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Alzheimer disease's (AD) is a neurodegenerative disorder characterized by cognitive and behavioral impairment. The central nervous system is an important target of thyroid hormones (TH). An inverse association between serum triiodothyronine (T3) levels and the risk of AD symptoms and progression has been reported. We investigated the effects of T3 treatment on the depression-like behavior in male transgenic 3xTg-AD mice. Animals were divided into 2 groups treated with daily intraperitoneal injections of 20 ng/g of body weight (b.w.) L-T3 (T3 group) or saline (vehicle, control group). The experimental protocol lasted 21 days, and behavioral tests were conducted on days 18-20. At the end of the experiment, the TH profile and hippocampal gene expression were evaluated. The T3-treated group significantly increased serum T3 and decreased thyroxine (T4) levels. When compared to control hippocampal samples, the T3 group exhibited attenuated glycogen synthase kinase-3 (GSK3), metalloproteinase 10 (ADAM10), amyloid-beta precursor-protein (APP), serotonin transporter (SERT), 5HT1A receptor, monocarboxylate transporter 8 (MCT8) and bone morphogenetic protein 7 (BMP-7) gene expression, whereas augmented superoxide dismutase 2 (SOD2) and Hairless gene expression. T3-treated animals also displayed reduced immobility time in both the tail suspension and forced swim tests, and in the latter presented a higher latency time compared to the control group. Therefore, our findings suggest that in an AD mouse model, T3 supplementation promotes improvements in depression-like behavior, through the modulation of the serotonergic related genes involved in the transmission mediated by 5HT1A receptors and serotonin reuptake, and attenuated disease progression.

Multiple sclerosis (MS) is an inflammatory disease of the CNS, characterized by demyelination, focal inflammatory infiltrates and axonal damage. Oxidative stress has been linked to MS pathology. Previous studies have suggested the involvement of NADPH oxidase 2 (Nox2), an enzyme that catalyzes the reduction of oxygen to produce reactive oxygen species, in the MS pathogenesis. The mechanisms of Nox2 activation on MS are unknown. The purpose of this study was to investigate the effect of Nox2 deletion on experimental autoimmune encephalomyelitis (EAE) onset and severity, on astrocyte activation as well as on pro-inflammatory and anti-inflammatory cytokine induction in striatum and motor cortex. Methodology: Subcutaneous injection of MOG35-55 emulsified with complete Freund’s adjuvant was used to evaluate the effect of Nox2 depletion on EAE-induced encephalopathy. Striatum and motor cortices were isolated and evaluated by immunoblotting and RT-PCR. Results: Nox2 deletion resulted in clinical improvement of the disease and prevented astrocyte activation following EAE induction. Nox2 deletion prevented EAE-induced induction of pro-inflammatory cytokines and stimulated the expression of the anti-inflammatory cytokines IL-4 and IL-10. Conclusions: Our data suggest that Nox2 is involved on the EAE pathogenesis. IL-4 and IL-10 are likely to be involved on the protective mechanism observed following Nox2 deletion.

Retinopathy, a common complication of diabetes, is characterized by an unbalanced production of nitric oxide (NO), a process regulated by nitric oxide synthase (NOS). We hypothesized that retinopathy might stem from changes in the insulin receptor substrate (IRS)/PI3K/AKT pathway and/or expression of NOS isoforms. Thus, we analysed the morphology and apoptosis index in retinas of obese rats in whom insulin resistance had been induced by a high-fat diet (HFD). Immunoblotting analysis revealed that the retinal tissue of HFD rats had lower levels of AKT(1) , eNOS and nNOS protein than those of samples taken from control animals. Furthermore, immunohistochemical analyses indicated higher levels of iNOS and 4-hydroxynonenal and a larger number of apoptotic nuclei in HFD rats. Finally, both the inner and outer retinal layers of HFD rats were thinner than those in their control counterparts. When considered alongside previous results, these patterns suggest two major ways in which HFD might impact animals: direct activity of ingested fatty acids and/or via insulin-resistance-induced changes in intracellular pathways. We discuss these possibilities in further detail and advocate the use of this animal model for further understanding relationships between retinopathy, metabolic syndrome and type 2 diabetes.

