Neurons In Rat Brain Research Articles

Histochemical Detection of 3β-Hydroxysteroid Dehydrogenase in Neurons of Rat Brain

Here we present the results of histochemical detection of 3β-hydroxysteroid dehydrogenase in neurons of the neocortex, cerebellum, and brainstem. The positive reaction was observed in a low number of neocortical, midbrain, and pontine neurons. The maximum number of positive neurons was detected in the cerebellar cortex (Purkinje cells) and hippocampus.

Cytoarchitectural characteristics of hippocampal CA1 pyramidal neurons of rats, four months after global cerebral ischemia and progesterone treatment

To analyze the cytoarchitectural characteristics of the remaining pyramidal neurons in the hippocampal CA1 subfield of rats, four months after global cerebral ischemia (GCI) and progesterone treatment. Dendritic arborization, and density and shape of the dendritic spines of CA1 pyramidal neurons in brains of intact rats, or rats submitted 120 days earlier to GCI and treatment with progesterone (8 mg/kg) or its vehicle, at 15 min, and 2, 6, 24, 48, and 72 h after the onset of reperfusion, were analyzed in samples processed by a modified Golgi method. Few impregnated CA1 pyramidal neurons were identified in the ischemic vehicle-treated rats, with a short apical dendrite devoid of bifurcations and dendritic spines. In contrast, the remaining CA1 pyramidal neurons sampled from ischemic progesterone-treated rats showed sinuously branched dendrites with similar number of bifurcations and whole density of spines, and higher proportional density of mushroom spines than those in the intact group. These cytoarchitectural characteristics may underlie the long-term preservation of place learning and memory functions seen after ischemia and progesterone neuroprotective treatment, possibly compensating for the severe reduction in neuronal population.

Sonic hedgehog distribution within mature hippocampal neurons

Sonic hedgehog (Shh) regulates neural progenitor cells in the adult brain but its role in postmitotic mature neurons is not well understood. Using immunoelectron microscopy, we have recently demonstrated the postsynaptic distribution of Patched (Ptch) and Smoothened (Smo), the receptors for Shh, in hippocampal neurons of the adult rat brain. In this study, we describe the distribution of Shh protein in these adult hippocampal neurons. We find that Shh is present in both presynaptic and postsynaptic terminals. In presynaptic terminals, Shh is located either at the center or on the side of the synaptic junction. In postsynaptic terminals, Shh is mostly located on the side of the synaptic junction. We also find Shh in dendrites. Synaptic and dendritic Shh often reside in or are associated with vesicular structures that include dense-cored vesicles, synaptic vesicles, and endosomes. Thus, our subcellular map of Shh and its receptors provides a foundation for elucidating the functional significance of Shh signaling in mature neurons.

Acetyl-l-carnitine and α-lipoic acid affect rotenone-induced damage in nigral dopaminergic neurons of rat brain, implication for Parkinson's disease therapy

Although the mechanisms of neurodegeneration in Parkinson's disease are not fully understood, mitochondrial dysfunction, oxidative stress and environmental toxins may be involved. The current research was directed to investigate the protective role of two bioenergetic antioxidants, acetyl-l-carnitine and α-lipoic acid, in rotenone-parkinsonian rats. Ninety six male rats were divided into five groups. Group I is the vehicle-injected group, group II is the disease control group and was injected with six doses of rotenone (1.5mg/kg/48h, s.c.). Groups III, IV and V received rotenone in addition to acetyl-l-carnitine (100mg/kg/day, p.o.), α-lipoic acid (50mg/kg/day, p.o.) or their combination, respectively. Results showed that rotenone-treated rats exhibited bradykinesia and motor impairment in the open-field and square bridge tests. In addition, ATP level was decreased whereas lipid peroxides and protein carbonyls increased in the striata of rotenone-treated rats as compared to vehicle-treated rats. Treatment with acetyl-l-carnitine or α-lipoic acid improved the motor performance and reduced the level of lipid peroxides in rat brains as compared to rotenone group. Further, ATP production was enhanced along with acetyl-l-carnitine treatments (p≤0.05). Taken together, our study reinforces the view that acetyl-l-carnitine and α-lipoic acid are promising candidates for neuroprotection in Parkinson's disease.

Transcription factor Phox2 upregulates expression of norepinephrine transporter and dopamine β-hydroxylase in adult rat brains.

