Brain-region specific responses of astrocytes to an in vitro injury and neurotrophins.

Astrocytes are a heterogeneous population of glial cells that react to brain insults through a process referred to as astrogliosis. Reactive astrocytes are characterized by an increase in proliferation, size, migration to the injured zone and release of a plethora of chemical mediators such as NGF and BDNF. The aim of this study was to determine whether there are brain region-associated responses of astrocytes to an injury and to the neurotrophins NGF and BDNF. We used the scratch injury model to study the closure of a wound inflicted on a monolayer of astrocytes obtained from cortex, hippocampus or striatum. Our results indicate that the response of astrocytes to a mechanical lesion differ according to brain regions. Astrocytes from the striatum proliferate and repopulate the injury site more rapidly than astrocytes from cortex or hippocampus. We found that the scratch injury induced the upregulation of neurotrophin receptor p75NTR and TrkB.t in astrocytes from all brain regions studied. When astrocytes from all regions were treated with NGF, the neurotrophin induced migration of the astrocytes (assessed in Boyden chambers) and induced wound closure but did not affect proliferation. In contrast, BDNF induced wound closure but only in astrocytes from striatum. Our overall findings show the heterogeneity in astrocyte functions based on their brain region of origin, and how this functional diversity may determine their responses to an injury and to neurotrophins.

Calpain-dependent truncated form of TrkB-FL increases in neurodegenerative processes.

Recent findings indicate that the mechanisms that drive reshaping of the nervous system are aberrantly activated in epilepsy and several neurodegenerative diseases. The recurrent seizures in epilepsy, particularly in the condition called status epilepticus, can cause permanent neurological damage, resulting in cognitive dysfunction and other serious neurological conditions. In this study, we used an in vitro model of status epilepticus to examine the role of calpain in the degeneration of hippocampal neurons. We grew neurons on a culture system that allowed studying the dendritic and axonal domains separately from the cell bodies. We found that a recently characterized calpain substrate, the neurotrophin receptor TrkB, is cleaved in the dendritic and axonal domain of neurons committed to die, and this constitutes an early step in the neuronal degeneration process. While the full-length TrkB (TrkB-FL) levels decreased, the truncated form of TrkB (Tc TrkB-FL) concurrently increased, leading to a TrkB-FL/Tc TrkB-FL imbalance, which is thought to be causally linked to neurodegeneration. We further show that the treatment with N-acetyl-Leu-Leu-norleucinal, a specific calpain activity blocker, fully protects the neuronal processes from degeneration, prevents the TrkB-FL/Tc TrkB-FL imbalance, and provides full neuroprotection. Moreover, the use of the TrkB antagonist ANA 12 at the time when the levels of TrkB-FL were significantly decreased, totally blocked neuronal death, suggesting that Tc TrkB-FL may have a role in neuronal death. These results indicate that the imbalance of these neurotrophins receptors plays a key role in neurite degeneration induced by seizures.

Enkephalin is essential for the molecular and behavioral expression of cocaine sensitization.

Behavioral sensitization to cocaine is associated to neuroadaptations that contribute to addiction. Enkephalin is highly expressed in mesocorticolimbic areas associated with cocaine-induced sensitization; however, their influence on cocaine-dependent behavioral and neuronal plasticity has not been explained. In this study, we employed a knockout (KO) model to investigate the contribution of enkephalin in cocaine-induced behavioral sensitization. Wild-type (WT) and proenkephalin KO mice were treated with cocaine once daily for 9 days to induce sensitization. Additionally, to clarify the observations in KO mice, the same procedure was applied in C57BL/6 mice, except that naloxone was administered before each cocaine injection. All animals received a cocaine challenge on days 15 and 21 of the treatment to evaluate the expression of locomotor sensitization. On day 21, microdialysis measures of accumbal extracellular dopamine, Western blotting for GluR1 AMPA receptor (AMPAR), phosphorylated ERK2 (pERK2), CREB (pCREB), TrKB (pTrkB) were performed in brain areas relevant for sensitization from KO and WT and/or naloxone- and vehicle pre-treated animals. We found that KO mice do not develop sensitization to the stimulating properties of cocaine on locomotor activity and on dopamine release in the nucleus accumbens (NAc). Furthermore, pivotal neuroadaptations such as the increase in pTrkB receptor, pERK/CREB and AMPAR related to sensitized responses were absent in the NAc from KO mice. Consistently, full abrogation of cocaine-induced behavioral and neuronal plasticity after naloxone pre-treatment was observed. We show for first time that the proenkephalin system is essential in regulating long-lasting pivotal neuroadaptations in the NAc underlying behavioral sensitization to cocaine.

