Areas Of Mouse Brain Research Articles

Abstract 2523: Isolation And Characterization Of Neural Stem/progenitor Cells From Infarct Area In Neonatal Rat Brains

Background and purpose In adult brain, it has been reported newly generated neural stem / progenitor cells survived and differentiated to mature neurons in the severely damaged area after stroke. In neonatal brain, however, it was demonstrated that neurogenesis was stimulated after hypoxia ischemia (HI) and provided neuronal progenitors to the infarct area, but only few new neurons could survive under an unfavorable microenvironment and lack of appropriate trophic support. Recently, neural stem / progenitor cells were isolated from the infarct area in adult mouse brain and elucidated its character as a neural stem / progenitor cell. However, it is still unclear whether the infarct area in neonatal brain has neural stem / progenitor cells as same feature as adult brain. Therefore, the present study was conducted to investigate whether neural stem / progenitor cells could be isolated from infarct area in neonatal rat brain, and to clear its feature. Methods HI brain injury was induced in 7-days-old rat pups by the left common carotid artery occlusion followed by 120 minutes exposure to 8% oxygen. Three days after HI, the brain is removed and sliced at 2 mm thickness, and then six columns are punched out from various infarct areas for cell culture using neurosphere method. The cell clusters are analyzed by immunocytochemistry and mRNA expression for neuron, astrocyte and oligodendrocyte. Also, the cell clusters are investigated the ability of induction for neuron, astrocyte and oligodendrocyte. Results The numbers of neurospere-like cell clusters from infarct areas at 3 days after HI are dramatically increased compared with those from sham control animals. Four and 30 days incubation of the cell clusters provided various types of cells positive for BrdU, Nestin, NG2, β III tubulin, GFAP, O4, Vimentin or Iba1. These cell clusters expressed mRNA such as Nanog, Sox2, Oct3/4 and Rex1 as same as ES cell. Also these cell clusters could be differentiated into neurons, astrocytes or oligodendrocytes, and expressed mRNA such as Nestin, βIIItubulin, GFAP and MBP. Conclusion We have isolated injury-induced neural stem / progenitor cells form ischemic area after neonatal HI. This cell has a potential to use as a source for new neurons driving CNS repair after neonatal HI.

Tau protein phosphorylation in diverse brain areas of normal and CRH deficient mice: up-regulation by stress.

Tau protein misfolding is a pathological mechanism, which plays a critical role in the etiopathogenesis of neurodegeneration. However, it is not entirely known what kind of stimuli can induce the misfolding. It is believed that physical and emotional stresses belong to such risk factors. Although the influence of stress on the onset and progression of Alzheimer's disease (AD) has already been proposed, the molecular links between stresses and AD are still unknown. We have therefore focused our attention on determination of the influence of acute immobilization stress (IMO) in normal mice and mice deficient in corticotropin-releasing hormone (CRH). Specifically, we have analyzed levels of hyperphosphorylated tau proteins, bearing the AD-specific phospho-epitopes (AT-8, pT181, and PHF-1), which are implicated in the pathogenesis of AD. We found that IMO induces transient hyperphosphorylation of tau proteins regardless of continuation of the stimulus. Concerning tau modifications, detailed analysis of the mouse brain revealed that neurons in different brain regions including frontal cortex, temporal cortex, hippocampal C1 and CA3 regions, dentate gyrus as well as nucleus basalis Meynert, and several brainstem nuclei such as locus coeruleus but also raphe nucleus and substantia nigra respond similarly to IMO. The strongest tau protein phosphorylation was observed after 30min of IMO stress. Stress lasting for 120min led either to the disappearance of tau hyperphosphorylation or to the induction of a second wave of hyperphosphorylation. Noteworthy is the magnitude of pathological phosphorylation of tau protein in CRH and glucocorticoids deficient mice, being much lower in comparison to that observed in wild-type animals, which suggests a critical role of CRH in the pathogenesis of AD. Thus, our results indicate that hyperphosphorylation of tau protein induced by stress may represent the pathogenic event upstream of tau protein misfolding, which leads to progression or eventually initiation of neurodegeneration. The data show that CRH plays an important role in stress induced hyperphosphorylation of tau protein, which might be either a direct effect of CRH innervations in the brain or an effect mediated via the hypothalamo-pituitary-adrenal axis.

