Exercise-induced plasticity of AMPA-type glutamate receptor subunits in the rat brain

The aim of this study was to analyze the plastic effects of moderate exercise upon the motor cortex (M1 and M2 areas), cerebellum (Cb), and striatum (CPu) of the rat brain This assessment was made by verifying the expression of AMPA type glutamate receptor subunits (GluR1 and GluR2/3) We used adult Wistar rats, divided into 5 groups based on duration of exercise training, namely 3 days (EX3), 7 days (EX7) 15 days (EX15) 30 days (EX30), and sedentary (S) The exercised animals were subjected to a treadmill exercise protocol at the speed of the 10 meters/min for 40 mm After exercise, the brains were subjected to immunohistochemistry and immunoblotting to analyze changes of GluR1 and GluR2/3, and plasma cortcosterone was measured by ELISA in order to verify potential stress induced by physical training Overall the results of immunohistochemistry and immunoblotting were similar and revealed that GluR subunits show distinct responses over the exercise periods and for the different structures analyzed In general, there was increased expression of GluR subunits after longer exercise periods (such as EX30) although some opposite effects were seen after short periods of exercise (Ex3) In a few cases biphasic patterns with decreases and subsequent increases of GluR expression were seen and may represent the outcome of exercise dependent, complex regulatory processes The data show that the protocol used was able to promote plastic GluR changes during exercise, suggesting a specific involvement of these receptors in exercise induced plasticity processes in the brain areas tested (C) 2010 Elsevier B V All rights reserved

Moderate exercise changes synaptic and cytoskeletal proteins in motor regions of the rat brain

Physical exercise is known to enhance brain function in several aspects. We evaluated the acute effects of a moderate forced exercise protocol on synaptic proteins, namely synapsin 1 (SYN) and synaptophysin (SYP), and structural proteins (neurofilaments, NFs) in rat brain regions related to motor function and often affected by neurodegenerative disorders. Immunohistochemistry, Western blotting and real-time PCR were used to analyze the expression of those proteins after 3, 7 and 15 days of exercise (EX3, EX7 and EX15). In the cerebellum, increase of SYN was observed at EX7 and EX15 and of NF68 at EX3. In the substantia nigra, increases of protein levels were observed for NF68 and NF160 at EX3. In the striatum, there was an increase of SYN at EX3 and EX7, of SYP at EX7 and of NF68 at EX3. In the cortex, decreased levels of NF68 and NF160 were observed at EX3, followed by an increase of NF68 at EX15. In the reticular formation, all NF proteins were increased at EX15. The mRNA data for each time-point and region also revealed significant exercise-related changes of SYN, SYP and NF expression. These results suggest that moderate physical exercise modulates synaptic and structural proteins in motor brain areas, which may play an important role in the exercise-dependent brain plasticity. (C) 2010 Elsevier B.V. All rights reserved.

cis-4-decenoic acid provokes mitochondrial bioenergetic dysfunction in rat brain

Aims: In the present work we investigated the in vitro effect of cis-4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods: Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings: cis-4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis-4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis-4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondria! membrane. Significance: It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis-4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients. (C) 2010 Elsevier Inc. All rights reserved.

Antioxidant defense in rat brain regions after developmental lead exposure

Oxidative stress is considered a possible molecular mechanism involved in Pb neurotoxicity. Considering the vulnerability of the developing brain to Pb neurotoxicity, this study was carried out to investigate the effects of low-level developmental Pb exposure on brain regions antioxidant enzymes activities. Wister dams were exposed to 500 ppm of Pb, as Pb acetate, or to 660 ppm Na acetate in the drinking water during pregnancy and lactation. The activities of superoxide dismutase (SOD), glutathione peroxidase and glutathione reductase were determined in the hypothalamus, hippocampus and striatum of male pups at 23 (weaned) or 70 days (adult) of age. In the Pb-exposed 23-day-old pups, the activity of SOD was decreased in the hypothalamus. Regarding adults, there was no significant treatment effect in any of the enzymes and regions evaluated. Based on the present results, it seems that oxidative stress due to decreased antioxidant function may occur in weaned rats but it is suggested that this should not be the main mechanism involved in the neurotoxicity of low-level Pb exposure. (C) 2001 Elsevier B.V. Ireland Ltd. All rights reserved.