Early exercise promotes positive hippocampal plasticity and improves spatial memory in the adult life of rats.

There is a great deal of evidence showing the capacity of physical exercise to enhance cognitive function, reduce anxiety and depression, and protect the brain against neurodegenerative disorders. Although the effects of exercise are well documented in the mature brain, the influence of exercise in the developing brain has been poorly explored. Therefore, we investigated the morphological and functional hippocampal changes in adult rats submitted to daily treadmill exercise during the adolescent period. Male Wistar rats aged 21 postnatal days old (P21) were divided into two groups: exercise and control. Animals in the exercise group were submitted to daily exercise on the treadmill between P21 and P60. Running time and speed gradually increased over this period, reaching a maximum of 18 m/min for 60 min. After the aerobic exercise program (P60), histological and behavioral (water maze) analyses were performed. The results show that early-life exercise increased mossy fibers density and hippocampal expression of brain-derived neurotrophic factor and its receptor tropomyosin-related kinase B, improved spatial learning and memory, and enhanced capacity to evoke spatial memories in later stages (when measured at P96). It is important to point out that while physical exercise induces hippocampal plasticity, degenerative effects could appear in undue conditions of physical or psychological stress. In this regard, we also showed that the exercise protocol used here did not induce inflammatory response and degenerating neurons in the hippocampal formation of developing rats. Our findings demonstrate that physical exercise during postnatal development results in positive changes for the hippocampal formation, both in structure and function.

Postweaning Enriched Environment Enhances Cognitive Function and Brain-Derived Neurotrophic Factor Signaling in the Hippocampus in Maternally Separated Rats.

Adverse environments during early life may lead to different neurophysiological and behavioral consequences, including depression and learning and memory deficits that persist into adulthood. Previously, we demonstrated that exposure to an enriched environment during adolescence mitigates the cognitive impairment observed after maternal separation in a task-specific manner. However, underlying neural mechanisms are still not fully understood. The current study examines the effects of neonatal maternal separation (MS) and postweaning environmental enrichment (EE) on spatial learning and memory performance in a short version of the Barnes Maze, active and passive behaviors in the forced swim test, and on TrkB/BDNF receptor expression in the hippocampus. Our results revealed that MS impaired acquisition learning and that enriched rats performed better than non-enriched rats in acquisition trials, regardless of early conditions. During the probe, enriched-housed rats demonstrated better performance than those reared in standard conditions. No significant differences between groups were found in the forced swim test. Both MS and EE increase full-length TrkB expression, and the combination of MS and EE treatment caused the highest levels of this protein expression. Similarly, truncated TrkB expression was higher in the MS/EE group. Animal facility rearing (AFR) non-enriched groups present the lowest activation of phosphorylated Erk, a canonical downstream kinase of TrkB signaling. Taken together, our results demonstrate the importance of enriched environment as an intervention to ameliorate the effects of maternal separation on spatial learning and memory. TrkB/BDNF signaling could mediate neuroplastic changes related to learning and memory during exposure to enriched environment.

Brain-derived neurotrophic factor facilitates TrkB down-regulation and neuronal injury after status epilepticus in the rat hippocampus.

