Shashi Wadhwa

Developmental expression of calretinin immunoreactivity in the human retina and a comparison with two other EF-hand calcium-binding proteins

This paper reports the localization pattern of calretinin, a calcium-binding protein, in the human retina during development, as studied by immunohistochemistry. A comparison is made of the cellular distribution of calretinin with two other calcium-binding proteins, calbindin and parvalbumin, recently reported by us in the human retina, and by parallel labeling with both antisera in the same tissues. At 11-12 weeks of gestation, calretinin immunoreactivity was expressed in many prospective ganglion cells of the central inner neuroblastic zone. At 16-17 weeks of gestation, the immunoreactivity was localized in the ganglion cell layer, inner plexiform layer, and in most differentiated amacrine, horizontal and cone cells located in the central (1-2 mm temporal from optic disc) to midperipheral parts of the retina. By midgestation (20-21 weeks), calretinin immunoreactivity was strongly developed in the cone photoreceptors. Parallel labeling with calbindin and parvalbumin antisera revealed that the calretinin-positive horizontal cells were somewhat smaller and less frequent and less intense than the calbindin- and parvalbumin-positive counterparts, at 16-21 weeks of gestation. No horizontal cells were calretinin immunopositive in the postnatal (four-month-old infant) and adult retinas examined. Also, at both stages, a few bipolar and cone cells were weakly immunoreactive. These observations suggest a critical role for calretinin in the development and maturation of a select class of horizontal cells. The widespread expression of immunoreactivity in the early ganglion cells indicates that calretinin may be involved in their differentiation. The weak immunoreactivity pattern noted in the adult photoreceptor and bipolar cells, and an apparent lack of immunoreactivity in the mature horizontal cells, tends to indicate that, unlike calbindin and parvalbumin, calretinin plays little role in the transport and physiological buffering of Ca2+ in these neurons of the human retina. It appears, however, that calretinin is predominantly involved in both processes in amacrine cells.

Expression of the neurotrophin receptors Trk A and Trk B in adult human astrocytoma and glioblastoma

Neurotrophins and their receptors of the Trk family play a critical role in proliferation, differentiation and survival of the developing neurons. There are reports on their expression in neoplasms too, namely, the primitive neuroectodermal tumours of childhood, and in adult astrocytic gliomas. The involvement of Trk receptors in tumour pathogenesis, if any, is not known. With this end in view, the present study has examined 10 tumour biopsy samples (identified as astrocytoma, pilocytic astrocytoma and glioblastoma) and peritumoral brain tissue of adult patients, for the presence of Trk A and Trk B receptors, by immunohistochemistry. The nature of the tumour samples was also confirmed by their immunoreactivity (IR) to glial fibrillary acidic protein. In the peritumoral brain tissue, only neurons showed IR for Trk A and Trk B. On the contrary, in the tumour sections, the IR to both receptors was localized in the vast majority of glia and capillary endothelium. There was an obvious pattern of IR in these gliomas: high levels of IR were present in the low-grade (type I and II) astrocytoma; whereas in the advanced malignant forms (WHO grade IV giant cell glioblastoma and glio-blastoma multiforme) the IR was very weak. These findings suggest that Trk A and Trk B are involved in tumour pathogenesis, especially in the early stage, and may respond to signals that elicit glial proliferation, and thus contribute to progression towards malignancy.

Effect of prenatal auditory enrichment on developmental expression of synaptophysin and syntaxin 1 in chick brainstem auditory nuclei.