Differential subcellular distribution of neurolysin (EC 3.4.24.16) and thimet oligopeptidase (EC 3.3.24.15) in the rat brain

Immunohistochemistry was used to analyze the rat brain distribution of thimet oligopeptidase and neurolysin. Both enzymes appear ubiquitously distributed within the entire rat brain. However, neuronal perikarya and processes stained for neurolysin, while intense nuclear labeling was only observed for thimet oligopeptidase. These data suggest that neurolysin and thimet oligopeptidase, endopeptidases sharing several functional and structural similarities, are present in distinctive intracellular compartments in neuronal cells. (C) 1999 Elsevier B.V. B.V. All rights reserved.

Mechanism for the uncoupling of oxidative phosphorylation by juliprosopine on rat brain mitochondria

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cloning and expression of the cDNA encoding rat granulocyte colony-stimulating factor

Granulocyte colony-stimulating factor (G-CSF) acts on precursor hematopoietic cells to control the production and maintenance of neutrophils. Recombinant G-CSF (re-G-CSF)is used clinically to treat patients with neutropenia and has greatly reduced the infection risk associated with bone marrow transplantation. Cyclic hematopoiesis, a stem cell defect characterized by severe recurrent neutropenia, occurs in man and grey collie dogs, and can be treated by administration of re-G-CSF. Availability of the rat G-CSF cDNA would benefit the use of rats as models of gene therapy for the treatment of cyclic hematopoiesis. In preliminary rat experiments, retroviral-mediated expression of canine G-CSF caused neutralizing antibody formation which precluded long-term increases in neutrophil counts. To overcome this problem we cloned the rat G-CSF cDNA from RNA isolated from skin fibroblasts. The rat G-CSF sequence shared a high degree of identity in both the coding and non-coding regions with both the murine G-CSF (85%) and human G-CSF (74%). The signal peptides of murine and human G-CSF both contained 30 amino acids (aa), whereas the deduced signal sequence for rat G-CSF possessed 21 aa. A retrovirus encoding the rat G-CSF cDNA synthesized bioactive G-CSF from transduced vascular smooth muscle cells.

Circadian-rhythm of the endopeptidase 22.19 (ec 3.4.22.19) in the rat-brain

The endopeptidase 22.19 (EC 3.4.22.19) has been associated with the metabolism of neuropeptides by its ability to convert small enkephalin-containing peptides (8 to 13 amino acids) into enkephalins. In addition, this enzyme cleaves the Arg8-Arg9 bond of neurotensin and the Phe5-Ser6 bond of bradykinin. We analyzed the circadian variation of endopeptidase 22.19 in the whole and individual areas of the rat brain. Endopeptidase 22.19 activity was analyzed by high-performance liquid chromatography (HPLC) using bradykinin as an operative substrate. Enzymatic specific activities were analyzed by rhythmometric methods and indicate a circadian fluctuation of endopeptidase 22.19 specific activity (mU of enzyme/mg of protein) in the whole brain [p < 0.001, mesor (M) = 7.62, amplitude (A) = 2.89, and acrophase (phi) = 23:08 h], striatum (p < 0.001, M = 2.92, A = 0.62, phi-23:03 h), hypothalamus (p < 0.001, M = 3.15, A = 0.86, phi-01:12 h), periaqueductal gray matter (p < 0.005, M = 2.62, A = 0.34, phi = 22:35 h), and cerebellum (p < 0.0 14, M = 4.27, A = 0.88, phi = 17:12 h). The circadian rhythmicity in endopeptidase 22.19 specific activity suggests that light may have an effect on the peptidase activity in whole brain and in areas of the central nervous system and may be essential for the mechanisms of circadian fluctuations of neuropeptides in the brain.

PARAVENTRICULAR AND SUPRAOPTIC NUCLEI OF THE HYPOTHALAMUS MEDIATE CARDIOVASCULAR RESPONSES EVOKED BY THE MICROINJECTION OF NORADRENALINE INTO THE MEDIAL AMYGDALOID NUCLEUS OF THE RAT BRAIN

The medial amygdaloid nucleus (MeA) is a part of the limbic system and is involved in cardiovascular modulation. We previously reported that microinjection of noradrenaline (NA) into the MeA of unanesthetized rats caused pressor and bradycardiac responses, which were mediated by acute vasopressin release into the systemic circulation. In the present study, we tested the possible involvement of magnocellular neurons of the paraventricular (PVN) and/or supraoptic (SON) of the hypothalamus that synthesize vasopressin in the cardiovascular pathway activated by the microinjection of NA into the MeA. Pressor and bradycardiac responses to the microinjection of NA (27 nmol/100 nL) into the MeA were blocked by pretreatment of either the PVN or the SON with cobalt chloride (CoCl2, 1 mM/100 nL), thus indicating that both hypothalamic nuclei mediate the cardiovascular responses evoked by microinjection of NA Into the MeA. Our results suggest that the pressor and bradycardiac response caused by the microinjection of NA into the MeA is mediated by magnocellular neurons in both the PVN and SON. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Different protocols of physical exercise produce different effects on synaptic and structural proteins in motor areas of the rat brain

