Biomedical subjects

J N Hokoç

Publications and source records attributed to J N Hokoç.

At least 19 recordsLinked to original sources

PubMed2006

GABAergic circuitry in the opossum retina: a GABA release induced by L-aspartate.

Glutamate and gamma-amino butyric acid (GABA) are the major excitatory and inhibitory neurotransmitters, respectively, in the central nervous system (CNS), including the retina. Although in a number of studies the retinal source of GABA was identified, in several species, as horizontal, amacrine cells and cells in the ganglion cell layer, nothing was described for the opossum retina. Thus, the first goal of this study was to determine the pattern of GABAergic cell expression in the South America opossum retina by using an immunohistochemical approach for GABA and for its synthetic enzyme, glutamic acid decarboxylase (GAD). GABA and GAD immunoreactivity showed a similar cellular pattern by appearing in a few faint horizontal cells, topic and displaced amacrine cells. In an effort to extend the knowledge of the opossum retinal circuitry, the possible influence of glutamatergic inputs in GABAergic cells was also studied. Retinas were stimulated with different glutamatergic agonists and aspartate (Asp), and the GABA remaining in the tissue was detected by immunohistochemical procedures. The exposure of retinas to NMDA and kainate resulted the reduction of the number of GABA immunoreactive topic and displaced amacrine cells. The Asp treatment also resulted in reduction of the number of GABA immunoreactive amacrine cells but, in contrast, the displaced amacrine cells were not affected. Finally, the Asp effect was totally blocked by MK-801. This result suggests that Asp could be indeed a putative neurotransmitter in this non-placental animal by acting on an amacrine cell sub-population of GABA-positive NMDA-sensitive cells.

Amacrine Cells↗

PubMed2005

Quantitative ultrastructural evidence of myelin malformation in optic nerves of rats submitted to a multideficient diet.

Pups were subjected to malnutrition by feeding the lactating mothers a multi-deficient (8% protein content) diet, known as regional basic diet (RBD), from birth up to weaning. The weanings were fed the same diet until 60 days of age. Ultrastructure of the optic nerve was analyzed at postnatal (P) day P8, P13, P21, P30 and P60. Electron microscopy revealed that at P8 the process of myelination has not started yet in neither groups. At P 13 different stages of myelination were observed and, in the experimental group, the optic nerve showed non-organized axon bundles and empty spaces. Control optic nerve at P21 exhibited axons with fully developed myelin sheath; whereas malnourished group had many axons being enveloped by myelin with anomalous alteration. These alterations were present in malnourished group at P30 and P60. Quantitative analysis showed statistically significant difference between control and malnourished groups when compared to the percentage of myelinated axons, axons with myelin anomalous alterations (MAA) and non-myelinated axons. Also, the myelin area was significantly smaller in malnourished groups when compared to control group. Finally, a high percentage of large non-myelinated fibers were found in the malnourished group. In conclusion, our results show that early malnutrition by a multideficient diet (RBD) affects permanently the optic nerve organization and myelination, indicating an impairment of nerve transmission and a probable dysfunction in the visual ability.

Aging↗

PubMed2002

Opposite roles of GABA and excitatory amino acids on the control of GAD expression in cultured retina cells.

The mechanism of control of GAD expression by GABA and excitatory amino acids (EAAs) was studied in chick and rat retina cultures using immunohistochemical and PAGE-immunoblot detection of the enzyme, as well as by measuring enzyme activity. Aggregate cultures were prepared with retina cells obtained from chick embryos at embryonic days 8-9 (E8-E9). Organotypical cultures were also prepared with retinas from E14 chick embryos, post-hatched chicken and P21 rats. GABA (1-20 mM) fully prevented GAD expression in aggregate and organotypical cultures from chick embryo retinas. A substantial, but not complete, reduction of GAD was also observed in organotypical cultures of post-hatched chicken and P21 rats, in which both forms of the enzyme (GAD65 and 67) were affected. The GABA effect was not mimicked by THIP (100 microM), baclofen (100 microM) or CACA (300 microM), agonists of GABAa, b and c receptors, respectively. NNC-711, a potent inhibitor of GABA transporters, reduced by 50% the inhibition of GAD activity promoted by GABA. Aggregates exposed to GABA and treated with glutamate (5 mM) or kainate (100 microM) displayed an intense GAD-like immunoreactivity in many cell bodies, but not in neurite regions. Immunoblot analysis revealed that the increase in GAD-like immunoreactivity by EAA corresponded to a 67-kDa protein. However, GAD activity was not detected. Treatment of aggregates or retina homogenates with SNAP, a NO producing agent (but not its oxidized form), reduced GAD activity by more than 60% indicating that the lack of enzyme activity in GAD-like immunoreactive cells, could be due to NO production by EAA stimulation.