Degeneration of the noradrenergic locus coeruleus (LC) in aging and neurodegenerative diseases is well documented. Slowing or reversing this effect may have therapeutic implications. Phox2a and Phox2b are homeodomain transcriptional factors that function as determinants of the noradrenergic phenotype during embryogenesis. In the present study, recombinant lentiviral eGFP-Phox2a and -Phox2b (vPhox2a and vPhox2b) were constructed to study the effects of Phox2a/2b over-expression on dopamine β-hydroxylase (DBH) and norepinephrine transporter (NET) levels in central noradrenergic neurons. Microinjection of vPhox2 into the LC of adult rats significantly increased Phox2 mRNA levels in the LC region. Over-expression of either Phox2a or Phox2b in the LC was paralleled by significant increases in mRNA and protein levels of DBH and NET in the LC. Similar increases in DBH and NET protein levels were observed in the hippocampus following vPhox2 microinjection. In the frontal cortex, only NET protein levels were significantly increased by vPhox2 microinjection. Over-expression of Phox2 genes resulted in a significant increase in BrdU-positive cells in the hippocampal dentate gyrus. The present study demonstrates an upregulatory effect of Phox2a and Phox2b on the expression of DBH and NET in noradrenergic neurons of rat brains, an effect not previously shown in adult animals. Phox2 genes may play an important role in maintaining the function of the noradrenergic neurons after birth, and regulation of Phox2 gene expression may have therapeutic utility in aging or disorders involving degeneration of noradrenergic neurons.

Neuroprotective effects of overexpressed cyclophilin B against Aβ-induced neurotoxicity in PC12 cells

Accumulated amyloid-β (Aβ) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aβ toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aβ25-35 mediates apoptotic cell death. Aβ25-35-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aβ25-35 toxicity. We showed that Aβ25-35-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aβ25-35-induced toxicity. Consequently, these findings suggest that CypB may protect against Aβ toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway.

Study of DNA damage via the comet assay and base excision repair activities in rat brain neurons and astrocytes during aging

Earlier we have used biochemical approach to assess the number of single (SSBs) and double (DSBs) strand breaks in brain cellular DNA. However, a quick method to obtain a reliable measure of DNA damage in cells was in need for population studies. Therefore, single cell gel electrophoresis technique (popularly known as “comet” assay) has been standardized using the Trevigen protocol. DNA damage was assessed in isolated neurons and astrocytes from the cortex of young (7 days), adult (6 months) and old (2 years). Marked increase is seen in DNA damage in terms SSBs and DSBs in both types of cells by 6 months of age, which increased further by 2 years of age. The number of 8-oxoguanine DNA glycosylase (OGG1) and uracil DNA glycosylase (UDG) sensitive sites also increased in DNA with age with the simultaneous decrease in OGG1, UDG and AP endonuclease (APE1) activities. Thus the comet assay adapted to our lab conditions has proven to be useful for a quick assessment of DNA damage in a large number of samples that constitute our future studies.

The effect of ethanol on the activity of central catecholamine neurones in rat brain.

The effect of ethanol on the activity of central catecholamine neurones in rat brain.

Replenishment by 5-hydroxytryptophan of the amine stores in the central 5-hydroxytryptamine neurons after depletion induced by reserpine or by an inhibitor of monoamine synthesis.

Abstract A combined biochemical and histochemical analysis of central 5-hydroxytryptamine distribution has produced further evidence to support the theory that 5-hydroxytryptophan is specifically taken up and decarboxylated in 5-hydroxytryptamine neurons of rat brain. After treatment with an inhibitor of monoamine synthesis, α-propyldopacetamide, but not after treatment with reserpine, the intraneuronal 5-hydroxytryptamine stores could be replenished by injected 5-hydroxytryptophan. This appears to indicate that the uptake-storage mechanism of amines in the 5-hydroxytryptamine neurons is intact after α-propyldopacetamide. Pretreatment with a monoamine oxidase inhibitor, nialamide, allowed repletion of the intraneuronal 5-hydroxytryptamine stores by 5-hydroxytryptophan even after treatment with reserpine. This further stresses the importance of monoamine oxidase in regulating the amine levels of the 5-hydroxytryptamine neurons.

Dynamic Changes in Neurexins' Alternative Splicing: Role of Rho-Associated Protein Kinases and Relevance to Memory Formation

The three neurexins genes (NRXN1/2/3) encode polymorphic synaptic membrane proteins that are involved in cognitive functioning. Neurexins' selectivity of function is presumably conferred through differential use of 2 promoters and 5 alternative splicing sites (SS#1/2/3/4/5). In day-old rat brain neurons grown in culture, activation (depolarization) induces reversible, calcium dependent, repression of NRXN2α SS#3 insert. The effects of depolarization on NRXN1/2/3α splicing and biochemical pathways mediating them were further studied in these neurons. NRXN1/2/3α splicing in the course of memory formation in vivo was also explored, using fear conditioning paradigm in rats in which the animals were trained to associate an aversive stimulus (electrical shock) with a neutral context (a tone), resulting in the expression of fear responses to the neutral context.In the cultured neurons depolarization induced, beside NRXN2α SS#3, repression of SS#3 and SS#4 exons in NRXN3α but not NRXN1α. The repressions were mediated by the calcium/protein kinase C/Rho-associated protein kinase (ROCK) pathway. Fear conditioning induced significant and transient repressions of the NRXN1/2/3α SS#4 exons in the rat hippocampus. ROCK inhibition prior to training attenuated the behavioral fear response, the NRXN1/2/3α splicing repressions and subsequent recovery and the levels of excitatory (PSD95) and inhibitory (gephyrin) synaptic proteins in the hippocampus. No such effects were observed in the prefrontal cortex. Significant correlations existed between the fear response and hippocampal NRXN3α and NRXN2α SS#4 inserts as well as PSD95 protein levels. Hippocampal NRXN1α SS#4 insert and gephyrin levels did not correlate with the behavioral response but were negatively correlated with each other.These results show for the first time dynamic, experience related changes in NRXN1/2/3α alternative splicing in the rat brain and a role for ROCK in them. Specific neurexins' transcripts may be involved in synaptic remodeling occurring at an intermediate (hours) time scale in the course of memory formation.