Monocrotaline: histological damage and oxidant activity in brain areas of mice.

This work was designed to study MCT effect in histopathological analysis of hippocampus (HC) and parahippocampal cortex (PHC) and in oxidative stress (OS) parameters in brain areas such as hippocampus (HC), prefrontal cortex (PFC), and striatum (ST). Swiss mice (25–30 g) were administered a single i.p. dose of MCT (5, 50, or 100 mg/kg) or 4% Tween 80 in saline (control group). After 30 minutes, the animals were sacrificed by decapitation and the brain areas (HC, PHC, PFC, or ST) were removed for histopathological analysis or dissected and homogenized for measurement of OS parameters (lipid peroxidation, nitrite, and catalase) by spectrophotometry. Histological evaluation of brain structures of rats treated with MCT (50 and 100 mg/kg) revealed lesions in the hippocampus and parahippocampal cortex compared to control. Lipid peroxidation was evident in all brain areas after administration of MCT. Nitrite/nitrate content decreased in all doses administered in HC, PFC, and ST. Catalase activity was increased in the MCT group only in HC. In conclusion, monocrotaline caused cell lesions in the hippocampus and parahippocampal cortex regions and produced oxidative stress in the HC, PFC, and ST in mice. These findings may contribute to the neurological effects associated with this compound.

Evidence for a role of inhibition of orexinergic neurons in the anxiolytic and sedative effects of diazepam: A c-Fos study

The classical benzodiazepine diazepam (DZ) induces anxiolysis at low doses and sedation and hypnosis at higher doses. Different brain areas and neuronal populations most likely mediate these different behavioral effects. We used c-Fos immunohistochemistry as an indirect way to study neuronal activation or inhibition induced by DZ at anxiolytic and sedative doses (0.5 and 5mg/kg, respectively) in various brain areas involved in anxiety, arousal, sedation and addiction in C57BL/6J mice. We also focused on the two neuronal populations, orexinergic and dopaminergic neuronal populations, with the help of double-immunohistochemistry using c-Fos and orexin-A antibodies and c-Fos and tyrosine hydroxylase antibodies. We found that different brain areas of unhabituated mice reacted differently to the mild stress induced by vehicle injection. Also the response to anxiolytic or sedative doses of DZ differed between the areas, suggesting that distinct brain areas mediate the behavioral effects of low and high DZ doses. Our findings propose a role for inhibition of orexin neurons in the anxiolytic and sleep-promoting effects of DZ. In addition, the activation of central amygdala neurons by DZ treatment was associated with anxiolytic and sedative effects. On the other hand, the ventral hippocampus, basolateral amygdala, ventral tegmental area and prefrontal cortex were sensitive even to the mild injection stress, but not to the anxiolytic dose of DZ.

Microglial activation, recruitment and phagocytosis as linked phenomena in ferric oxide nanoparticle exposure

Microglia as the resident macrophage-like cells in the central nervous system (CNS) play a pivotal role in the innate immune responses of CNS. Understanding the reactions of microglia cells to nanoparticle exposure is important in the exploration of neurobiology of nanoparticles. Here we provide a systemic mapping of microglia and the corresponding pathological changes in olfactory-transport related brain areas of mice with Fe 2O 3-nanoparticle intranasal treatment. We showed that intranasal exposure of Fe 2O 3 nanoparticle could lead to pathological alteration in olfactory bulb, hippocampus and striatum, and caused microglial proliferation, activation and recruitment in these areas, especially in olfactory bulb. Further experiments with BV2 microglial cells showed the exposure to Fe 2O 3 nanoparticles could induce cells proliferation, phagocytosis and generation of ROS and NO, but did not cause significant release of inflammatory factors, including IL-1β, IL-6 and TNF-α. Our results indicate that microglial activation may act as an alarm and defense system in the processes of the exogenous nanoparticles invading and storage in brain.