Animals↗

PubMed2001

Comparative study of glutamate mediated gamma-aminobutyric acid release from nitric oxide synthase and tyrosine hydroxylase immunoreactive cells of the Cebus apella retina.

The effects of excitatory amino acids (EAAs) upon transporter-mediated gamma-aminobutyric acid (GABA) release were investigated in cells containing tyrosine hydroxylase (TH) or nitric oxide synthase (NOS) in retina of the primate Cebus apella. Retinas were treated in vitro with 50 microM Kainate (KA) or 5 mM L-Glutamate (L-Glu), for 30 min at 37 degrees C, in an Mg2+-free Locke's solution with or without Ca2+. The effects of EAAs were measured immunocytochemically by determining the GABA content in TH or NOS-immunoreactive cells in the inner retina, after stimulation. L-Glu and KA induced a Ca2+-independent GABA release from most GABA-immunoreactive cells of the inner retina. Double label experiments indicated that this release occurs in NOS+/GABA+ cells, but not in TH+/GABA+ cells suggesting that these cell subpopulations may be differentiated in some functional aspects.

Animals↗

PubMed2001

Dopaminergic retinal cell differentiation in culture: modulation by forskolin and dopamine.

We examined the effects of dopamine and cAMP on the differentiation of dopaminergic retinal cells in the chick retina, using an in vitro system and tyrosine hydroxylase immunocytochemistry. Tyrosine hydroxylase-positive cells were detected in cultures prepared from embryonic day 10 retinas. These increased in number as a function of time in vitro and by treatment for 4 days with forskolin. Besides causing a 3.4-fold increase in the tyrosine hydroxylase-positive population, forskolin also caused these cells to developed morphogenetic features of more mature cells. As opposed to forskolin, cultures treated with dopamine exhibited a 55% reduction of the tyrosine hydroxylase-positive cell population, as compared to untreated cultures. Quinpirole was able to mimic the dopamine effect. This dopamine effect could only be blocked by clozapine, whereas raclopride and eticlopride were ineffective. Our results suggest the existence of a narrow window during development when undifferentiated dopaminergic cells are capable of being influenced by specific signals, possibly via cAMP production. The data also indicate that dopamine may act as a regulatory factor limiting the tyrosine hydroxylase-positive population in the retina.

Animals↗

PubMed2001

The GAbAergic and cholinergic systems in the retina are differentially affected by postnatal malnutrition during the suckling period.

Malnutrition by severe protein deprivation induces deleterious consequences in the nervous system particularly in the initial period of development. These deficits can alter several important events during development, such as the expression of neurotransmitters. The induction of nutritional deficiency by using low protein diet, similar to that consumed by low income populations in Brazil, was applied in rats to investigate the effect of malnutrition on cells containing gamma-aminobutyric acid (GABA) and acetylcholine in the retina. GABA immunoreactivity was present in cells in the inner nuclear and ganglion cell layers and in processes in the inner and outer plexiform layers in retinas of control and malnourished animals. At postnatal day 8, there is a decrease (ca. 40%) of the GABAergic neurons in malnourished animals. At P13 and P21 the percentage of these neurons increased and was equivalent to control animals in the adult. Glutamic acid decarboxylase activity did not show significant changes between the two groups along development. Choline acetyltransferase immunoreactivity was localized in amacrine cells in the inner nuclear and ganglion cell layers and their processes in the inner plexiform layer. The percentage of cholinergic cells was always higher in malnourished animals than that observed in the control until postnatal day 30, when the same proportion of cholinergic neurons was found in the retinas of both groups. Choline acetyltransferase activity did not show significant changes between the two groups along development. In conclusion, our results show that despite the extreme somatic and behavioral changes observed the neurotransmitter systems studied were at a certain extent shielded from the insult.