Anti-high Mobility Group Box-1 Monoclonal Antibody Protects the Blood–Brain Barrier From Ischemia-Induced Disruption in Rats

High mobility group box-1 (HMGB1) exhibits inflammatory cytokine-like activity in the extracellular space. We previously demonstrated that intravenous injection of anti-HMGB1 monoclonal antibody (mAb) remarkably ameliorated brain infarction induced by middle cerebral artery occlusion in rats. In the present study, we focused on the protective effects of the mAb on the marked translocation of HMGB1 in the brain, the disruption of the blood-brain barrier (BBB), and the resultant brain edema. Middle cerebral artery occlusion in the rat was used as the ischemia model. Rats were treated with anti-HMGB1 mAb or control IgG intravenously. BBB permeability was measured by MRI. Ultrastructure of the BBB unit was observed by transmission electron microscope. The in vitro BBB system was used to study the direct effects of HMGB1 in BBB components. HMGB1 was time-dependently translocated and released from neurons in the ischemic rat brain. The mAb reduced the edematous area on T2-weighted MRI. Transmission electron microscope observation revealed that the mAb strongly inhibited astrocyte end feet swelling, the end feet detachment from the basement membrane, and the opening of the tight junction between endothelial cells. In the in vitro reconstituted BBB system, recombinant HMGB1 increased the permeability of the BBB with morphological changes in endothelial cells and pericytes, which were inhibited by the mAb. Moreover, the anti-HMGB1 mAb facilitated the clearance of serum HMGB1. These results indicated that the anti-HMGB1 mAb could be an effective therapy for brain ischemia by inhibiting the development of brain edema through the protection of the BBB and the efficient clearance of circulating HMGB1.

Effects of paradoxical sleep deprivation on behavioral reactions and ultrastructure of the brain neurons in rats

The aim of this work was morpho-functional analysis of the effect of paradoxical sleep deprivation of different duration on ultrastructure of the brain neurons, as well as on rearing, grooming, sexual activity and food and water consumption. It was revealed that, in early terms (36–48 hours) of the paradoxical sleep deprivation, the neuronal reparative changes were accompanied by intensification of all behavioral reactions. Under 60- hours paradoxical sleep deprivation, reparative processes in neurons were slightly reduced, while dystrophic changes covered a large number of neurons, which was accompanied by decrease in the number of main behavioral reactions.

ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.

Expression of cystic fibrosis transmembrane conductance regulator in paracervical gangliaThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an important protein that acts as a chloride channel and regulates many physiological functions, including salt transport and fluid flow. Mutations in the gene encoding the CFTR protein cause cystic fibrosis. CFTR is expressed in the epithelial cells of the lungs, pancreas, intestines, and other organs. In the peripheral and central nervous system, CFTR expression has been detected in the neurons of rat brains, ganglion cells of rat hearts, human hypothalamus, human spinal cord, and human spinal and sympathetic ganglia. However, CFTR has not been identified in other parts of the nervous system. In this study, we used immunohistochemistry, in situ hybridization, and laser-assisted microdissection (LMD) followed by reverse transcriptase (RT) PCR to identify CFTR proteins and messenger RNA in human and rat paracervical ganglion cells. CFTR and its gene expression were both detected in paracervical ganglion cells, a finding that might link this important protein to the neuronal regulation of female urogenital function. These findings could provide new insights into the symptoms related to the reproductive system frequently observed in female cystic fibrosis patients.

Mammalian Numb-interacting Protein 1/Dual Oxidase Maturation Factor 1 Directs Neuronal Fate in Stem Cells

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate.

