Retinal development impairment and degenerative alterations in adult rats subjected to post-natal malnutrition.

BACKGROUND: The early stages of central nervous system (CNS) development are extremely important. Key events such as neurogenesis, gliogenesis, synaptogenesis, and ontogenesis occur. Malnutrition promotes alterations in CNS development, including the retinal development. During retinal development, malnutrition can induce a delay in some important events, such as neurotransmitter expression and neurogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Postpartum Wistar rats were fed either a commercial diet or a multideficient diet. Pups were breastfed by these rats, and from PND21 were kept with the same diet until PND45. We investigated the effects of malnutrition on adult retinal tissue with regard to (1) endogenous gamma-amino butyric acid (GABA) release induced by excitatory amino acids (EAAs) and (2) the expression of cellular markers related to degenerative events, such as reactive gliosis, microglial activation, cell proliferation and cell death. Endogenous GABA release induced by EAAs was higher in the retina of malnourished rats. The Müller cell population was reduced and displayed alterations in their phenotype profile compatible with reactive gliosis. The expression of glutamine synthetase and markers of cellular proliferation were higher in the retina of malnourished rats. Additionally, retinal dysplasia-like structures were present, indicating disturbance in the cell cycle machinery. CONCLUSION/SIGNIFICANCE: The current study provides evidence that the adult retina shows degenerative processes induced by long-term malnutrition during the postnatal development. These findings have high clinical significance with regard to the identification of possible targets for interventions in malnourished patients.

Hippocampal biomarkers of fear memory in an animal model of generalized anxiety disorder.

Generalized anxiety disorder (GAD) is highly prevalent and incapacitating. Here we used the Carioca High-Conditioned Freezing (CHF) rats, a previously validated animal model for GAD, to identify biomarkers and structural changes in the hippocampus that could be part of the underlying mechanisms of their high-anxiety profile. Spatial and fear memory was assessed in the Morris water maze and passive avoidance test. Serum corticosterone levels, immunofluorescence for glucocorticoid receptors (GR) in the dentate gyrus (DG), and western blotting for hippocampal brain derived neurotrophic factor (BDNF) were performed. Immunohistochemistry for markers of cell proliferation (bromodeoxiuridine/Ki-67), neuroblasts (doublecortin), and cell survival were undertaken in the DG, along with spine staining (Golgi) and dendritic arborization tracing. Hippocampal GABA release was assessed by neurochemical assay. Fear memory was higher among CHF rats whilst spatial learning was preserved. Serum corticosterone levels were increased, with decreased GR expression. No differences were observed in hippocampal cell proliferation/survival, but the number of newborn neurons was decreased, along with their number and length of tertiary dendrites. Increased expression of proBDNF and dendritic spines was observed; lower ratio of GABA release in the hippocampus was also verified. These findings suggest that generalized anxiety/fear could be associated with different hippocampal biomarkers, such as increased spine density, possibly as a compensatory mechanism for the decreased hippocampal number of neuroblasts and dendritic arborization triggered by high corticosterone. Disruption of GABAergic signaling and BDNF impairment are also proposed as part of the hippocampal mechanisms possibly underlying the anxious phenotype of this model.

Translational findings on brain-derived neurotrophic factor and anxiety: contributions from basic research to clinical practice.

BACKGROUND/AIMS: Anxious responses are evolutionarily adaptive, but excessive fear can become disabling and lead to anxiety disorders. Translational models of anxiety might be useful sources for understanding the neurobiology of fear and anxiety and can contribute to future proposals of therapeutic intervention for the disorders studied. Brain-derived neurotrophic factor (BDNF), which is known for its importance on neuroplasticity and contextual memory, has emerged as a relevant element for emotional memory. Recent studies show that the Val(66)Met BDNF polymorphism correlates with various psychiatric disorders, including anxiety, but there are several differences between experimental and clinical studies. METHODS: In this work, we review the literature focused on the BDNF Val(66)Met polymorphism and anxiety, and discuss biological findings from animal models to clinical studies. RESULTS: As occurs with other psychiatric disorders, anxiety correlates with anatomical, behavioral and physiological changes related to the BDNF polymorphism. In animal studies, it has been shown that a significant decrease in regulated secretion from both BDNFVal/Met and BDNFMet/Met neurons represented a significant decrease in available BDNF. CONCLUSION: These studies suggest that developing pharmacological strategies facilitating the release of BDNF from synapses or prolongation of the half-life of secreted BDNF may improve the therapeutic responses of humans expressing the BDNF polymorphism.