Nitric oxide (NO) seems to act as a retrograde messenger in the establishment and refinement of synaptic connections during development and in neural plasticity processes in adult life. Previous studies have shown that the expression of NO synthase (NOS) in the optic tectum of developing chicks is regulated by the retinal innervation. The aim of this study was to observe the effects of unilateral retinal lesions upon the expression of NOS in central visual areas of the adult chick brain. After different survival times (1-30 days), the chick brains were submitted to immunohistochemical, immunoblotting, and NO imaging procedures to evaluate NOS expression and activity. Our results indicate that NOS expression in visual areas is also regulated by retinal innervation in the adult chick. However, differently from the case in the developing animal, the deafferentation seems to generate an increase of the NOS expression in retinorecipient visual areas. Our results suggest that NOS expression in visual structures of the adult chick brain may be down-regulated by the retinal innervation. Alternatively, the increase of NOS expression observed after retinal removal could be an indicative of a role of the NO system in plasticity processes.

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.

The purpose of this study was to investigate the effects of topical application of 3 M KCl for 20 minutes to the surface of the rat visual cortex upon the expression of presumptive neuroprotective molecules. Conventional immunohistochemical and immunoblotting techniques revealed a marked reduction of the expression of calbindin and parvalbumin after application of KCl, whereas calretinin, nitric oxide synthase, and the alpha7 subunit of the nicotinic acetylcholine receptor clearly increased following that procedure. Such effects were quantified both in terms of the number of immunoreactive perikarya and optical density of immunoblottings, and were observed as soon as 1 h, and to last at least until 15 days, following KCl application. These results suggest that calbindin and parvalbumin may not be able to exert an appreciable neuroprotective effect in the presence of KCl. On the other hand, calretinin, nitric oxide synthase, and the alpha7-containing nicotinic receptors are possibly upregulated to protect the neurons in which they are expressed.

Anterograde transport of cholera toxin subunit B (CTb) was used to study the retinal projections in birds, with an emphasis on retinohypothalamic connections. Pigeons (Columba livia) were deeply anesthetized and received unilateral intraocular injections of CTb. In addition to known contralateral retinorecipient regions, CTb-immunoreactive fibers and presumptive terminals were found in several ipsilateral regions, such as the nucleus of the basal optic root, ventral lateral geniculate nucleus, intergeniculate leaflet, nucleus lateralis anterior, area pretectalis, and nucleus pretectalis diffusus. In the hypothalamus, CTb-immunoreactive fibers were observed in at least two contralateral cell groups, a medial hypothalamic retinorecipient nucleus, and a lateral hypothalamic retinorecipient nucleus. To compare retinorecipient hypothalamic nuclei in pigeons with the mammalian suprachiasmatic nucleus, double-label experiments were conducted to study the existence of neurophysin-like immunoreactivity in the retinorecipient avian hypothalamus. The results showed that only cell bodies in the medial hypothalamic nucleus contained neurophysin-like immunoreactivity. The results demonstrate CTb to be a sensitive anterograde tracer and provide further anatomical information on the avian equivalent of the mammalian suprachiasmatic nucleus.