Efficacy of a Combined Intracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder

Multiple sulfatase deficiency (MSD), a severe autosomal recessive disease is caused by mutations in the sulfatase modifying factor 1 gene (Sumf1). We have previously shown that in the Sumf1 knockout mouse model (Sumf1(-/-)) sulfatase activities are completely absent and, similarly to MSD patients, this mouse model displays growth retardation and early mortality. The severity of the phenotype makes MSD unsuitable to be treated by enzyme replacement or bone marrow transplantation, hence the importance of testing the efficacy of novel treatment strategies. Here we show that recombinant adeno-associated virus serotype 9 (rAAV9) vector injected into the cerebral ventricles of neonatal mice resulted in efficient and widespread transduction of the brain parenchyma. In addition, we compared a combined, intracerebral ventricles and systemic, administration of an rAAV9 vector encoding SUMF1 gene to the single administrations-either directly in brain, or systemic alone -in MSD mice. The combined treatment resulted in the global activation of sulfatases, near-complete clearance of glycosaminoglycans (GAGs) and decrease of inflammation in both the central nervous system (CNS) and visceral organs. Furthermore, behavioral abilities were improved by the combined treatment. These results underscore that the "combined" mode of rAAV9 vector administration is an efficient option for the treatment of severe whole-body disorders.

Localization of nesfatin-1 neurons in the mouse brain and functional implication

Nesfatin-1 reduces food intake when injected centrally in rodents. We recently described wide distribution of nucleobindin2 (NUCB2)/nesfatin-1 immunoreactivity in rat brain autonomic nuclei activated by various stressors. We used C57BL/6 mice to localize brain NUCB2/nesfatin-1 immunoreactivity and assessed activation of NUCB2/nesfatin 1 neurons after water avoidance stress (WAS). Gastric emptying of a non-nutrient liquid was also determined. NUCB2/nesfatin-1 immunoreactivity was detected in cortical areas including piriform, insular, cingulate and somatomotor cortices, the limbic system including amygdaloid nuclei, hippocampus and septum, the basal ganglia, bed nucleus of the stria terminalis, the thalamus including paraventricular and parafascicular nuclei, the hypothalamus including supraoptic, periventricular, paraventricular (PVN), arcuate nuclei and ventromedial and lateral hypothalamic areas. Intensely labeled NUCB2/nesfatin-1 neurons were detected in a previously undefined region which we named intermediate dorsomedial hypothalamus. In the brainstem, NUCB2/nesfatin-1 immunoreactivity was detected in the raphe nuclei, Edinger–Westphal nucleus, locus coeruleus (LC), lateral parabrachial nucleus, ventrolateral medulla (VLM) and dorsal vagal complex. WAS induced Fos expression in 35% of NUCB2/nesfatin-1-immunoreactive neurons in the PVN, 50% in the LC, 54% in the rostral raphe pallidus, 58% in the VLM, 39% in the middle part of the nucleus of the solitary tract (NTS) and 33% in the caudal NTS. Nesfatin-1 injected intracerebroventricularly significantly decreased gastric emptying. These data showed that NUCB2/nesfatin-1 immunoreactivity is distributed in mouse brain areas involved in the regulation of stress response and visceral functions activated by an acute psychological stressor suggesting that nesfatin-1 might play a role in the efferent component of the stress response.

Alterations in BDNF and phospho-CREB levels following chronic oral nicotine treatment and its withdrawal in dopaminergic brain areas of mice

Neuronal changes induced by chronic nicotine in the brain dopaminergic circuits are thought to lead to compulsive nicotine use. When nicotine is given to mice chronically in their drinking water, its intake and effects mimic human smoking. Previously, we have reported that this treatment in mice induces several neurochemical and behavioural changes that are associated with nicotine addiction. Here we studied the effects of chronic oral nicotine treatment and nicotine treatment cessation on two well-characterised markers of neuronal plasticity, brain-derived neurotrophic factor (BDNF) and phosphorylated cAMP-responsive element-binding protein (pCREB), in several dopaminergic brain areas. BDNF levels were not altered by chronic nicotine treatment, but they were significantly increased in the nucleus accumbens (NAc) after 24 h and 29 days of nicotine abstinence and in the ventral tegmental area (VTA) and substantia nigra after 29 days of nicotine abstinence. These findings suggest that nicotine abstinence promotes long-lasting neuroadaptations in dopaminergic neurocircuits by inducing BDNF production. Withdrawal from chronic nicotine treatment oppositely affected pCREB levels in the NAc and in the VTA. Thus, in the NAc, the pCREB levels were significantly elevated and in the VTA significantly decreased as compared with the pCREB levels during the nicotine treatment. These alterations could be compensatory and related to increased dopaminergic signalling during nicotine treatment. In conclusion, the current results suggest the involvement of BDNF- and CREB-related neuronal processes in nicotine-induced neurochemical, behavioural, and neuroplastic changes in dopaminergic neurocircuits.