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of neuronal biology and hippocampal physiology. Status epilepticus (SE) is a condition in which prolonged seizures lead to neuronal degeneration. SE-induced in rodents serves as a model of Temporal Lobe Epilepsy with hippocampal sclerosis, the most frequent epilepsy in humans. We have recently described a strong correlation between TrkB decrease and p75ntr increase with neuronal degeneration (Neuroscience 154:978, 2008). In this report, we report that local, acute intra-hippocampal infusion of function-blocking antibodies against BDNF prevented both early TrkB down-regulation and neuronal degeneration after SE. Conversely, the infusion of recombinant human BDNF protein after SE greatly increased neuronal degeneration. The inhibition of BDNF mRNA translation by the infusion of antisense oligonucleotides induced a rapid decrease of BDNF protein levels, and a delayed increase. If seizures were induced at the time endogenous BDNF was decreased, SE-induced neuronal damage was prevented. On the other hand, if seizures were induced at the time endogenous BDNF was increased, SE-induced neuronal damage was exacerbated. These results indicate that under a pathological condition BDNF exacerbates neuronal injury.

Enriched environment protects the nigrostriatal dopaminergic system and induces astroglial reaction in the 6-OHDA rat model of Parkinson's disease.

Enriched environment (EE) is neuroprotective in several animal models of neurodegeneration. It stimulates the expression of trophic factors and modifies the astrocyte cell population which has been said to exert neuroprotective effects. We have investigated the effects of EE on 6-hydroxydopamine (6-OHDA)-induced neuronal death after unilateral administration to the medial forebrain bundle, which reaches 85-95% of dopaminergic neurons in the substantia nigra after 3 weeks. Continuous exposure to EE 3 weeks before and after 6-OHDA injection prevents neuronal death (assessed by tyrosine hydroxylase staining), protects the nigrostriatal pathway (assessed by Fluorogold retrograde labeling) and reduces motor impairment. Four days after 6-OHDA injection, EE was associated with a marked increase in glial fibrillary acidic protein staining and prevented neuronal death (assessed by Fluoro Jade-B) but not partial loss of tyrosine hydroxylase staining in the anterior substantia nigra. These results robustly demonstrate that EE preserves the entire nigrostriatal system against 6-OHDA-induced toxicity, and suggests that an early post-lesion astrocytic reaction may participate in the neuroprotective mechanism.

Loss of TrkB Signaling Due to Status Epilepticus Induces a proBDNF-Dependent Cell Death.

Neurotrophins (NTs) are secretory proteins that bind to target receptors and influence many cellular functions, such as cell survival and cell death in neurons. The mammalian NT brain-derived neurotrophic factor (matBDNF) is the C-terminal mature form released by cleavage from the proBDNF precursor. The binding of matBDNF to the tyrosine kinase receptor B (TrkB) activates different signaling cascades and leads to neuron survival and plasticity, while the interaction of proBDNF with the p75 NT receptor (p75NTR)/sortilin receptor complex has been highly involved in apoptosis. Many studies have demonstrated that prolonged seizures such as status epilepticus (SE) induce changes in the expression of NT, pro-NT, and their receptors. We have previously described that the blockage of both matBDNF and proBDNF signaling reduces neuronal death after SE in vivo (Unsain et al., 2008). We used an in vitro model as well as an in vivo model of SE to determine the specific role of TrkB and proBDNF signaling during neuronal cell death. We found that the matBDNF sequestering molecule TrkB-Fc induced an increase in neuronal death in both models of SE, and it also prevented a decrease in TrkB levels. Moreover, SE triggered the interaction between proBDNF and p75NTR, which was not altered by sequestering matBDNF. The intra-hippocampal administration of TrkB-Fc, combined with an antibody against proBDNF, prevented neuronal degeneration. In addition, we demonstrated that proBDNF binding to p75NTR exacerbates neuronal death when matBDNF signaling is impaired through TrkB. Our results indicated that both the mature and the precursor forms of BDNF may have opposite effects depending on the scenario in which they function and the signaling pathways they activate.

Protection of dopaminergic neurons by electroconvulsive shock in an animal model of Parkinson's disease.