Acetylcholine↗

PubMed2001

Watery and dark axons in Wallerian degeneration of the opossum's optic nerve: different patterns of cytoskeletal breakdown?

In this paper we report a qualitative morphological analysis of Wallerian degeneration in a marsupial. Right optic nerves of opossums Didelphis marsupialis were crushed with a fine forceps and after 24, 48, 72, 96 and 168 hours the animals were anaesthetized and perfused with fixative. The optic nerves were immersed in fixative and processed for routine transmission electron microscopy. Among the early alterations typical of axonal degeneration, we observed nerve fibers with focal degeneration of the axoplasmic cytoskeleton, watery degeneration and dark degeneration, the latter being prevalent at 168 hours after crush. Our results point to a gradual disintegration of the axoplasmic cytoskeleton, opposed to the previous view of an "all-or-nothing" process (Griffin et al 1995). We also report that, due to an unknown mechanism, fibers show either a dark or watery pattern of axonal degeneration, as observed in axon profiles. We also observed fibers undergoing early myelin breakdown in the absence of axonal alterations.

Animals↗

PubMed2000

Transporter-mediated GABA release induced by excitatory amino acid agonist is associated with GAD-67 but not GAD-65 immunoreactive cells of the primate retina.

The release of GABA from amacrine and interplexiform cells after exposure to excitatory amino acids (EAAs) agonists was investigated by immunohistochemistry. Cebus monkey retinas were treated in vitro with 50 microM kainate (KA) or 5 mM L-Glutamate (L-Glu), for 30 min at 37 degrees C. The effects of the EAAs were measured by detecting immunocytochemically the GABA remaining in the tissue after stimulation. L-Glu and KA reduced the number of GABA-immunoreactive perikarya in the innermost part of the inner nuclear layer by approximately 60% and 80%, respectively, as compared to controls. The cell processes in the inner plexiform layer (IPL) were restricted to only three defined bands in the strata 1, 3 and 5, as compared to an intense and homogeneous labeling in the IPL of the untreated retinas. The effect of KA was inhibited by 100 microM CNQX, 100 microM NNC-711, or when Na(+) was replaced by choline. The release of GABA was Ca(2+)-independent, suggesting the mobilization of GABA from the cytoplasmic pool of this neurotransmitter. At least two subsets of retinal neurons including amacrine and interplexiform cells retained GABA-immunoreactivity after stimulation with EAAs, as revealed by glutamic acid decarboxylase (GAD) immunocytochemistry. Our results suggest that non-NMDA receptor activation by KA and glutamate are associated with the efflux of GABA from cells of the inner retina (amacrine and interplexiform cells). The data also show that cells containing GAD-67 released GABA via its transporter, while cells containing exclusively GAD-65 apparently did not release the neurotransmitter by the reversal of the transporter.

Animals↗

PubMed2000

Retinal ganglion cells in the South American opossum (Didelphis aurita).

By using the Golgi technique, the authors investigated the morphology of ganglion cells in the retinas of South American opossums. In flat-mount preparations of the retinas, cell bodies, entire dendritic fields, and the stratification level of ganglion cells were studied. Fractal dimensions of dendritic trees, an objective quantitative measure of morphological complexity, were included as a morphological parameter of classification. Based on these characteristics, nineteen types of ganglion cells were described. A great number of opossum ganglion cell types had dendrites stratifying in both sublaminae of the inner plexiform layer (IPL) in five different ways (S1-S3 [G9], S1-S4 [G17 and G22], S2/S3 [G19], S2-S4 [G15, G16, G21 and G221, and S2-S5 [G61), and only two types (G8, and G10) showed narrow field dendritic trees ramifying in S4 only. Morphological types of opossum ganglion cells were compared to their counterparts in cat retina. The distribution pattern of large cell bodies on the ganglion cell layer was analyzed employing the Nissl staining method, immunocytochemistry for neurofilaments, and the reduced silver neurofibrillar staining method. The results showed a random pattern of distribution.