The plastic brain responses generated by the training with acrobatic exercise (AE) and with treadmill exercise (TE) may be different. We evaluated the protein expression of synapsin I (SYS), synaptophysin (SYP), microtubule-associated protein 2 (MAP2) and neurofilaments (NF) by immunohistochemistry and Western blotting in the motor cortex, striatum and cerebellum of rats subjected to TE and AE. Young adult male Wistar rats were divided into 3 groups: sedentary (Sed) (n=15), TE (n=20) and AE (n=20). The rats were trained 3 days/week for 4 weeks on a treadmill at 0.6 km/h, 40 min/day (TE), or moved through a circuit of obstacles 5 times/day (AE). The rats from the TE group exhibited a significant increase of SYS and SYP in the motor cortex, of NF68, SYS and SYP in the striatum, and of MAP2, NF and SYS in the cerebellum, whereas NF was decreased in the motor cortex and the molecular layer of the cerebellar cortex. On the other hand, the rats from the AE group showed a significant increase of MAP2 and SYP in the motor cortex, of all four proteins in the striatum, and of SYS in the cerebellum. In conclusion, AE induced changes in the expression of synaptic and structural proteins mainly in the motor cortex and striatum, which may underlie part of the learning of complex motor tasks. TE, on the other hand, promoted more robust changes of structural proteins in all three regions, especially in the cerebellum, which is involved in learned and automatic tasks. (C) 2012 Elsevier B.V. All rights reserved.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 mu M Ca2+, C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. (C) 2012 Elsevier Inc. All rights reserved.

Acute cocaine treatment increases thimet oligopeptidase in the striatum of rat brain

Many studies indicate that thimet oligopeptidase (EC3.4.24.15; TOP) can be implicated in the metabolism of bioactive peptides, including dynorphin 1-8, alpha-neoendorphin, beta-neoendorphin and GnRH. Furthermore, the higher levels of this peptidase are found in neuroendocrine tissue and testis. In the present study, we have evaluated the effect of acute cocaine administration in male rats on TOP specific activity and mRNA levels in prosencephalic brain areas related with the reward circuitry; ventral striatum, hippocampus, and frontal cortex. No significant differences on TOP specific activity were detected in the hippocampus and frontal cortex of cocaine treated animals compared to control vehicle group. However, a significant increase in activity was observed in the ventral striatum of cocaine treated-rats. The increase occurred in both, TOP specific activity and TOP relative mRNA amount determined by real time RT-PCR. As TOP can be implicated in the processing of many neuropeptides, and previous studies have shown that cocaine also alters the gene expression of proenkephalin and prodynorphin in the striatum, the present findings suggest that TOP changes in the brain could play important role in the balance of neuropeptide level correlated with cocaine effects. (C) 2012 Elsevier Inc. All rights reserved.

Chronic infusion of amyloid-β peptide and sustained attention altered α7 nicotinic receptor density in the rat brain

It is already known that progressive degeneration of cholinergic neurons in brain areas such as the hippocampus and the cortex leads to memory deficits, as observed in Alzheimer's disease. This work verified the effects of the infusion of amyloid-beta (A beta) peptide associated to an attentional rehearsal on the density of alpha 7 nicotinic cholinergic receptor (nAChR) in the brain of male Wistar rats. Animals received intracerebroventricular infusion of A beta or vehicle (control - C) and their attention was stimulated weekly (Stimulated A beta group: S-A beta and Stimulated Control group: SC) or not (Non-Stimulated A beta group: N-SA beta and Non-Stimulated Control group: N-SC), using an active avoidance apparatus. Conditioned avoidance responses (CAR) were registered. Chronic infusion of A beta caused a 37% reduction in CAR for N-SA beta. In S-A beta, this reduction was not observed. At the end, brains were extracted and autoradiography for alpha 7 nAChR was conducted using [I-125]-alpha-bungarotoxin. There was an increase in alpha 7 density in hippocampus, cortex and amygdala of SA beta animals, together with the memory preservation. In recent findings from our lab using mice infused with A beta and the alpha 7 antagonist methyllycaconitine, and stimulated weekly in the same apparatus, it was observed that memory maintenance was abolished. So, the increase in alpha 7 density in brain areas related to memory might be related to a participation of this receptor in the long-lasting change in synaptic plasticity, which is important to improve and maintain memory consolidation.