Regulation of GABA content by glucose in the chick retina.

Some visual information is processed in the retina by γ-aminobutyric acid (GABA) signaling. Once retinal degeneration and visual impairment caused by diabetic retinopathy (DR) are affecting an increasing number of people worldwide, and the disease is characterized by hyper- and hypoglycemic events, the authors aimed to investigate how retinal GABA cell content is affected by variations in glucose availability. Using the ex vivo chick retinas exposed to different glucose concentrations, we observed that amacrine cells from both inner nuclear layer (INL) and ganglion cell layer (GCL) as well as their processes in the inner plexiform layer (IPL) released GABA through GABA transporter-1 (GAT-1) after 30 min of glucose deprivation. Extending this insult to 60 min triggered a permanent loss of GABA-positive amacrine cells, caused swelling of IPL and cell death. High glucose (35 mM) for 30 min induced an increment in GABA immunolabeling in both outer and inner retina. Further, glucose deprivation effects could not be reverted by basal glucose levels and high glucose did not prevent GABA loss upon a glucose deprivation insult. Therefore, GABA cell content is differently affected by short-term variations in glucose availability. While high glucose modulates outer and inner GABAergic circuits, glucose deprivation affects mainly the inner retina. Also, consecutive alteration in glucose supply was not able to rescue basal GABA content. Therefore, glucose oscillations interfering with GABAergic retinal functioning during early stages of retinopathies should be further investigated.

The role of dietary polyphenols on adult hippocampal neurogenesis: molecular mechanisms and behavioural effects on depression and anxiety.

Although it has been long believed that new neurons were only generated during development, there is now growing evidence indicating that at least two regions in the brain are capable of continuously generating functional neurons: the subventricular zone and the dentate gyrus of the hippocampus. Adult hippocampal neurogenesis (AHN) is a widely observed phenomenon verified in different adult mammalian species including humans. Factors such as environmental enrichment, voluntary exercise, and diet have been linked to increased levels of AHN. Conversely, aging, stress, anxiety and depression have been suggested to hinder it. However, the mechanisms underlying these effects are still unclear and yet to be determined. In this paper, we discuss some recent findings addressing the effects of different dietary polyphenols on hippocampal cell proliferation and differentiation, models of anxiety, and depression as well as some proposed molecular mechanisms underlying those effects with particular focus on those related to AHN. As a whole, dietary polyphenols seem to exert positive effects on anxiety and depression, possibly in part via regulation of AHN. Studies on the effects of dietary polyphenols on behaviour and AHN may play an important role in the approach to use diet as part of the therapeutic interventions for mental-health-related conditions.

Current methodological designs of fMRI studies of panic disorder: can data be compared?

Panic disorder (PD) is a pluridimensional condition that leads to psychological suffering. Due to advances in neuroimaging techniques, important contributions have been made in the understanding of the neurobiological basis of PD. However, because of diverging research designs and protocols, more conclusive data concerning the neurocircuitry of PD remain difficult to achieve. To address this issue, a bibliographical search was performed using the Institute for Scientific Information Web of Science and Medline/PubMed databases. Fifteen articles were found, and their research methodology including sample, comorbidity, gender, and pharmacological criteria were explored. Although current functional magnetic resonance imaging studies of PD constitute fundamental tools for health sciences, more uniform research protocols must be implemented to provide more consistent and conclusive data concerning the neural substrates of PD.(AU)

Current methodological designs of fMRI studies of panic disorder: can data be compared?

Panic disorder (PD) is a pluridimensional condition that leads to psychological suffering. Due to advances in neuroimaging techniques, important contributions have been made in the understanding of the neurobiological basis of PD. However, because of diverging research designs and protocols, more conclusive data concerning the neurocircuitry of PD remain difficult to achieve. To address this issue, a bibliographical search was performed using the Institute for Scientific Information Web of Science and Medline/PubMed databases. Fifteen articles were found, and their research methodology including sample, comorbidity, gender, and pharmacological criteria were explored. Although current functional magnetic resonance imaging studies of PD constitute fundamental tools for health sciences, more uniform research protocols must be implemented to provide more consistent and conclusive data concerning the neural substrates of PD.