Animals↗

PubMed2000

Photoreceptor topography of the retina in the New World monkey Cebus apella.

The number and topographical distribution of photoreceptors was studied in whole-mounted retinas of Cebus apella. It was estimated a total of 48 million rods and 3.8 million cones. The average peak foveal cone density and the Nyquist Limit at the foveola were estimated as 169, 127 cells/mm(2) and 46.77+/-7.98 cyc/deg, respectively. A cone-enriched rim was found near the ora serrata, more noticeable in the nasal retina. Rod distribution was asymmetrical along horizontal and vertical meridians with a higher density in the dorsal retina. The rod/cone ratio was variable and asymmetrical along both meridians.

Animals↗

PubMed2000

Neurogenesis of GABAergic cells in the chick retina.

Two classes of retinal neurons in the chick retina, the horizontal and the amacrine cells, are GABAergic. This study evaluates the neurogenesis of glutamic acid decarboxylase immunoreactive cells in the chick retina. Twenty-five microCi [3H]thymidine was injected into eggs of 2-10 days and the embryos were sacrificed at embryonic day 18 (E18). Glutamic acid decarboxylase immunohistochemistry was revealed by avidin-biotin complex method followed by autoradiography of thymidine. We used the cumulative method for counting autoradiographic grains. At E3, 10% of the amacrine cells were thymidine negative/glutamic acid decarboxylase positive and this rate remained constant until E6. From E6 to E8 about 80% of the amacrine cells were thymidine negative/glutamic acid decarboxylase positive. At E9, 100% of these neurons had been generated. On the other hand, at E3 only 1.5% of the horizontal cells had been generated (thymidine negative/glutamic acid decarboxylase positive) while at E6 this number increased to 10%. From E6 to E9 the neurogenesis pattern was similar to that found for amacrine cells. Our data show that the great majority (80%) of glutamic acid decarboxylase positive amacrine and horizontal cells proliferate between E6 and E9, i.e. the last 3 days of the neurogenesis period. From E3 to E6 only 20% of the glutamic acid decarboxylase positive amacrine and horizontal cells are generated, which suggests that glutamic acid decarboxylase positive cells may require a specific signal at about E6, which triggers their withdrawal from the cell cycle.

Animals↗

PubMed1999

Ontogeny of cholinergic amacrine cells in the oppossum (Didelphis aurita) retina.

Immunocytochemistry for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, was used to determine the onset and to follow the maturation of the cholinergic cells in the retina of a marsupial, the South American opossum (Didelphis aurita). ChAT-immunoreactivity was first detected in amacrine cells in the ganglion cell layer by postnatal day 15 (P15) and in the inner nuclear layer by P35. Much later, at P50 a second sub-population of ChAT-immunoreactive cell bodies was evident in the inner nuclear layer. Processes from ChAT-immunoreactive amacrine cells were detected in the two bands of the inner plexiform layer before synaptogenesis. In the adult retina, these two bands correspond to sublamina 2 and 4 of the inner plexiform layer. In flat whole-mounted preparations, cholinergic cell density was 263 +/- 13 cells/mm2 in the ganglion cell layer and it was estimated a total of 24,000 cholinergic neurons. ChAT-immunoreactive somata showed a random pattern of distribution.

Age Factors↗

PubMed1997

GABAergic retinocollicular projection in the New World monkey Cebus apella.

GABA immunoreactivity was examined in the retina of the New World monkey Cebus apella. Labeled cell bodies were identified as horizontal, bipolar, interplexiform, amacrine and a population of putative ganglion cells. To determine whether ganglion cells were immunoreactive to GABA, double-labeling experiments were performed using Fast Blue as retrograde neuronal tracer injected into the superior colliculus. Retinas containing FB-labeled ganglion cells were subsequently incubated with antiserum against GABA. Although retinocollicular ganglion cells were found in three different layers (ganglion cell layer, inner nuclear layer and inner plexiform layer), our experiments revealed GABA-positive ganglion cells only in the outer half of the ganglion cell layer.

Amidines↗

PubMed1997

On the functional anatomy of the nucleus of the optic tract-dorsal terminal nucleus commissural connection in the opossum (Didelphis marsupialis aurita).