Electroconvulsive shock (ECS) improves motor function in Parkinson's disease. In rats, ECS stimulates the expression of various factors some of which have been proposed to exert neuroprotective actions. We have investigated the effects of ECS on 6-hydroxydopamine (6-OHDA)-injected rats. Three weeks after a unilateral administration of 6-OHDA, 85-95% nigral dopaminergic neurons are lost. Chronic ECS prevented this cell loss, protect the nigrostriatal pathway (assessed by FloroGold retrograde labeling) and reduce motor impairment in 6-OHDA-treated animals. Injection of 6-OHDA caused loss of expression of glial cell-line derived neurotrophic factor (GDNF) in the substantia nigra. Chronic ECS completely prevented this loss of GDNF expression in 6-OHDA-treated animals. We also found that protected dopaminergic neurons co-express GDNF receptor proteins. These results strongly suggest that endogenous changes in GDNF expression may participate in the neuroprotective mechanism of ECS against 6-OHDA induced toxicity.

An alkaloid extract obtained from Phlegmariurus Saururus induces neuroprotection after status epilepticus.

BACKGROUND: The brain is exposed to many excitotoxic insults that can lead to neuronal damage. Among these, Epilepsy is a neurological disease that affects a large percentage of world population and is commonly associated with cognitive disorders and excitotoxic neuronal death. Most experimental strategies are focused on preventing Status Epilepticus (SE), but once it has already occurred, the key question is whether it is possible to save neurons. PURPOSE: The aim of this study was to determine if a purified alkaloid extract (AE) obtained from Phlegmariurus saururus, a genus of Lycophyte plants (sometimes known as firmossesor fir club mosses) could induce neuroprotection following SE. METHODS: In vitro and in vivo techniques were applied for this purpose. Protein levels were measured by western blotting procedures. Neuronal death analysis was performed by calcein-ethidium staining and the presence of the NeuN protein as a marker for presence or absence of cells (in vitro experiments) and by Fluoro Jade B staining for the in vivo experiments. RESULTS: The effect of AE in the hippocampal neurons culture was the first determination, where we found an increase in neuronal survival and in the level of pErk and TrkB activation, 24 h after the addition of AE. In a well-established in vitro model of SE, we found that 24 h after being added to the hippocampal neuron-astrocyte co-culture, the AE induces a significant increase in neuronal survival. In addition to this, in the in vivo Li-pilocarpine model of SE, the AE induced a remarkable neuroprotection in areas such as the entorhinal cortex and hippocampal CA1 area. CONCLUSION: These results make the AE an excellent candidate for potential clinical use in neurological disorders where memory impairment and neuronal death occurs.

Cdk5 Is Essential for Amphetamine to Increase Dendritic Spine Density in Hippocampal Pyramidal Neurons.

Psychostimulant drugs of abuse increase dendritic spine density in reward centers of the brain. However, little is known about their effects in the hippocampus, where activity-dependent changes in the density of dendritic spine are associated with learning and memory. Recent reports suggest that Cdk5 plays an important role in drug addiction, but its role in psychostimulant's effects on dendritic spines in hippocampus remain unknown. We used in vivo and in vitro approaches to demonstrate that amphetamine increases dendritic spine density in pyramidal neurons of the hippocampus. Primary cultures and organotypic slice cultures were used for cellular, molecular, pharmacological and biochemical analyses of the role of Cdk5/p25 in amphetamine-induced dendritic spine formation. Amphetamine (two-injection protocol) increased dendritic spine density in hippocampal neurons of thy1-green fluorescent protein (GFP) mice, as well as in hippocampal cultured neurons and organotypic slice cultures. Either genetic or pharmacological inhibition of Cdk5 activity prevented the amphetamine-induced increase in dendritic spine density. Amphetamine also increased spine density in neurons overexpressing the strong Cdk5 activator p25. Finally, inhibition of calpain, the protease necessary for the conversion of p35 to p25, prevented amphetamine's effect on dendritic spine density. We demonstrate, for the first time, that amphetamine increases the density of dendritic spine in hippocampal pyramidal neurons in vivo and in vitro. Moreover, we show that the Cdk5/p25 signaling and calpain activity are both necessary for the effect of amphetamine on dendritic spine density. The identification of molecular mechanisms underlying psychostimulant effects provides novel and promising therapeutic approaches for the treatment of drug addiction.

