Belmira Lara da Silveira Andrade da Costa

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.

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.

Nutritional restriction of omega-3 fatty acids alters topographical fine tuning and leads to a delay in the critical period in the rodent visual system

The development and maturation of sensory systems depends on the correct pattern of connections which occurs during a critical period when axonal elimination and synaptic plasticity are involved in the formation of topographical maps. Among the mechanisms involved in synaptic stabilization, essential fatty acids (EFAs), available only through diet, appear as precursors of signaling molecules involved in modulation of gene expression and neurotransmitter release. Omega-3 fatty acids, such as docosahexaenoic acid (DHA), are considered EFAs and are accumulated in the brain during fetal period and neonatal development. In this study, we demonstrated the effect of omega-3/DHA nutritional restriction in the long-term stabilization of connections in the visual system. Female rats were fed 5 weeks before mating with either a control (soy oil) or a restricted (coconut oil) diet. Litters were fed until postnatal day 13 (PND13), PND28 or PND42 with the same diets when they received an intraocular injection of HRP. Another group received a single retinal lesion at the temporal periphery at PND21. Omega-3 restriction induced an increase in the optical density in the superficial layers of the SC, as a result of axonal sprouting outside the main terminal zones. This effect was observed throughout the SGS, including the ventral and intermediate sub-layers at PND13 and also at PND28 and PND42. The quantification of optical densities strongly suggests a delay in axonal elimination in the omega3(-) groups. The supplementation with fish oil (DHA) was able to completely reverse the abnormal expansion of the retinocollicular projection. The same pattern of expanded terminal fields was also observed in the ipsilateral retinogeniculate pathway. The critical period window was studied in lesion experiments in either control or omega-3/DHA restricted groups. DHA restriction induced an increased sprouting of intact, ipsilateral axons at the deafferented region of the superior colliculus compared to the control group, revealing an abnormal extension of the critical period. Finally, in omega-3 restricted group we observed in the collicular visual layers normal levels of GAP-43 with decreased levels of its phosphorylated form, p-GAP-43, consistent with a reduction in synaptic stabilization. The data indicate, therefore, that chronic dietary restriction of omega-3 results in a reduction in DHA levels which delays axonal elimination and critical period closure, interfering with the maintenance of terminal fields in the visual system.

Differential vulnerability of substantia nigra and corpus striatum to oxidative insult induced by reduced dietary levels of essential fatty acids

Oxidative stress (OS) has been implicated in the etiology of certain neurodegenerative disorders. Some of these disorders have been associated with unbalanced levels of essential fatty acids (EFA). The response of certain brain regions to OS, however, is not uniform and a selective vulnerability or resilience can occur. In our previous study on rat brains, we observed that a two-generation EFA dietary restriction reduced the number and size of dopaminergic neurons in the substantia nigra (SN) rostro-dorso-medial. To understand whether OS contributes to this effect, we assessed the status of lipid peroxidation (LP) and anti-oxidant markers in both SN and corpus striatum (CS) of rats submitted to this dietary treatment for one (F1) or two (F2) generations. Wistar rats were raised from conception on control or experimental diets containing adequate or reduced levels of linoleic and α-linolenic fatty acids, respectively. LP was measured using the thiobarbituric acid reaction method (TBARS) and the total superoxide dismutase (t-SOD) and catalase (CAT) enzymatic activities were assessed. The experimental diet significantly reduced the docosahexaenoic acid (DHA) levels of SN phospholipids in the F1 (~28%) and F2 (~50%) groups. In F1 adult animals of the experimental group there was no LP in both SN and CS. Consistently, there was a significant increase in the t-SOD activity (p < 0.01) in both regions. In EF2 young animals, degeneration in dopaminergic and non-dopaminergic neurons and a significant increase in LP (p < 0.01) and decrease in the CAT activity (p < 0.001) were detected in the SN, while no inter-group difference was found for these parameters in the CS. Conversely, a significant increase in t-SOD activity (p < 0.05) was detected in the CS of the experimental group compared to the control. The results show that unbalanced EFA dietary levels reduce the redox balance in the SN and reveal mechanisms of resilience in the CS under this stressful condition.