Neural activity plays an important role in shaping the developing brain. We have determined the consequence of increased auditory stimulation on the developmental profile of synaptic proteins, synaptophysin and syntaxin 1, in the chick brainstem auditory nuclei, nucleus magnocellularis and nucleus laminaris, by immunohistochemistry and western blotting techniques. The chick embryos were provided with patterned sounds of species-specific calls or musical notes of a sitar, a stringed instrument, in a graded manner from embryonic day 10 (E10) through hatching, for 15 min every hour. During normal synaptogenesis of nucleus magnocellularis and nucleus laminaris, synaptophysin immunoreactivity increased significantly from E8 to E20, in parallel with synapse formation, and reduced at hatching. The embryos receiving species-specific sound stimuli exhibited a similar pattern with higher levels of immunoreactivity, though the difference between the study groups was not statistically significant. The music stimulated embryos showed an earlier peak at E16, followed by a gradual decline until hatching. In all three groups studied, syntaxin immunoreactivity showed a surge at E12, followed by a decline at E16 and subsequent stabilization. The stimulated groups continually expressed higher amounts of syntaxin immunoreactivity. The results suggest that prenatal sound stimulation enhances the normal pattern of synaptic protein expression in these auditory nuclei.

Quantitative study of plasticity in the auditory nuclei of chick under conditions of prenatal sound attenuation and overstimulation with species specific and music sound stimuli

Morphological effects of prenatal sound attenuation and sound overstimulation by species specific and music sounds on the brainstem auditory nuclei of chick have been evaluated quantitatively. Changes in length, volume, neuron number, size of neuronal nuclei and glial numbers of second and third order auditory nuclei, n. magnocellularis (NM) and n. laminaris (NL), were determined from thionine‐stained serial sections of control and experimental groups on posthatch day 1 using stereological methods.

Ultrastructure of the human retina in aging and various pathological states

Vision is hampered in aging and diseases, such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy and glaucoma. This review collates the fine structural alterations of the human retina in aging and various pathological situations and their links to the disease pathogenesis. It transpires that most changes occur at the level of the retinal pigment epithelium -Bruchs membrane and the photoreceptor layer, causing visual problems to the sufferers. These changes include loss of normal, essential features of these cells and their gradual disappearance. It is important to understand in depth the selective vulnerability of this retinal region to alterations in aging and diseases. Evidence indicates that some of these changes may be mediated by the effects of oxidative stress, inflammation, and chronic light exposure. There are changes also in the inner retinal layers, wherein hypertension, auto-immunity, hypoxia and ischemia could play significant roles in disease pathogenesis. Results of extensive research utilizing animal models have broadened our idea about photoreceptor pathology. However, equivalent knowledge on various changes in aging human retina and in dystrophies that affect the macula is not complete. Since cone photoreceptor and ganglion cell death are a potential problem, it is imperative to know about the basic facts on how they are affected and the mechanisms involved in their death. Thus, prevention of cone and ganglion cell loss should be the target of choice. This review also highlights the significant role played by electron microscopy in understanding such ultrastructural changes and future strategies utilizing it and other techniques to fill some of the existing lacunae and advance our knowledge.

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.

Neurotrophin receptors (Trk A, Trk B, and Trk C) in the developing and adult human retina

In this study, the ontogeny and distribution patterns of three neurotrophin receptors (Trk A, Trk B, and Trk C) were examined in the human retinas. Immunohistochemistry was performed on sections of retina and optic nerve from fetuses (11-24 weeks of gestation, wg), one infant (4-month-old) and two adult (35- and 65-years-old) subjects. At 11 wg, Trk A was expressed in the nerve fiber and inner plexiform layers, while Trk B and Trk C were expressed in many neuroblastic cells. By 16-17 wg, the photoreceptors showed immunoreactivity for all three receptors. The ganglion cell layer and amacrine cells were conspicuously immunoreactive for Trk A and Trk C, but labeled diffusely for Trk B. The horizontal cells were labeled for Trk A and Trk B. The pattern was same in the retinas at midgestation (20-21 wg). Shortly after this period, there was an apparent decrease in receptor immunoreactivity in the fetal retinas. In the infant retina, Trk A immunoreactivity was absent from horizontal cells. The photoreceptors were immunopositive for Trk B and Trk C, in infant and adult retinas. In the adults, few cells of the ganglion cell layer and inner nuclear layer were clearly labeled for Trk A and Trk C, and diffusely for Trk B. The glial cells of the retina and optic nerve immunoreacted for Trk A only, right from fetal 16 wg. The early expression of Trk B and Trk C on neuroblastic cells suggests that both play a role in cell proliferation. The developmental distribution pattern of Trk A, on the other hand, provides evidence for its involvement in differentiation of the inner plexiform layer, horizontal cells and neuroglia. The results strongly suggest that photoreceptor development is mediated by Trk receptors. The novel localization of Trk B and Trk C on adult photoreceptors points to a possible therapeutic potential for BDNF and NT-3, respectively, in photoreceptor diseases.