A Simple and Highly Sensitive HPLC Method with Fluorescent Detection for Determination of Pipecolic Acid in Mouse Brain Areas

A simple and highly sensitive high-performance liquid chromatography method for the determination of pipecolic acid in mouse brain areas was developed. After homogenization of brain and pretreatment with o-phthalaldehyde, the pipecolic acid and (2S,3S)-3-methylpyrrolidine-2-carboxylic acid (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 °C for 15 min in basic medium (pH 9.0). The fluorescent derivatives of pipecolic acid and internal standard were separated on a reversed-phase column by stepwise elution using acetic acid (30 mM)-acetonitrile at 50 °C and detected by fluorescence measurement at 316 nm (excitation) and 403 nm (emission). The detection limit (signal-to-noise ratio=3) of pipecolic acid was 13 fmol per injection. The recovery was about 106.7%. The precision (relative standard deviation) was 3.2%.

Effect of some cement components on ion contents in different brain areas of adult male albino mice

The aim of this study is to investigate the chronic effect of some cement components on the content of ions in different brain areas in adult male albino mice. It is clear that chronic intraperitoneal administration of 0.0013mg/g aluminum ion caused a significant increase in aluminum, calcium and sodium ions and significant decrease in iron ions, the chronic intraperitoneal administration of 0.00065mg/g iron caused a significant increase in iron, calcium, and sodium ions but No significant change in potassium and aluminum ions. Chronic intraperitoneal administration of 0.0013mg/g silicon caused no significant change in calcium, potassium, sodium, aluminum and iron. Chronic intraperitoneal administration of 0.0013mg/g aluminum, 0.0013mg/g silicon and 0.00065mg/g iron, respectively, – using separating time interval 30min between each – caused a higher elevation in calcium, sodium, aluminum and iron concentrations than the elevation in other groups and no significant change in potassium ions. This may be due to the elevation in glutamate which leads to increase in the intracellular of calcium concentration and the inhibition of membrane-bound Na+, K+, Ca2+ ATPase activity which lead to cellular alterations and may be death. So long-term exposure to cement components as environmental pollutants may lead to neurodegenerative diseases.

Spatiotemporal expression of PSD-95 in Fmr1 knockout mice brain

To investigate and compare the spatial and temporal expression of post-synaptic density-95 (PSD-95) in Fmr1 knockout mice (the animal model of fragile X syndrome, FXS) and wild-type mice brain, on postnatal day 7 (P7), P14, P21, P28 and P90, mice from each group were decapitated, and three principal brain regions (cerebral cortex, hippocampus and cerebellum) were obtained and stored for later experiments. PSD-95 mRNA in the three brain areas was analyzed with quantitative RT-PCR. PSD-95 protein was measured by immunohistochemical staining and Western blot. In the three principal brain areas of Fmr1 knockout mice and wild-type mice, the expression of PSD-95 mRNA and protein were detected at the lowest levels on P7, and then significantly increased on P14, reaching the peak levels in adolescents or adults. Moreover, it was found that PSD-95 mRNA and protein in the hippocampus were significantly decreased in Fmr1 knockout mice during the developmental period (P7, P14, P21 and P28) as well as at adulthood (P90) (P < 0.05, and P < 0.01, respectively). However, there was no significant difference of expression of PSD-95 in the cortex and cerebellum between Fmr1 knockout and wild mice. The expression of PSD-95 in the hippocampus might be regulated by fragile X mental retardation protein (FMRP) during mice early developmental and adult periods. It is suggested that impairment of PSD-95 is possibly involved in hippocampal-dependent learning defects, which are common in people with FXS.