Coexistence of GAD-65 and GAD-67 with tyrosine hydroxylase and nitric oxide synthase in amacrine and interplexiform cells of the primate, Cebus apella.

The expression of glutamate decarboxylase forms, GAD-65 and GAD-67, in GABAergic cells was studied by immunocytochemistry in the retina of the New World monkey, Cebus apella. In the innermost rows of the inner nuclear layer (INL), somata that express GABA correspond to about 45% of the total number of cells in the central retina and about 25% on the periphery. Three subsets of GABAergic amacrine cells were identified along the horizontal meridian: about 5% express only GAD-65 and 40% GAD-67, and approximately 50% contain both forms of GAD. In the INL, GAD-65 immunoreactivity was detected in broad bands around strata 1, 3, and 5. GAD-67 immunoreactivity was observed throughout all strata. Somata that expressed GAD-67 exclusively stratified only in narrow bands around strata 2 and 4 of the INL and colocalized with beta2 and beta3 subunits of GABA-A receptor. Interplexiform and amacrine cells that express GABA also express tyrosine hydroxylase (TH) or nitric oxide synthase (NOS). GAD-67 colocalized with TH or NOS in presumed amacrine cells of the inner plexiform layer (IPL) and ganglion cell layer (GCL). GAD-65 was expressed in the TH- and NOS-immunoreactive interplexiform and amacrine cells, respectively. Different from what has been described in other mammals, TH and NOS were coexpressed in some neurons, indicating a partial overlap in retinal cell populations containing dopamine or nitric oxide in this primate.

Neonatal treatment with monosodium glutamate lastingly facilitates spreading depression in the rat cortex

AIMS Monosodium glutamate (MSG) is a neuroexcitatory amino acid used in human food to enhance flavor. MSG can affect the morphological and electrophysiological organization of the brain. This effect is more severe during brain development. Here, we investigated the electrophysiological and morphological effects of MSG in the developing rat brain by characterizing changes in the excitability-related phenomenon of cortical spreading depression (CSD) and microglial reaction. MAIN METHODS From postnatal days 1-14, Wistar rat pups received 2 or 4 g/kg MSG (groups MSG-2 and MSG-4, respectively; n=9 in each group), saline (n=10) or no treatment (naïve group; n=5) every other day. At 45-60 days, CSD was recorded on two cortical points for 4h. The CSD parameters velocity, and amplitude and duration of the negative potential change were calculated. Fixative-perfused brain sections were immunolabeled with anti-IBA-1 antibodies to identify and quantify cortical microglia. KEY FINDINGS MSG-4 rats presented significantly higher velocities (4.59 ± 0.34 mm/min) than the controls (saline, 3.84 ± 0.20mm/min; naïve, 3.71 ± 0.8mm/min) and MSG-2 group (3.75 ± 0.10mm/min). The amplitude (8.8 ± 2.2 to 11.2 ± 1.9 mV) and duration (58.2 ± 7.1 to 73.6 ± 6.0s) of the negative slow potential shift was similar in all groups. MSG-treatment dose-dependently increased the microglial immunolabeling. SIGNIFICANCE The results demonstrate a novel, dose-dependent action of MSG in the developing brain, characterized by acceleration of CSD and significant microglial reaction in the cerebral cortex. The CSD effect indicates that MSG can influence cortical excitability, during brain development, as evaluated by CSD acceleration. Data suggest caution when consuming MSG, especially in developing organisms.

Topographic analysis of the ganglion cell layer in the retina of the four-eyed fish Anableps anableps.