Immunocytochemical methods revealed the presence of GABA in cell bodies and terminals in the nucleus of the optic tract-dorsal terminal nucleus, the medial terminal nucleus, the lateral terminal nucleus and the interstitial nucleus of the superior fasciculus of the opossum (Didelphis marsupialis aurita). Moreover, after unilateral injections of rhodamine beads in the nucleus of the optic tract-dorsal terminal nucleus complex and processing for GABA, double-labelled cells were detected in the ipsilateral complex, up to 400 microns from the injected site, but not in the opposite. Analysis of the distributions of GABAergic and retrogradely-labelled cells throughout the contralateral nucleus of the optic tract-dorsal terminal nucleus showed that the highest density of GABAergic and rhodamine-labelled cells overlapped at the middle third of the complex. Previous electrophysiological data obtained in the opossum had suggested the existence, under certain conditions, of an inhibitory action between the nucleus of the optic tract-dorsal terminal nucleus of one side over the other. The absence of GABAergic commissural neurons may imply that this inhibition is mediated by an excitatory commissural pathway that activates GABAergic interneurons.

Animals↗

PubMed1997

Retinal ganglion cell depletion alters the phenotypic expression of GABA and GAD in the rat retina.

We have looked at the phenotypic expression of gamma-aminobutyric acid (GABA) and the two isoforms of its synthetic enzyme [glutamic acid decarboxylase (GAD)-65 and -67] in adult rat retinas that had the superior colliculus, pretectum and optic tract lesioned unilaterally at birth. It has been shown previously that this type of manipulation induces retrograde degeneration of retinal ganglion cells presumably without affecting other intraretinal neurons. We present evidence that GABAergic amacrine cells are affected by such manipulation. The number of cells immunoreactive for GABA, GAD-65 and GAD-67 decreased in the inner nuclear layer. In the retinal ganglion cell layer, however, the number of GABA- and GAD-65-labelled cells increased, while the number of GAD-67-labelled cells did not change. Biochemical assay showed that overall GAD activity was not altered in retinas of lesioned animals. Our results support the notion that, while neonatal lesion reorganizes the expression of GABA and GAD in the retina, enzyme activity is maintained within normal levels.

Animals↗

PubMed1996

Neurogenesis of cholinoceptive neurons in the chick retina.

Immunocytochemistry and [3H]thymidine autoradiography were combined in this study to determine the neurogenesis of cholinoceptive cells in the chick retina. After injections of [3H]thymidine between embryonic days 1 and 11, the time of birth of retinal neurons containing either the alpha 3 or the alpha 8 subunit of the nicotinic acetylcholine receptors was determined. The results indicate that the alpha 3-positive neurons in the ganglion cell layer leave the cell cycle from E2 through E7, and those in the inner nuclear layer (amacrine and displaced ganglion cells) from E2 through E9. The alpha 8-positive cells in the ganglion cell layer were born from E1 through E7, and those in the inner nuclear layer (amacrine and bipolar cells) from E2 through E11. These data suggest that the time of birth of cholinoceptive neurons in the chick retina follows the general pattern of cell generation in the chick retina, and that alpha 8-positive cells in the ganglion cell layer start to leave the cell cycle almost one day earlier than the alpha 3-positive cells in the same layer.

Acetylcholine↗

PubMed1996

Development of NADPH-diaphorase in the avian retina: regulation by calcium ions and relation to nitric oxide synthase.

Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit approximately 65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 mM calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by NG-nitro-L-arginine or L-NG-(iminoethyl) ornithine is concentration dependent with IC50 values of approximately 1 mM at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC50 values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.

Animals↗

PubMed1996

Neurotransmitter release in aggregate cultures of chick embryo retina cells.

The study of neurotransmitter release using aggregate cell cultures has been limited by the fact that cell aggregates are obtained only when cells are maintained in suspension with rotatory agitation. In this report we describe a simple and easy method that uses aggregate cell cultures to study the release of neurotransmitters. The results demonstrate that this relatively simple technique can be of great value to address the problem of neurotransmitter release and study the mechanisms of action of natural and synthetic compounds on the differentiation of functional synapses in the CNS.

Acetylcholine↗