Co-localization of PRiMA with acetylcholinesterase in cholinergic neurons of rat brain: An immunocytochemical study

In the central nervous system, acetylcholinesterase (AChE) is present in a tetrameric form that is anchored to membranes via a proline-rich membrane anchor (PRiMA). Previously it has been found that principal cholinergic neurons in the brain express high concentrations of AChE enzymic activity at their neuronal membranes. The aim of this study was to use immunocytochemical methods to determine the distribution of PRiMA in these neurons in the rat brain. Confocal laser and electron microscopic investigations showed that PRiMA immunoreactivity is associated with the membranes of the somata, dendrites and axons of cholinergic neurons in the basal forebrain, striatum and pedunculopontine nuclei, i.e. the neurons that innervate forebrain and brainstem structures. In these neurones, PRiMA also co-localizes with AChE immunoreactivity at the plasma membrane. PRiMA label was absent from neighboring GABAergic neurons, and from other neurons of the brain known to express high levels of AChE enzymic activity including cranial nerve motor neurons and dopaminergic neurons of the substantia nigra zona compacta. A strong association of AChE with PRiMA at the plasma membrane is therefore a feature specific to principal cholinergic neurons that innervate the central nervous system.

Phosphorylation status of pyruvate dehydrogenase distinguishes metabolic phenotypes of cultured rat brain astrocytes and neurons

Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria, and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons versus astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture, but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDH alpha). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorylated PDH alpha. Dephosphorylation of astrocytic PDH alpha restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed.

Regulation of FGF1 Gene Promoter through Transcription Factor RFX1

Fibroblast growth factor 1 (FGF1) has been suggested to have an important role in cell growth, proliferation, and neurogenesis. Human FGF1 gene 1B promoter (−540 to +31)-driven green fluorescence (F1BGFP) has been shown to monitor endogenous FGF1 expression. F1BGFP could also be used to isolate neural stem/progenitor cells from embryonic, neonatal, and adult mouse brains or to isolate glioblastoma stem cells (GBM-SCs) from human glioblastoma tissues. Here, we present evidence that transcription factor RFX1 could bind the 18-bp cis-elements (−484 to −467) of the F1B promoter, modulate F1BGFP expression and endogenous FGF1 expression, and further regulate the maintenance of GBM-SCs. These observations were substantiated by using yeast one-hybrid assay, electrophoretic mobility shift assay, chromatin immunoprecipitation assay, gain- and loss-of-function assays, and neurosphere assays. Overexpression of RFX1 was shown to down-regulate FGF-1B mRNA expression and neurosphere formation in human glioblastoma cells, whereas RNA interference knockdown of RFX1 demonstrated the opposite effects. Our findings provide insight into FGF1 gene regulation and suggest that the roles of FGF1 and RFX1 in the maintenance of GBM-SCs. RFX1 may negatively regulate the self-renewal of GBM-SCs through modulating FGF-1B and FGF1 expression levels by binding the 18-bp cis-elements of the F1B promoter.

Involvement of the NO Synthase System in Stress-Mediated Brain Reactions

The neurosis-like state developing as a result of chronic stress in animals and accompanied by transient cerebral hypoxia can lead to significant impairments to many brain structures. The effects of the humoral components of the stress reaction on the brain are mediated by both extra- and intracellular signal pathways, among which nitric oxide (NO) is of great importance. We report here immunohistochemical studies of the expression of the constitutive neuronal (nNOS) and inducible (iNOS) isoforms of NO synthase in neurons in the brains of white rats in conditions of chronic stress leading to the development of a neurosis- like state. Chronic stress was found to induce increases in nNOS and iNOS expression in many parts of the brain, predominantly in the neocortex and hippocampus. Administration of the nonspecific NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg) led to increases in the animals' depression, which was accompanied by reductions in motor and investigative activity assessed using traditional tests. This NOS inhibitor produced a minor increase in the expression of iNOS only. Thus, NO was shown to be involved in mediating the effects of stress with development of a neurosis-like state.

9-Aminoacridine blocks NMDA and AMPA receptors by different mechanisms

Tricyclic mono- and dicationic compounds (derivatives of 9-aminoacridine) antagonize AMPA and NMDA glutamate receptors. The aim of the present study was to compare mechanisms of the 9-aminoacridine action on AMPA and NMDA receptors. Experiments were carried out by whole-cell patch-clamp technique on native receptors from rat brain neurons. An important peculiarity of the 9-aminoacridine action on NMDA receptors is the large slope of the concentration dependence, which suggests the binding of two molecules in the channel. AMPA receptors blockade also demonstrated interesting features. In contrast to the NMDA receptor channel block, inhibition of AMPA receptors is voltage-independent. 9-Aminoacridine and its dicationic analog demonstrated similar anti-AMPA activity. For classical AMPA-receptor channel blockers (derivatives of adamantane and phenylcyclohexyl) it was demonstrated that dicationic analogs are much more potent than monocationic analogs. We conclude that 9-aminoacridine binds to a specific site in AMPA receptors. This finding opens a possibility to develop a new family of non-competitive antagonists of AMPA receptors.