In birds, neurons of the isthmo-optic nucleus (ION), as well as “ectopic” neurons, send axons to the retina, where they synapse on cells in the inner nuclear layer (INL). Previous work has shown that centrifugal axons can be divided into two anatomically distinct types depending on their mode of termination: either “convergent” or “divergent” (Ramon y Cajal, 1889; Maturana & Frenk, 1965). We show that cytochrome-oxidase histochemistry specifically labels “convergent” centrifugal axons and target neurons which appear to be amacrine cells, as well as three “types” of ganglion cells: two types found in the INL (displaced ganglion cells) and one in the ganglion cell layer. Labeled target amacrine cells have distinct darkly labeled “nests” of boutons enveloping the somas, are associated with labeled centrifugal fibers, and are confined to central retina. Lesions of the isthmo-optic tract abolish the cytochrome-oxidase labeling in the centrifugal axons and in the target amacrine cells but not in the ganglion cells. Cytochrome-oxidase-labeled ganglion cells in the INL are large; one type is oval and similar to the classical displaced ganglion cells of Dogiel, which have been reported to receive centrifugal input; the other type is rounder. Rhodamine beads injected into the accessory optic system results in retrograde label in both types of cells, showing that two distinct types of displaced ganglion cells project to the accessory optic system in chickens. The ganglion cells in the ganglion cell layer that label for cytochrome oxidase also project to the accessory optic system. These have proximal dendrites that ramify in the outer inner plexiform layer. Neither the target amacrine cells nor either of the displaced ganglion cells are immunoreactive for the inhibitory transmitter gamma aminobutyric acid. At least some of the target amacrine cells may, however, be cholinoceptive: we found that the antibody to the alpha-7 subunit of the nicotinic ACh receptor labels a population of cells in the INL that are similar in location, size, and the presence of labeled bouton-like structures to those we find labeled with cytochrome oxidase. This antibody also labels neurons in the ION proper but not ectopic cells. In conclusion, it appears that cytochrome oxidase may be a marker for “convergent” centrifugal axons and at least one of their target cells in the INL.

Immunohistochemical and tracing techniques were used in combination to reveal the source of a neuropeptide Y-like immunoreactive (NPY-LI) plexus in the nucleus of the basal optic root (nBOR) of the pigeon accessory optic system. Injections of rhodamine-labeled latex microspheres into nBOR produced retrograde labeling of a population of neurons interposed between the principal optic nucleus of the dorsolateral thalamus (equivalent to the mammalian dorsal lateral geniculate nucleus) and the ventral lateral geniculate nucleus. The retrogradely labeled neurons were distributed mainly in the immediate vicinity of the lateral, dorsal, and ventral aspects of the nucleus rotundus. Immunohistochemical methods revealed many NPY-containing somata within the same intergeniculate thalamic area. Double-labeling immunohistochemical and retrograde tracing experiments evidenced that many NPY-LI neurons in the intergeniculate area contained rhodamine microspheres that had been previously injected into the ipsilateral nBOR. The projection of that general thalamic area to the nBOR was then confirmed by means of anterograde transport of Phaseolus vulgaris leucoagglutinin. In these experiments, the intergeniculate region was demonstrated to project to all divisions of the nBOR and to every other retino-recipient structure, including the suprachiasmatic nucleus. Finally, electrolytic lesions of the intergeniculate area produced a dramatic reduction in the number of NPY-LI axons and terminals within the ipsilateral nBOR and also within other retino-recipient structures. These data indicate the existence of a thalamic NPY-LI projection to the pigeon nBOR of the accessory optic system. This chemically specific projection originates from the intergeniculate area, which was shown in this study to project to all other retino-recipient structures. Thus, NPY may have a role in the functional organization of the accessory optic system and also of the avian visual system as a whole.

An antiserum directed against tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of dopamine, was used to study the pigeon retina. Labeled cells were observed in both the inner nuclear layer (INL) and ganglion cell layer (GCL). Two populations of TH-immunoreactive neurons were observed in the INL. Some of these cells were 7−10 μ in diameter and gave rise to processes that arborized in three Layers of the inner plexiform layer (IPL). These cells appeared similar to the dopaminergic amacrine cells described previously (Marc, 1988). Other labeled cells in the INL were 12−20 μ in diameter and were recognizable as a previously described subpopulation of TH-immunoreactive displaced ganglion cells (Britto et al., 1988).A population of labeled cells was observed in the GCL. Counts of these cells in two retinae revealed 5000 and 7000 cells, respectively. They ranged in size from 8−15 μ in diameter in the central retina and from 8−20 m in diameter in the peripheral retina. The density of labeled cells was highest in the central retina and red field and lowest in the retinal periphery. The difference in cell size and cell density as a function of eccentricity is characteristic of the total population of ganglion cells in the avian retina (Ehrlich, 1981; Hayes, 1982). Some of the TH-positive cells in the GCL could be classified as ganglion cells for two reasons: (1) The axons of many of the TH-positive cells in the GCL were TH-immunoreactive as well and could be followed to the optic nerve head. (2) The injection of rhodamine-labeled microspheres into the nucleus geniculatus lateralis, pars ventralis (GLv), resulted in the retrograde labeling of many of the TH-positive cells in the contralateral retina.

