degeneration.

Metabolic correlates of optokinetic stimulation in the central visual system of the frog, Rana pipiens.

The [14C]2-deoxyglucose (2-DG) method was used to identify those structures in the central visual system of Rana pipiens showing increased metabolic activity during binocular and monocular optokinetic stimulation at two pattern velocities (2 deg/sec and 9 deg/sec). Analysis of autoradiograms made with computer-assisted microdensitometry and pseudocolor image-enhancement techniques revealed that the greatest uptake of 2-DG occurred in the pretectal region, which included the large-celled nucleus lentiformis mesencephali (nLM), nucleus pretectalis, and pretectal gray. Both temporal-to-nasal (T-N) and nasal to temporal (N-T) directions of pattern motion were correlated with high levels of 2-DG uptake. However, the nucleus of the basal optic root (nBOR) showed the greatest uptake of 2-DG for the N-T direction, which evokes little or no optokinetic (OKN) response in this species. These results suggest that the major efferent projection from nBOR to nLM may exert its greatest effect upon the ipsilateral pretectum during N-T stimulation. Other regions showing substantial 2-DG uptake included a large region of the ventral thalamus, for all stimulus conditions tested, including those cell groups associated with the retinorecipient neuropil, corpus geniculatum. The pattern of uptake was less closely associated with specific parameters of optokinetic stimulation than was observed in the pretectum and accessory optic nucleus. Like nBOR, the auricular lobe of the cerebellum also showed greatest uptake of 2-DG for the N-T, monocular stimulus condition. The neural circuitry that underlies OKN and its directional asymmetry during monocular stimulation appears to involve a number of structures whose functional interrelationships are yet to be described.

Organization of ascending projections from the optic tectum and mesencephalic pretectal gray in Rana pipiens.

The ascending projections from the dorsal mesencephalon to the thalamus and pretectum in Rana pipiens were investigated by using the anterograde and retrograde transport of HRP with regard to two major issues: (1) the degree of tectotopic organization in the projections, and (2) their cells of origin. The results indicate that the spatial organization of the tecto-thalamic tract is specifically related to the laminar organization of the contributing tectal efferent neurons. Axons of neurons in the superficial portion of tectal layer 8 exit the tectum through layer 9 and travel in the superficial portion of the dorsal and ventral tecto-thalamic tracts and innervate the nucleus lentiformis mesencephali, the posterior lateral dorsal nucleus, and corpus geniculatum. The distribution of terminals within these structures varied with the tectal HRP-injection site. HRP injections in the ventral tecto-thalamic tract retrogradely labeled neurons in the superficial portion of tectal layer 8 across the lateral and caudal portion of the tectal lobe. HRP injections into the dorsal tecto-thalamic tract, at the level of the pretectum, retrogradely labeled pyriform neurons in the superficial portion of tectal layer 8 in the rostral and medial portions of the tectal lobe. With regard to the deep tectal layers, axons from pyramidal neurons in layer 6 and ganglionic neurons in layer 8 leave the tectum through layer 7, travel in both the dorsal and ventral tecto-thalamic tracts, and are located internal to the axons of the pyriform neurons of superficial tectal layer 8. The majority of the ganglionic neurons project to the posterior lateral ventral nucleus and the anterior lateral nucleus. The distribution of terminals within these nuclei did not display a tectotopic organization. A second major projection to the thalamus originates from the mesencephalic pretectal gray and innervates the nucleus lentiformis mesencephali, the posterior lateral dorsal nucleus, the anterior lateral nucleus, dorsal and ventral divisions of the ventral lateral thalamus, and the nucleus of Bellonci. Other axons from the mesencephalic pretectal gray terminate in the contralateral, medial portions of the posterior lateral dorsal thalamus, the ventral lateral thalamus, and the anterior lateral nucleus. The isthmo-tectal projection was also retrogradely labeled following tectal injections of HRP. This pathway travels in the most ventral portion of the ventral tecto-thalamic tract; its axons passed over the lateral margin of the endopeduncular nucleus bilaterally, and crossed the midline in the caudal portion of the optic chiasm. Extensive, bead-like varicosities were observed on these axons both in the endopeduncular nucleus and in the posterior optic chiasm.(ABSTRACT TRUNCATED AT 400 WORDS)

Anatomical evidence for an intergeniculate leaflet in Rana pipiens.

The projections of the nucleus of Bellonci and the anterior thalamic nucleus in Rana pipiens appear to be remarkably similar to those that have been described for the mammalian intergeniculate leaflet. The connections of these nuclei were examined using both the anterograde and retrograde transport of horseradish peroxidase. Afferents to the neuropil of Bellonci and its nucleus include bilateral projections from the retina, the contralateral nucleus of Bellonci, and anterior thalamic nucleus as well as bilateral projections from the pretectum and the ipsilateral suprachiasmatic nucleus. Efferent projections observed following HRP injections in the anterior thalamus consist of three components: (1) a ventral hypothalamic-suprachiasmatic and commissural projection, (2) a dorsal descending tract to the pretectum and tectum, and (3) a ventral descending tract to the somatomotor brainstem.

Age-dependent changes in visual acuity and retinal morphology in pigeons.

The visual acuities of 17 pigeons that ranged in age from 2 to 17 years were tested with high-contrast, square-wave gratings. A systematic decline in visual acuity was observed that was well described by a logarithmic function. Pupillary diameter also declined with age, which decreased retinal illumination, but increased depth of focus. A small amount of presbyopia also was observed. Both the decrease in retinal illumination and the presbyopia accounted for only a trivial proportion of the acuity loss. No relationship between corneal or lenticular density and age was observed. Ophthalmoscopic examination of the optic media revealed no abnormalities associated with age. Microscopic examination of the area dorsalis of the retina (the high-density region specialized for frontal vision) revealed age-related losses of up to 33% of photoreceptors and 23% of cells in the ganglion-cell layer. A study of the photoreceptor layer within area dorsalis indicated that single-cone densities were unaffected by aging whereas the double-cone densities, which are the predominant photoreceptor type in the area dorsalis, were reduced in number by about one third. Calculation of the Nyquist limit both for photoreceptors and ganglion cells suggested that the decreased retinal density together with the decreased retinal illumination and presbyopia could not account for all of the observed acuity loss.