Current concept of anxiety: implications from Darwin to the DSM-V for the diagnosis of generalized anxiety disorder.

This article proposes a revision of the historical evolution of the concepts of generalized anxiety disorder (GAD). Currently, Darwin's evolutionary theory is the hegemonic paradigm for modern science and influences research on mental disorders. Throughout the 20th Century, the editions of the Diagnostic and Statistical Manual of Mental Disorders (DSM; American Psychiatric Association) have changed the diagnostic criteria for GAD, reflecting the prevailing psychiatric understanding of this disorder. The prevalence and symptoms of major depression and GAD show the fragility of the categorical conception of these conditions. Differences in cultural views towards anxiety disorders also suggest that anxiety cannot have a uniform definition. This article provides contributions for reflecting future guidelines concerning the diagnostic criteria for GAD in DSM-V.

Current findings of fMRI in panic disorder: contributions for the fear neurocircuitry and CBT effects.

Thanks to brain imaging great advances have been made concerning the comprehension of neural substrates related to panic disorder (PD). This article aims to: review the recent functional MRI (fMRI) studies concerning PD; correlate the PD fMRI neurobiological findings with the fear neurocircuitry hypothesis; discuss the fear neurocircuitry hypothesis and link it to cognitive-behavior therapy findings; and comment on fMRI study limitations and suggest methodological changes for future research. As a whole, there is increasing evidence that brain structures such as the prefrontal cortex, the anterior cingulate cortex and limbic areas (hippocampus and amygdala) might play a major role in the panic response.

Behavioral profile and dorsal hippocampal cells in carioca high-conditioned freezing rats.

Selection for contextual fear conditioning is an important behavioral paradigm for studying the role of genetic variables and their interaction with the surrounding environment in the etiology and development of anxiety disorders. Recently, a new line of animals selectively bred for high levels of freezing in response to contextual cues previously associated with footshock was developed from a Wistar population. The purpose of the present study was to evaluate the emotional and cognitive aspects of this new line of animals, which has been named Carioca High-Freezing (CHF). For the characterization of anxious behavior, CHF and control animals were tested in the elevated plus-maze (EPM) and the social interaction test. CHF animals were significantly more anxious than control rats in terms of both the number of entries into EPM open arms and the percentage of time spent in these arms. The time spent in social interaction behavior was also significantly decreased. No statistical differences were found in locomotor activity, as measured by both the number of entries into the closed arms of the EPM and the number of crossings into the social interaction test arena. No differences between CHF and control groups were found in the depression forced swimming test, suggesting that the anxiety trait selected in the CHF line did not interact with affective disorders traits such as those for depression. Cognitive aspects of the CHF rats were evaluated in the object recognition task. Results from this test indicated no difference between the two groups. The present study also encompassed histological analysis of the dorsal hippocampus from CHF and control animals. Results revealed an absence of qualitative and quantitative differences between these two groups of animals in cells located in the dentate gyrus, CA1, and CA3 areas. Therefore, future studies are required to further investigate the possible neural mechanisms involved in the origin and development of the anxious phenotype observed in this model.

Differential immunodetection of L-DOPA decarboxylase and tyrosine hydroxylase in the vertebrate retina.

The immunocytochemical staining of L-DOPA decarboxylase (DDC) and tyrosine hydroxylase (TH) in cells of the developing avian retina and in cells of the retina of adult rats and opossum were compared. DDC was identified at embryonic day 8 in the chick, in cells in the inner nuclear layer (INL). At embryonic day 13, two types of DDC positive cells were observed; type 1, with the soma located in the innermost layer of the INL; and type 2, with the soma located two cell rows from the innermost part of the INL. Immunolabeling for DDC in the presumptive outer plexiform layer was more clearly defined at embryonic day 19 and at post-hatched day 7. Processes of DDC labeled cells extended into the inner plexiform layer, supporting the amacrine identity of these cells. Dot-blot analysis revealed that DDC could be detected at embryonic day 4. Confocal microscopy showed that at embryonic day 10, DDC positive cells, but not TH positive cells, were found. After embryonic day 13, cells immunolabeled for DDC and DDC plus TH were detected. The mean density of DDC positive cells quantified in whole-mounted chick retinas showed that in all stages the density of DDC positive cells exceeded that of TH positive cells by 10-13-fold. As for the avian retina, density of DDC positive cells in opossum and rat retinas exceeded the density of TH positive cells. In opossum, Müller fibers were also clearly labeled for DDC but not for TH. We propose the hypothesis that the dopamine synthesis in the developing avian retina as well as in the mature rat and opossum tissue is greater than would be expected from TH staining alone.

Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: late expression of beta1-adrenergic receptor shifts norepinephrine specificity in the adult tissue.

Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC(50)=10microM) and norepinephrine (EC(50)=30microM), but not the beta(1)-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2microM SCH23390, a D(1) receptor antagonist, but not by alprenolol (beta-adrenoceptor antagonist). In P15 retinas, 100microM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10microM), but not by 2microM SCH23390. Embryonic and adult retina display beta(1) adrenergic receptor mRNA as detected by RT-PCR, but the beta(1) adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D(1) dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D(1) receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via beta(1)-like adrenergic receptors in the mature tissue.

Pituitary adenylate cyclase-activating polypeptide (PACAP) can act as determinant of the tyrosine hydroxylase phenotype of dopaminergic cells during retina development.

In the chick retina, dopaminergic cells are generated between embryonic days 3 and 7 (E3/E7). However, the expression of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, is only detected after E11/E12. During the interval comprising E7 to E12, signals conveyed by cAMP are important to determine the TH phenotype. The present study shows that pituitary adenylyl cyclase-activating polypeptide (PACAP), via cAMP, is a major endogenous component in defining the TH phenotype of retina dopaminergic cells during development. PACAP type 1 receptor and its mRNA were detected in retinas since E6. PACAP was also immunodetected in cells localized in the inner nuclear layer of retinas since E8. This peptide promoted greater than 10-fold increase in cAMP accumulation of retinas obtained from embryos since E8, an effect that was blocked by PACAP6-38 (PAC1 receptor antagonist). In cultured retina cells from E8 and E9, maintained for 6 days in vitro with 10 nM PACAP (for 5 days), the number of dopaminergic cells expressing tyrosine hydroxylase increased 2.4-fold. The cAMP analog, 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) also increased the number of tyrosine hydroxylase-positive cells by 4- to 6-fold. IBMX plus PACAP treatment resulted in 17-fold increase in the number of cells positive for tyrosine hydroxylase. Under this condition the amount of tyrosine hydroxylase expression, as detected by western blot analysis, was also increased. The protein kinase-A inhibitor, rp-cAMPS, significantly reduced the effect of PACAP. Our data show that this peptide is an important factor influencing the definition of the tyrosine hydroxylase phenotype of retina dopaminergic cells within a narrow window of development.

Local differences in GABA release induced by excitatory amino acids during retina development: selective activation of NMDA receptors by aspartate in the inner retina.

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.

GABA(Abeta2-3) immunoreactive cells in the developing chick retina.

Gama-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). It has been shown that GABA is an important factor for CNS maturation and that its functions are mainly mediated by GABA(A) receptors. Thus, in order to fully comprehend the role of GABA during development, it is essential to establish the developmental features of the catalytic subunits (beta) of GABA(A) receptor. Here, we determine the ontogenesis and neurogenesis of cells expressing beta2-3 subunits of GABA(A) receptor (GABA(Abeta2-3)) in the chick retina. In the ontogenetic experiments, only the immunohistochemistry for GABA(Abeta2-3) approach was employed. For neurogenesis a double-labeling method (autoradiography and immunohistochemistry) was applied. [H(3)]-thymidine was injected into eggs (2-11 days) and the embryos were sacrificed at embryonic day 19 (E19). GABA(Abeta2-3) immunohistochemistry was processed and then autoradiography was performed. We used a cumulative counting method to quantify the autoradiographic grains. The ontogenesis study revealed that at E9, GABA(Abeta2-3) immunoreactivity was restricted to the inner plexiform layer and the first cell bodies immunoreactive to GABA(Abeta2-3) were seen at E14. Thereafter, the number of cell bodies and the intensity of GABA(Abeta2-3) immunoreactivity increased until the adult pattern was established. The neurogenesis study showed that cells that will express GABA(Abeta2-3) were generated between E6 and E9. In addition, from E7 to E9 the rate of neurogenesis of GABA(Abeta2-3) immunoreactive cells quickly increases. Therefore, the detection of GABA(Abeta2-3) occurred only after the end of generation period of this cell population.