Neuroanatomical distribution and neurochemical characterization of cells expressing adenylyl cyclase isoforms in mouse and rat brain

Transmembrane adenylyl cyclases (Adcy) are involved in the regulation of multiple brain processes such as synaptic plasticity, learning and memory. They synthesize intracellular cyclic adenosine monophosphate (cAMP) following activation by G-protein coupled receptors. We examined the neuroanatomical distribution of the nine Adcy isoforms in rat and mouse brain by in situ hybridization, as well as their location in glutamatergic, GABAergic and cholinergic neurons in several mouse brain areas by double in situ hybridization. The Adcys are widely distributed throughout the brain in both rat and mouse, being especially abundant in cortex, hippocampus, thalamic nuclei, the olfactory system and the granular layer of the cerebellum. Double-labeling experiments showed that Adcy isoforms are differently expressed in glutamatergic, GABAergic and cholinergic neuronal cell populations. We report the neuroanatomical distribution of the nine known Adcy isoforms in rat and mouse brain and their cellular localization.

Flavin-Containing Monooxygenase mRNA Levels are Up-Regulated in ALS Brain Areas in SOD1-Mutant Mice

Flavin-containing monooxygenases (FMOs) are a family of microsomal enzymes involved in the oxygenation of a variety of nucleophilic heteroatom-containing xenobiotics. Recent results have pointed to a relation between Amyotrophic Lateral Sclerosis (ALS) and FMO genes. ALS is an adult-onset, progressive, and fatal neurodegenerative disease. We have compared FMO mRNA expression in the control mouse strain C57BL/6J and in a SOD1-mutated (G93A) ALS mouse model. Fmo expression was examined in total brain, and in subregions including cerebellum, cerebral hemisphere, brainstem, and spinal cord of control and SOD1-mutated mice. We have also considered expression in male and female mice because FMO regulation is gender-related. Real-Time TaqMan PCR was used for FMO expression analysis. Normalization was done using hypoxanthine-guanine phosphoribosyl transferase (Hprt) as a control housekeeping gene. Fmo genes, except Fmo3, were detectably expressed in the central nervous system of both control and ALS model mice. FMO expression was generally greater in the ALS mouse model than in control mice, with the highest increase in Fmo1 expression in spinal cord and brainstem. In addition, we showed greater Fmo expression in males than in female mice in the ALS model. The expression of Fmo1 mRNA correlated with Sod1 mRNA expression in pathologic brain areas. We hypothesize that alteration of FMO gene expression is a consequence of the pathological environment linked to oxidative stress related to mutated SOD1.

Acetylcholinesterase inhibition in cognition-relevant brain areas of mice treated with a nootropic Amazonian herbal (Marapuama)

The goal of acetylcholinesterase inhibitors (AChEIs) used to treat Alzheimer's patients is an improvement in cholinergic transmission. While currently available AChEIs have limited success, a huge impediment to the development of newer ones is access to the relevant brain areas. Promnesic, anti-amnesic and AChEI properties were identified in a standardized ethanol extract from Ptychopetalum olacoides (POEE), a medicinal plant favored by the elderly in Amazon communities. The purpose of this study was to provide conclusive evidence that orally given POEE induces AChE inhibition in brain areas relevant to cognition. Histochemistry experiments confirmed that the anticholinesterase compound(s) present in POEE are orally bioavailable, inducing meaningful AChE inhibition in the hippocampus CA1 (∼33%) and CA3 (∼20%), and striatum (∼17%). Ellman's colorimetric analysis revealed that G1 and G4 AChE isoforms activities were markedly inhibited (66 and 72%, respectively) in hippocampus and frontal cortex (50 and 63%, respectively), while G4 appeared to be selectively inhibited (72%) in the striatum. Western blotting showed that POEE did not induce significant changes in the AChE immunocontent suggesting that its synthesis is not extensively modified. This study provides definitive proof of meaningful anticholinesterase activity compatible with the observed promnesic and anti-amnesic effects of POEE in mice, reaffirming the potential of this extract for treating neurodegenerative conditions where a hypofunctioning cholinergic neurotransmission is prominent. Adequate assessment of the safety and efficacy of this extract and/or its isolated active compound(s) are warranted.