Functional reduction of SK3-mediated currents precedes AMPA-receptor-mediated excitotoxicity in dopaminergic neurons.

In primary cultures of mesencephalon small-conductance calcium-activated potassium channels (SK) are expressed in dopaminergic neurons. We characterized SK-mediated currents (I(SK)) in this system and evaluated their role on homeostasis against excitotoxicity. I(SK) amplitude was reduced by the glutamatergic agonist AMPA through a reduction in SK channel number in the membrane. Blockade of I(SK) for 12 h with apamin or NS8593 reduced the number of dopaminergic neurons in a concentration-dependent manner. The effect of apamin was not additive to AMPA toxicity. On the other hand, two I(SK) agonists, 1-EBIO and CyPPA, caused a significant reduction of spontaneous loss of dopaminergic neurons. 1-EBIO reversed the effects of both AMPA and apamin as well. Thus, I(SK) influences survival and differentiation of dopaminergic neurons in vitro, and is part of protective homeostatic responses, participating in a rapidly acting negative feedback loop coupling calcium levels, neuron excitability and cellular defenses. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

The same cellular signaling pathways mediate survival in sensory neurons that switch their trophic requirements during development.

A distinct subpopulation of rat dorsal root sensory (DRG) neurons, termed P-neurons, switch their trophic requirements for survival during development from nerve growth factor (NGF) at embryonic stages to basic fibroblast growth factor (bFGF) just after birth. We investigated in cultured P-neurons the intracellular signaling pathways mediating survival before and after this switch. The NGF-induced survival was completely blocked by either wortmannin (100 nM) or PD98059 (25-50 nM), which selectively inhibit the phosphatidylinositol 3-kinase-AKT (PI3 kinase-AKT) and mitogen-activated kinase kinase extracellular regulated kinase (MEK-ERKs) pathways, respectively. NGF activated AKT and ERKs in single embryonic P-neurons, as assayed by immunofluorescence of phosphorylated proteins. In concordance with the survival assays, wortmannin and PD98059 blocked AKT and ERKs activation, respectively. Following the trophic switch, bFGF used the same signaling pathways to promote survival of post-natal P-neurons, as either wortmannin or PD98059 blocked its effect. Also, bFGF activated AKT and ERKs in single P-neurons, and this activation was blocked by the same inhibitors. These results strongly suggest that both pathways concurrently mediate the action of NGF and bFGF during embryonic and post-natal periods, respectively. Thus, we report the novel result that the switch in trophic requirements occurs with conservation of the signaling pathways mediating survival.

Nuclear translocation of nuclear transcription factor-kappa B by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors leads to transcription of p53 and cell death in dopaminergic neurons.

We describe a new molecular mechanism of cell death by excitotoxicity mediated through nuclear transcription factor kappa B (NF kappa B) in rat embryonic cultures of dopaminergic neurons. Treatment of mesencephalic cultures with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) resulted in a number of changes that occurred selectively in dopaminergic neurons, including persistent elevation in intracellular Ca(2+) monitored with Fura-2, and a significant increase in intramitochondrial oxidation of dihydrorhodamine 123, probably associated with transient increase of mitochondrial permeability, cytochrome c release, nuclear translocation of NF kappa B, and transcriptional activation of the oncogene p53. Interruption of any of these steps by specific antagonists prevented neurite pruning and programmed cell death. In contrast, cell death was not prevented by caspase antagonists and only partly prevented by nitric-oxide synthase inhibitors. This signal transduction pathway might be a contributing mechanism in ongoing neuronal death in Parkinson disease.