Single units within the medial terminal nucleus of the accessory optic system were recorded and examined for their responses to a moving pattern, in both intact and decorticated urethane-anesthetized rats. The preferred directions of motion in control rats were mainly upward with a temporal component and downward with a nasal component. The responses to upward motion were almost absent after cortical ablation, with most units now preferring temporal or downward-nasal directions. These data suggest that cortical structures modulate the directional selectivity of accessory optic neurons in the rat.

Immunohistochemical and retrograde tracing techniques were combined to study the retinal ganglion cells which project to the pars ventralis of the lateral geniculate nucleus (GLv) in the pigeon. Using two different fluorescent tracers, two histochemically-distinct populations of ganglion cells were found to project to both the GLv and the optic tectum. The first population of ganglion cells exhibited tyrosine hydroxylase-like immunoreactivity and represented about 20% of all ganglion cells which were retrogradely labeled from the GLv. The second population of ganglion cells showed substance P-like immunoreactivity and represented about 13% of all ganglion cells projecting to the GLv. These results confirm earlier suggestions that the retinal axons projecting to the GLv also project elsewhere and demonstrate that heterogeneity of retinal ganglion cells transmitters is evident even within a single retino-recipient nucleus such as the GLv.

In birds, displaced ganglion cells (DGCs) constitute the exclusive source of retinal input to the nucleus of the basal optic root (nBOR) of the accessory optic system. Tyrosine-hydroxylase (TH) immunoreactivity was examined in the pigeon retina after injections of rhodamine-labeled microspheres into the nBOR. A population of about 400 DGCs was observed in each case to exhibit both TH immunoreactivity and rhodamine bead fluorescence. This corresponded to about 10-15% of the total number of identified DGCs in each retina. Double-labeled cells were medium- to large-size (12 to 20 microns in the largest axis) and were always located at the border between the inner nuclear and the inner plexiform layers. Their dendrites could be followed horizontally in lamina 1 of the inner plexiform layer for up to 300 microns from the cell body. The distribution of double-labeled DGCs appeared to be mostly peripheral, matching the overall distribution of identified DGCs. Larger DGCs (21-28 microns) were never seen to contain TH immunoreactivity. Examination of brain sections revealed plexuses of thin varicose TH-positive axons in all subdivisions of the nBOR. Unilateral enucleation produced an almost complete elimination of TH immunoreactivity in the contralateral nucleus. Such results suggest the existence of a population of catecholaminergic DGCs projecting into the accessory optic system of the pigeon. They also support the emerging hypothesis concerning the neurotransmitter heterogeneity of ganglion cells in the vertebrate retina.

The directional selectivity of units within the nucleus of the basal optic root (nBOR) of the accessory optic system (AOS) was studied before and after lesions of the visual telencephalon (visual Wulst) in urethane-anesthetized pigeons. In intact pigeons, most nBOR units preferred upward motion with a temporal component or downward motion with a nasal component. The ipsilateral and bilateral telencephalic lesions generated a dramatic reduction in the number of cells with optimal responses to upward motion. The overall distribution of preferred directions was still bimodal following ipsilateral or bilateral Wulst lesions, with most units showing best responses to a straight temporal or to downward-nasal directions. The contralateral Wulst lesions produced, instead, a marked reduction in downward preferences. The nBOR units which were studied in these cases showed mainly upward-temporal and upward-nasal responses. These data suggest an involvement of the visual Wulst in the determination of the dictional selectivity of nBOR neurons in the pigeon. Specifically, the responses of nBOR units to upward motion appeared to depend on the integrity of the telencephalic descending systems which impinge, in both direct and indirect ways, upon that AOS nucleus. Taken together with data for the mammalian AOS, the present results indicate that nonretinal afferents to AOS nuclei have an important role in the functional organization of that subcortical visual pathway.