A miniaturized chronic microelectrode drive for awake behaving head restrained mice and rats

The present work introduces an electrode microdrive system small enough to be placed into two distinct brain areas in head-fixed mice. To meet the space constraint imposed by the size of mice and the additional presence of a head post, the size and weight of the components were minimized. In the current version, an individual microdrive moves an array of four Reitboeck type electrodes. We report about successful implantation in rats and mice using one or two microdrives. Using two of these devices in individual mice/rats, the recording of parallel single and multi-unit as well as local field potential from prefrontal, motor, somatosensory cortex and hippocampus is demonstrated. The system can be easily constructed with machinery and equipment present in most neurophysiology labs.

Fragile X Mental Retardation Protein Regulates the Levels of Scaffold Proteins and Glutamate Receptors in Postsynaptic Densities

Functional absence of fragile X mental retardation protein (FMRP) causes the fragile X syndrome, a hereditary form of mental retardation characterized by a change in dendritic spine morphology. The RNA-binding protein FMRP has been implicated in regulating postsynaptic protein synthesis. Here we have analyzed whether the abundance of scaffold proteins and neurotransmitter receptor subunits in postsynaptic densities (PSDs) is altered in the neocortex and hippocampus of FMRP-deficient mice. Whereas the levels of several PSD components are unchanged, concentrations of Shank1 and SAPAP scaffold proteins and various glutamate receptor subunits are altered in both adult and juvenile knock-out mice. With the exception of slightly increased hippocampal SAPAP2 mRNA levels in adult animals, altered postsynaptic protein concentrations do not correlate with similar changes in total and synaptic levels of corresponding mRNAs. Thus, loss of FMRP in neurons appears to mainly affect the translation and not the abundance of particular brain transcripts. Semi-quantitative analysis of RNA levels in FMRP immunoprecipitates showed that in the mouse brain mRNAs encoding PSD components, such as Shank1, SAPAP1-3, PSD-95, and the glutamate receptor subunits NR1 and NR2B, are associated with FMRP. Luciferase reporter assays performed in primary cortical neurons from knock-out and wild-type mice indicate that FMRP silences translation of Shank1 mRNAs via their 3'-untranslated region. Activation of metabotropic glutamate receptors relieves translational suppression. As Shank1 controls dendritic spine morphology, our data suggest that dysregulation of Shank1 synthesis may significantly contribute to the abnormal spine development and function observed in brains of fragile X syndrome patients.

Induction of cerebral β-amyloidosis: Intracerebral versus systemic Aβ inoculation

Despite the importance of the aberrant polymerization of Aβ in the early pathogenic cascade of Alzheimer's disease, little is known about the induction of Aβ aggregation in vivo. Here we show that induction of cerebral β-amyloidosis can be achieved in many different brain areas of APP23 transgenic mice through the injection of dilute Aβ-containing brain extracts. Once the amyloidogenic process has been exogenously induced, the nature of the induced Aβ-deposition is determined by the brain region of the host. Because these observations are reminiscent of a prion-like mechanism, we then investigated whether cerebral β-amyloidosis also can be induced by peripheral and systemic inoculations or by the intracerebral implantation of stainless steel wires previously coated with minute amounts of Aβ-containing brain extract. Results reveal that oral, intravenous, intraocular, and intranasal inoculations yielded no detectable induction of cerebral β-amyloidosis in APP23 transgenic mice. In contrast, transmission of cerebral β-amyloidosis through the Aβ-contaminated steel wires was demonstrated. Notably, plasma sterilization, but not boiling of the wires before implantation, prevented the induction of β-amyloidosis. Our results suggest that minute amounts of Aβ-containing brain material in direct contact with the CNS can induce cerebral β-amyloidosis, but that systemic cellular mechanisms of prion uptake and transport to the CNS may not apply to Aβ.

Anabolic‐androgenic steroid treatment induces behavioral disinhibition and downregulation of serotonin receptor messenger RNA in the prefrontal cortex and amygdala of male mice

