Andréa S. Torrão

Hypoactivity of the Central Dopaminergic System and Autistic-Like Behavior Induced by a Single Early Prenatal Exposure to Lipopolysaccharide

The aim of the present study was to evaluate the behavioral patterns associated with autism and the prevalence of these behaviors in males and females, to verify whether our model of lipopolysaccharide (LPS) administration represents an experimental model of autism. For this, we prenatally exposed Wistar rats to LPS (100 μg/kg, intraperitoneally, on gestational day 9.5), which mimics infection by gram‐negative bacteria. Furthermore, because the exact mechanisms by which autism develops are still unknown, we investigated the neurological mechanisms that might underlie the behavioral alterations that were observed. Because we previously had demonstrated that prenatal LPS decreases striatal dopamine (DA) and metabolite levels, the striatal dopaminergic system (tyrosine hydroxylase [TH] and DA receptors D1a and D2) and glial cells (astrocytes and microglia) were analyzed by using immunohistochemistry, immunoblotting, and real‐time PCR. Our results show that prenatal LPS exposure impaired communication (ultrasonic vocalizations) in male pups and learning and memory (T‐maze spontaneous alternation) in male adults, as well as inducing repetitive/restricted behavior, but did not change social interactions in either infancy (play behavior) or adulthood in females. Moreover, although the expression of DA receptors was unchanged, the experimental animals exhibited reduced striatal TH levels, indicating that reduced DA synthesis impaired the striatal dopaminergic system. The expression of glial cell markers was not increased, which suggests that prenatal LPS did not induce permanent neuroinflammation in the striatum. Together with our previous finding of social impairments in males, the present findings demonstrate that prenatal LPS induced autism‐like effects and also a hypoactivation of the dopaminergic system.

BDNF RECEPTOR BLOCKADE HINDERS THE BENEFICIAL EFFECTS OF EXERCISE IN A RAT MODEL OF PARKINSON'S DISEASE

Physical exercise is known to produce beneficial effects to the nervous system. In most cases, brain-derived neurotrophic factor (BDNF) is involved in such effects. However, little is known on the role of BDNF in exercise-related effects on Parkinsons disease (PD). The aim of this study was to investigate the effects of intermittent treadmill exercise-induced behavioral and histological/neurochemical changes in a rat model of unilateral PD induced by striatal injection of 6-hydroxydopamine (6-OHDA), and the role of BDNF in the exercise effects. Adult male Wistar rats were divided into two main groups: (1) injection of K252a (a blocker of BDNF receptors), and (2) without BDNF receptor blockade. These groups were then subdivided into four groups: control (CLT), sedentary (SED, non-exercised with induction of PD), exercised 3×/week during four weeks before and four weeks after the induction of PD (EXB+EXA), and exercised 3×/week during four weeks after the induction of PD (EXA). One month after 6-OHDA injections, the animals were subjected to rotational behavioral test induced by apomorphine and the brains were collected for immunohistochemistry and immunoblotting assays, in which we measured BDNF and tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNc) and the striatum (caudate-putamen, CPu). Our results showed a significant reduction of rotational asymmetry induced by apomorphine in the exercised parkinsonian rats. BDNF decreased in the SNc of the SED group, and exercise was able to revert that effect. Exercised groups exhibited reduced damage to the dopaminergic system, detected as a decreased drop of TH levels in SNc and CPu. On the other hand, BDNF blockade was capable of substantially reducing TH expression postlesion, implying enhanced dopaminergic cell loss. Our data revealed that physical exercise is capable of reducing the damage induced by 6-OHDA, and that BDNF receptors are involved in that effect.

Prenatal LPS exposure reduces olfactory perception in neonatal and adult rats

Prenatal lipopolysaccharide (LPS) exposure causes reproductive, behavioral and neurochemical defects in both dams and pups. The present study evaluated male rats prenatally treated with LPS for behavioral and neurological effects related to the olfactory system, which is the main sensorial path in rodents. Pregnant Wistar rats received 100 μg/kg of LPS intraperitoneally (i.p.) on gestational day (GD) 9.5, and maternal behavior was evaluated. Pups were evaluated for (1) maternal odor preference, (2) aversion to cat odor, (3) monoamine levels and turnover in the olfactory bulb (OB) and (4) protein expression (via immunoblotting) within the OB dopaminergic system and glial cells. Results showed that prenatal LPS exposure impaired maternal preference and cat odor aversion and decreased dopamine (DA) levels in the OB. This dopaminergic impairment may have been due to defects in another brain area given that protein expression of the first enzyme in the DA biosynthetic pathway was unchanged in the OB. Moreover, there was no change in the protein expression of the DA receptors. The fact that the number of astrocytes and microglia was not increased suggests that prenatal LPS did not induce neuroinflammation in the OB. Furthermore, given that maternal care was not impaired, abnormalities in the offspring were not the result of reduced maternal care.

Increased expression of nitric oxide synthase in visual structures of the chick brain after retinal removal.

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.

Expression of cholinergic system molecules during development of the chick nervous system

There are suggestions of the participation of nicotinic acetylcholine receptors (nAChRs), the acetylcholine degradation enzyme, acetylcholinesterase (AChE), and the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), in the development of the nervous system. In this study, we aimed at comparing the development of some subunits of the nAChRs, AChE, and ChAT in the chick nervous system by standard immunohistochemical methods. The expression of all molecules investigated here appeared very early in ganglia (embryonic day 3.5-4), persisting into posthatching, except for ChAT, which is not detected after hatching in ganglia. A differential development was observed for nAChR subunits, with these receptors appearing around embryonic day 6 in some sites. The time-course of development of different nAChR subunits revealed several instances of transient expression (such as in the cerebellum), increasing expression (such as in the nucleus spiriformis lateralis), and diminishing expression into posthatching stages (such as in the oculomotor and throclear nuclei). Expression of AChE and ChAT also starts around embryonic day 6 in some structures and follows mainly increasing time-courses in the chick brain. The results of this study reveal a developmentally regulated expression of cholinergic system-related molecules in the chick nervous system and characterize differential time-courses of expression for nAChR subunits, AChE, and ChAT during development.