Fish of the genus Anableps (Anablepidae, Cyprinodontiformes) have eyes that are adapted for simultaneous aerial and aquatic vision. In this study we investigate some of the corresponding retinal specializations of the adult Anableps anableps eye using retinal transverse sections and wholemounts. The linear dimensions of the retina were found to be asymmetric with a greater representation of the dorsal compared to the ventral visual field. The total number of neurons in the ganglion cell layer of the ventral hemiretina was on average 3.6 times greater than the values obtained in the dorsal hemiretina. Isodensity contour maps revealed a prominent horizontal visual streak in the ventral hemiretina with an average peak cell density of 18,286 cells/mm(2). A second less-well-developed horizontal visual streak was also observed in the dorsal hemiretina. A sub-population of large cells with soma areas between 74 and 188 microm(2) was identified and found to be distributed evenly across both hemiretinas. Together, these results show that the sampling gain of the ventral retina is significantly greater than the dorsal segment, that retinal specializations important for mediating acute vision are present in the parts of the visual field immediately above and below the surface of the water, and that visual functions related with the large ganglion cells require more even sampling across the visual field. The relevance of these retinal specializations to the feeding and other behavioral strategies adopted by Anableps is discussed.

GFAP Expression in Astrocytes of Suprachiasmatic Nucleus and Medial Preoptic Area are Differentially Affected by Malnutrition during Rat Brain Development

Abstract The aim of the present study was investigate, in young rats, the effects of malnutrition on astrocyte distribution of two hypothalamic regions, the circadian pacemaker suprachiasmatic nucleus (SCN) and the medial preoptic area (MPA). Control rats were born from mothers fed on commercial diet since gestation and malnourished rats from mothers fed on multideficient diet, from the beginning of gestation (GLA group) or from the onset of lactation (LA group). After weaning, pups received ad libitum the same diet as their mothers, and were maintained under a 12/12 h light/dark cycle. The animals were analyzed either at 30-33, or 60-63 days of life. Brain coronal sections (50 μm) were processed to visualize glial fibrillary acidic protein (GFAP) immunoreactivity. Compared to control rats, both malnourished groups of 30 and 60 days exhibited a reduced number of GFAP-immunoreactive astrocytes in the SCN. The total GFAP-immunoreactive area in the SCN of the GLA group differed from the control group at both age ranges analyzed. The GFAP expression as measured by the relative optical density (ROD) exhibited a 50-60% reduction in the MPA in both malnourished groups, compared to controls. The results suggest that malnutrition early in life leads to alterations in gliogenesis or glial cell proliferation in both nuclei, being these alterations greater in the MPA. Compensatory plasticity mechanisms in the GFAP-expression seem to be developed in the astrocyte differentiation process in the SCN, especially when the malnutrition is installed from the lactation.

Comparative study of glutamate mediated γ-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 Lockes 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.

Resilience in migraine brains: decrease of coherence after photic stimulation

Background: During migraine attacks, patients generally have photophobia and phonophobia and seek for environments with less sensorial stimulation. Present work aimed to quantify cortical partial directed coherence (PDC) of electroencephalographic (EEG) recordings from migraine patients and controls in occipital, parietal, and frontal areas with or without photic stimulation. Our hypothesis is that migraine patients with visual aura might have neuronal networks with higher coherence than controls even in interictal periods due to a predisposition in sensory cortical processing. Methods: Eleven adult women with migraine with visual aura (at least 48 h without previous attacks) and seven healthy adult woman were submitted to EEG recording in basal state and during photic stimulation. Results: When compared to healthy volunteers, migraine patients show different coherence profiles. Migraine patients had greater coherence than controls during the basal period (without photic stimulation), showing predisposition for sensory processing in many frequency ranges. After photic stimulation, patients showed a decrease in cortical coherence while controls had an increase. Conclusions: When compared to healty subjects, migraineurs show increased cortical coherence before photic stimulation, but a decrease when stimulation starts. This may be the expression of a resilience mechanism that allows migraineurs the interictal period. The PDC analysis permits to address a patient coherence profile, or “coherence map,” that can be utilized for management of the headache disorder or following up treatments.