Nandrolone is an anabolic-androgenic steroid (AAS) that is highly abused by individuals seeking enhanced physical strength or body appearance. Supraphysiological doses of this synthetic testosterone derivative have been associated with many physical and psychiatric adverse effects, particularly episodes of impulsiveness and overt aggressive behavior. As the neural mechanisms underlying AAS-induced behavioral disinhibition are unknown, we investigated the status of serotonergic system-related transcripts in several brain areas of mice receiving prolonged nandrolone administration. Male C57BL/6J mice received 15 mg/kg of nandrolone decanoate subcutaneously once daily for 28 days, and different sets of animals were used to investigate motor-related and emotion-related behaviors or 5-HT-related messenger RNA (mRNA) levels by real-time quantitative polymerase chain reaction. AAS-injected mice had increased body weight, were more active and displayed anxious-like behaviors in novel environments. They exhibited reduced immobility in the forced swim test, a higher probability of being aggressive and more readily attacked opponents. AAS treatment substantially reduced mRNA levels of most investigated postsynaptic 5-HT receptors in the amygdala and prefrontal cortex. Interestingly,the 5-HT(1B) mRNA level was further reduced in the hippocampus and hypothalamus. There was no alteration of 5-HT system transcript levels in the midbrain. In conclusion,high doses of AAS nandrolone in male mice recapitulate the behavioral disinhibition observed in abusers. Furthermore, these high doses downregulate 5-HT receptor mRNA levels in the amygdala and prefrontal cortex. Our combined findings suggest these areas as critical sites for AAS-induced effects and a possible role for the 5-HT(1B) receptor in the observed behavioral disinhibition.

Evidence for Excitatory and Inhibitory Amino Acids Participation in the Neuropharmacological Activity of Alpha- and Beta-Amyrin Acetate

We evaluated the neuropharmacological profile of acetylated alpha- and beta-amyrin (AcAMY) obtained by the acetylation of the isomeric mixture of alpha- and beta-amyrin isolated from Protium heptaphyllum. Male Swiss mice were administered with AcAMY (2.5, 5, 10 and 25 mg/kg, i.p.), and anticonvulsant (pentylenetrazole- and pilocarpine-induced convulsions), sedative (barbiturate-induced sleep and open field tests) and anxiolytic (elevated plus maze test) activities were studied. Results showed that AcAMY administered intraperitoneally or orally, protected the animals against penty- lenetetrazole- but not against pilocarpine-induced convulsions. The drug increased both the latency to the 1 st convulsion and the latency to death. The barbiturate-induced sleeping time was also increased, as well as the ethyl ether-induced sleeping time, confirming the sedative nature of AcAMY. The acute administration of AcAMY also produced an anx- iolytic effect. After the sub-chronic administration, both sedative and anxiolytic effects were manifested, at the two higher doses. Amino acids measurements in brain areas of mice treated with AcAMY (25 mg/kg, i.p., for 7 days) showed an 89% increase in tyrosine levels, in the hippocampus. In the striatum, tyrosine and taurine were increased by 97 and 79%, re- spectively, while decreases in the levels of aspartate, GABA and glutamate of 72, 55 and 60%, respectively were ob- served. In conclusion, our results showed that AcAMY presents sedative, anxiolytic and anticonvulsant properties. Al- though the drug mechanism of action is not completely clarified, it seems to involve a decrease in excitatory amino acids and an increase of inhibitory amino acids. Furthermore, the GABAergic system may also play a role.

CDKL5 Expression Is Modulated during Neuronal Development and Its Subcellular Distribution Is Tightly Regulated by the C-terminal Tail

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with Rett syndrome (RTT), West syndrome, and X-linked infantile spasms, sharing the common feature of mental retardation and early seizures. CDKL5 is a rather uncharacterized kinase, but its involvement in RTT seems to be explained by the fact that it works upstream of MeCP2, the main cause of Rett syndrome. To understand the role of this kinase for nervous system functions and to address if molecular mechanisms are involved in regulating its distribution and activity, we studied the ontogeny of CDKL5 expression in developing mouse brains by immunostaining and Western blotting. The expression profile of CDKL5 was compared with that of MeCP2. The two proteins share a general expression profile in the adult mouse brain, but CDKL5 levels appear to be highly modulated at the regional level. Its expression is strongly induced in early postnatal stages, and in the adult brain CDKL5 is present in mature neurons, but not in astroglia. Interestingly, the presence of CDKL5 in the cell nucleus varies at the regional level of the adult brain and is developmentally regulated. CDKL5 shuttles between the cytoplasm and the nucleus and the C-terminal tail is involved in localizing the protein to the cytoplasm in a mechanism depending on active nuclear export. Accordingly, Rett derivatives containing disease-causing truncations of the C terminus are constitutively nuclear, suggesting that they might act as gain of function mutations in this cellular compartment.