Retinal removal up-regulates cannabinoid CB1 receptors in the chick optic tectum

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration and neuroprotection. The aim of this study was to evaluate the effects of unilateral retinal ablation on the expression of the cannabinoid receptor subtype 1 (CB1) at both protein and mRNA levels in the optic tectum of the adult chick brain. After different survival times postlesion (2–30 days), the chick brains were subjected to immunohistochemical, immunoblotting, and real‐time PCR procedures to evaluate CB1 expression. TUNEL and Fluoro‐Jade B were used to verify the possible occurrence of cell death, and immunostaining for the microtubule‐associated protein MAP‐2 was performed to verify possible dendritic remodeling after lesions. No cell death could be observed in the deafferented tectum, at least up to 30 days postlesion, although Fluoro‐Jade B could reveal degenerating axons and terminals. Retinal ablation seems to generate an increase of CB1 protein in the optic tectum and other retinorecipient visual areas, which paralleled an increase in MAP‐2 staining. On the other hand, CB1 mRNA levels were not changed after retinal ablation. Our results reveal that CB1 expression in visual structures of the adult chick brain may be negatively regulated by the retinal innervation. The increase of CB1 receptor expression observed after retinal removal indicates that these receptors are not presynaptic in retinal axons projecting to the tectum and suggests a role of the cannabinoid system in plasticity processes ensuing after lesions.

ERK3 associates with MAP2 and is involved in glucose-induced insulin secretion

The adaptation of pancreatic islets to pregnancy includes increased beta cell proliferation, expansion of islet mass, and increased insulin synthesis and secretion. Most of these adaptations are induced by prolactin (PRL). We have previously described that in vitro PRL treatment increases ERK3 expression in isolated rat pancreatic islets. This study shows that ERK3 is also upregulated during pregnancy. Islets from pregnant rats treated with antisense oligonucleotide targeted to the PRL receptor displayed a significant reduction in ERK3 expression. Immunohistochemical double-staining showed that ERK3 expression is restricted to pancreatic beta cells. Transfection with antisense oligonucleotide targeted to ERK3 abolished the insulin secretion stimulated by glucose in rat islets and by PMA in RINm5F cells. Therefore, we examined the participation of ERK3 in the activation of a cellular target involved in secretory events, the microtubule associated protein MAP2. PMA induced ERK3 phosphorylation that was companied by an increase in ERK3/MAP2 association and MAP2 phosphorylation. These observations provide evidence that ERK3 is involved in the regulation of stimulus-secretion coupling in pancreatic beta cells.

Neurotransmitter regulation of neural development: acetylcholine and nicotinic receptors

Several neurotransmitter systems have been related to developmental processes during the past decade. In this review, we discuss the evidence that the nicotinic acetylcholine receptors could have an additional function during development that may be unrelated to their role in cholinergic neurotransmission in the vertebrate brain. Both temporal expression data and in vitro and in vivo studies with nicotinic agonists and antagonists have provided direct support for a role of nicotinic receptors in neural developmental processes such as neurite outgrowth and differentiation. A similar picture has emerged for other neurotransmitter and receptor systems as well, which generates a new view of neural processes during both development and mature life.

Modulation of Pineal Melatonin Synthesis by Glutamate Involves Paracrine Interactions between Pinealocytes and Astrocytes through NF-κB Activation

The glutamatergic modulation of melatonin synthesis is well known, along with the importance of astrocytes in mediating glutamatergic signaling in the central nervous system. Pinealocytes and astrocytes are the main cell types in the pineal gland. The objective of this work was to investigate the interactions between astrocytes and pinealocytes as a part of the glutamate inhibitory effect on melatonin synthesis. Rat pinealocytes isolated or in coculture with astrocytes were incubated with glutamate in the presence of norepinephrine, and the melatonin content, was quantified. The expression of glutamate receptors, the intracellular calcium content and the NF-κB activation were analyzed in astrocytes and pinealocytes. TNF-αs possible mediation of the effect of glutamate was also investigated. The results showed that glutamates inhibitory effect on melatonin synthesis involves interactions between astrocytes and pinealocytes, possibly through the release of TNF-α. Moreover, the activation of the astrocytic NF-κB seems to be a necessary step. In astrocytes and pinealocytes, AMPA, NMDA, and group I metabotropic glutamate receptors were observed, as well as the intracellular calcium elevation. In conclusion, there is evidence that the modulation of melatonin synthesis by glutamate involves paracrine interactions between pinealocytes and astrocytes through the activation of the astrocytic NF-κB transcription factor and possibly by subsequent TNF-α release.

Nicotine and alpha-bungarotoxin modify the dendritic growth of cholinoceptive neurons in the developing chick tectum

Nicotinic acetylcholine receptors have been suggested to participate in morphogenetic processes during development of the nervous system. In this study, nicotine applied both in ovo and in vitro produced a reduction of the neuritic length of cholinoceptive neurons of the developing chick tectum, whereas alpha-bungarotoxin produced the opposite effect. Taken together with previous data, our results are indicative of a role of the alpha-bungarotoxin-sensitive nicotinic receptors in neural development.