Calbindin D-28K and parvalbumin expression in embryonic chick hippocampus is enhanced by prenatal auditory stimulation

Calcium-binding proteins (CaBPs) buffer excess of cytosolic Ca(2+), which accompanies neuronal activity following external stimuli. Prenatal auditory stimulation by species-specific sound and music influences early maturation of the auditory pathway and the behavioral responses in chicks. In this study, we determined the volume, total number of neurons, proportion of calbindin D-28K and parvalbumin-positive neurons along with their levels of expression in the developing chick hippocampus following prenatal auditory stimulation. Fertilized eggs of domestic chicks were exposed to sounds of either species-specific calls or sitar music at 65 dB for 15 min/h round the clock from embryonic day (E) 10 until hatching. Hippocampi of developmental stages (E12, E16 and E20) were examined. With an increase in embryonic age during normal development, the hippocampus showed an increase in its volume, total number of neurons as well as in the neuron proportions and levels of expression of calbindin D-28K and parvalbumin. A significant increase of volume at E20 was noted only in the music-stimulated group compared to that of their age-matched control (p<0.05). On the other hand, both auditory-stimulated groups showed a significant increase in the proportion of immunopositive neurons and the levels of expression of calbindin D-28K and parvalbumin as compared to the control at all developmental stages studied (p<0.003). The increase in proportions of CaBP neurons during development and in the sound-enriched groups suggests an activity-dependent increase in Ca(2+) influx. The enhanced expression of CaBPs may help in cell survival by preventing excitotoxic death of neurons during development and may also be involved in long-term potentiation.

Age-related decrease in rod bipolar cell density of the human retina: an immunohistochemical study

During normal ageing, the rods (and other neurones) undergo a significant decrease in density in the human retina from the fourth decade of life onward. Since the rods synapse with the rod bipolar cells in the outer plexiform layer, a decline in rod density (mainly due to death) may ultimately cause an associated decline of the neurones which, like the rod bipolar cells, are connected to them. The rod bipolar cells are selectively stained with antibodies to protein kinase C-α. This study examined if rod bipolar cell density changes with ageing of the retina, utilizing donor human eyes (age: 6–91 years). The retinas were fixed and their temporal parts from the macula to the mid-periphery sectioned and processed for protein kinase C-α immunohistochemistry. The density of the immunopositive rod bipolar cells was estimated in the mid-peripheral retina (eccentricity: 3–5 mm) along the horizontal temporal axis. The results show that while there is little change in the density of the rod bipolar cells from 6 to 35 years (2.2%), the decline during the period from 35 to 62 years is about 21% and between seventh and tenth decades, it is approximately 27%.

Calbindin and parvalbumin immunoreactivity in the developing and adult human retina

We report the expression and pattern of two calcium-binding proteins (CBPs), calbindin (CALB) and parvalbumin (PV), in the fetal (13-25 weeks of gestation), postnatal (5 months) and adult human retina, as studied by immunohistochemistry. Both CBPs appear prenatally in different neurones as well as in the nerve fibre, inner and outer plexiform layers. The cones do not show immunoreactivity for both CBPs up to 25 weeks, the last fetal stage studied; however, they are immunopositive in the postnatal and adult retina. Of the two CBPs, CALB appears first, followed by PV. The immunoreactivity for both CALB and PV in the fetal retina follows a centroperipheral gradient and vitreal to scleral pattern of retinal differentiation. The CALB immunoreactivity shows a gradual increase in intensity with age. A spurt in intensity of PV immunoreactivity in the 24-25 week fetal retina and increased intensity in the 60 year normal adult retina when compared to the light-deprived retina of a 34-year-old staphyloma patient suggests an activity-dependent functional role for PV.