Inhomogeneity in optical properties of rat brain: a study for LLLT dosimetry.

Over the last few years, low-level light therapy (LLLT) has shown an incredible suitability for a wide range of applications for central nervous system (CNS) related diseases. In this therapeutic modality light dosimetry is extremely critical so the study of light propagation through the CNS organs is of great importance. To better understand how light intensity is delivered to the most relevant neural sites we evaluated optical transmission through slices of rat brain point by point. We experimented red (λ = 660 nm) and near infrared (λ = 808 nm) diode laser light analyzing the light penetration and distribution in the whole brain. A fresh Wistar rat (Rattus novergicus) brain was cut in sagittal slices and illuminated with a broad light beam. A high-resolution digital camera was employed to acquire data of transmitted light. Spatial profiles of the light transmitted through the sample were obtained from the images. Peaks and valleys in the profiles show sites where light was less or more attenuated. The peak intensities provide information about total attenuation and the peak widths are correlated to the scattering coefficient at that individual portion of the sample. The outcomes of this study provide remarkable information for LLLT dose-dependent studies involving CNS and highlight the importance of LLLT dosimetry in CNS organs for large range of applications in animal and human diseases.

Study on the regulation of mRNA expression of GABA transporters (GAT-1 and GAT-3) in rat brain

The submitted work concentrated on the study of mRNA expression of two distinct GABA transporters, GAT-1 and GAT-3, in the rat brain. For the detection and quantification of the chosen mRNAs, appropriate methods had to be established. Two methods, ribonuclease protection assay (RPA) and competitive RT-PCR were emloyed in the present study. Competitive RT-PCR worked out to be 20 times more sensitive as RPA. Unlike the sensitivity, the fidelity of both techniques was comparable with respect to their intra- and inter-assay variability.The basal mRNA levels of GAT-1 and GAT-3 were measured in various brain regions. Messenger RNAs for both transporters were detected in all tested brain regions. Depending on the region, the observed mRNA level for GAT-1 was 100-300 higher than for GAT-3. The GAT-1 mRNA levels were similar in all tested regions. The distribution of GAT-3 mRNA seemed to be more region specific. The strongest GAT-3 mRNA expression was detected in striatum, medulla oblongata and thalamus. The lowest levels of GAT-3 were in cortex frontalis and cerebellum.Furthermore, the mRNA expression for GAT-1 and GAT-3 was analysed under altered physiological conditions; in kindling model of epilepsy and also after long-term treatment drugs modulating GABAergic transmission. In kindling model of epilepsy, altered GABA transporter function was hypothesised by During and coworkers (During et al., 1995) after observed decrease in binding of nipecotic acid, a GAT ligand, in hippocampus of kindled animals. In the present work, the mRNA levels were measured in hippocampus and whole brain samples. Neither GAT-1 nor GAT-3 showed altered transcription in any tested region of kindled animals compared to controls. This leads to conclusion that an altered functionality of GABA transporters is involved in epilepsy rather than a change in their expression.The levels of GAT-1 and GAT-3 mRNAs were also measured in the brain of rats chronically treated with diazepam or zolpidem, GABAA receptor agonists. Prior to the molecular biology tests, behavioural analysis was carried out with chronically and acutely treated animals. In two tests, open field and elevated plus-maze, the basal activity exploration and anxiety-like behaviour were analysed. Zolpidem treatment increased exploratory activity. There were observed no differencies between chronically and acutely treated animals. Diazepam increased exploratory activity and decresed anxiety-like behaviour when applied acutely. This effect disappeard after chronic administration of diazepam. The loss of effect suggested a development of tolerance to effects of diazepam following long-term administration. Double treatment, acute injection of diazepam after chronic diazepam treatment, confirmed development of a tolerance to effects of diazepam. Also, the mRNAs for GAT-1 and GAT-3 were analysed in cortex frontalis, hippocampus, cerebellum and whole brain samples of chronically treated animals. The mRNA levels for any of tested GABA transporters did not show significant changes in any of tested region neither after diazepam nor zolpidem treatment. Therefore, changes in GAT-1 and GAT-3 transcription are probably not involved in adaptation of GABAergic system to long-term benzodiazepine administration and so in development of tolerance to benzodiazepines.