Neurons In Rat Central Nervous System Research Articles

Optimization of a 96-Well Electroporation Assay for Postnatal Rat CNS Neurons Suitable for Cost–Effective Medium-Throughput Screening of Genes that Promote Neurite Outgrowth

Following an injury, central nervous system (CNS) neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterized by their failure to express key regeneration-associated genes (RAGs) and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimized a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons (CGNs) cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth inhibitory chondroitin sulfate proteoglycans. Optimal electroporation parameters resulted in 28% transfection efficiency and 51% viability for postnatal rat CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates.

Repulsive guidance molecule b inhibits neurite growth and is increased after spinal cord injury

Neuronal axons are guided by attractive and repulsive cues in their local environment. Since the identification of the repulsive guidance molecule (RGM) a (RGMa) as an axon repellent in the visual system, diverse functions, as part of the developing and adult central nervous system (CNS), have been ascribed to it. The binding of RGMa to its receptor neogenin has been shown to induce RhoA activation, leading to inhibitory/repulsive behavior and the collapse of the neuronal growth cone. In this paper, we provide evidence to suggest the involvement of RGMb, another member of the RGM family, in the rat CNS. RGMb inhibits neurite outgrowth in postnatal cerebellar granule neurons (CGNs) in vitro. RGMb is expressed by oligodendrocytes and neurons in the adult rat CNS, and the expression of this molecule is upregulated around the site of spinal cord injury. RGMb is present in myelin isolated from an adult rat brain. RGMb and neogenin are coexpressed in CGNs and entorhinal cortex neurons. These findings suggest that RGMb is a myelin-derived inhibitor of axon growth in the CNS. Inhibition of RGMb may provide an alternative approach for the treatment of spinal injuries.

A novel diol-derivative of chalcone produced by bioconversion, 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one, activates PKA/MEK/ERK signaling and antagonizes Aβ-inhibition of the cascade in cultured rat CNS neurons

Chalcone compounds have been widely studied for their anti-inflammatory, anti-pyretic, anti-invasive and anti-proliferative activities in various cell lines. However, their effects on the central nervous system (CNS) are still largely unexplored. We have recently developed a bioconversion system using a recombinant Escherichia coli that enables us to produce chemical compounds that are naturally rare and usually difficult to chemically synthesize. One such compound is 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one, a novel chalcone-diol. Here we show, for the first time, that the chalcone-diol enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) in a time- and concentration-dependent manner in cultured cortical neurons. Also, this chalcone-diol increased intracellular cyclic AMP (cAMP) concentration, thereby enhancing phosphorylation of ERK and cAMP-response element-binding protein (CREB), and CRE-mediated transcription via the cAMP-dependent protein kinase (PKA)/mitogen-activated protein kinase/ERK kinase (MEK) pathway in cultured rat hippocampal neurons. Recent studies have demonstrated that PKA/CREB-dependent signaling, which is required for long-term potentiation, is inhibited by sublethal concentrations of amyloid β-peptide (Aβ) in cultured hippocampal neurons. After treatment with the chalcone-diol at 50 μM prior to treatment with a sublethal concentration of Aβ 1–42, the Aβ 1–42-induced inhibition of phosphorylation of PKA substrates and CREB was prevented in cultured hippocampal neurons, indicating the potential for protection against the Aβ-induced impairment of PKA/CREB signaling observed in Alzheimer's disease. Therefore, these results suggest that our present study provides a new approach for discovering novel lead compounds for the treatment of neurodegenerative CNS diseases associated with impaired PKA/CREB signaling, including Alzheimer's disease.

Calcium-permeable Acid-sensing Ion Channel Is a Molecular Target of the Neurotoxic Metal Ion Lead

Acid-sensing ion channels (ASICs) are emerging as fundamental players in the regulation of neural plasticity and in pathological conditions. Here we showed that lead (Pb2+), a well known neurotoxic metal ion, reversibly and concentration-dependently inhibited ASIC currents in the acutely dissociated spinal dorsal horn and hippocampal CA1 neurons of rats. In vitro expression of ASIC subunits in combination demonstrated that both ASIC1 and -3 subunits were sensitive to Pb2+. Mechanistically, Pb2+ reduced the pH sensitivity of ASICs independent of membrane voltage change. Moreover, Pb2+ inhibited the ASIC-mediated membrane depolarization and the elevation of intracellular Ca2+ concentration. In addition, we compared the effect of Pb2+ with that of Ca2+ or amiloride to explore the possible interactions of Pb2+ and Ca2+ in regulating ASICs, and we found that Pb2+ inhibited ASIC currents independent of the amiloride/Ca2+ blockade. Because ASIC1b and -3 subunits are mainly expressed in peripheral neurons, our data identified ASIC1a-containing Ca2+-permeable ASIC as a novel central target of Pb2+ action, which may contribute to Pb2+ neurotoxicity.

Biosynthesis of NAAG by an enzyme-mediated process in rat central nervous system neurons and glia.

The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.

Corticotropin-releasing factor (CRF) and related peptides confer neuroprotection via type 1 CRF receptors

Corticotropin-releasing factor (CRF) receptors are members of the superfamily of G-protein coupled receptors that utilise adenylate cyclase and subsequent production of cAMP for signal transduction in many tissues. Activation of cAMP-dependent pathways, through elevation of intracellular cAMP levels is known to promote survival of a large variety of central and peripheral neuronal populations. Utilising cultured primary rat central nervous system neurons, we show that stimulation of endogenous cAMP signalling pathways by forskolin confers neuroprotection, whilst inhibition of this pathway triggers neuronal death. CRF and the related CRF family peptides urotensin I, urocortin, and sauvagine, which also induced cAMP production, prevented the apoptotic death of cerebellar granule neurons triggered by inhibition of phosphatidylinositol kinase-3 pathway activity with LY294002. These effects were negated by the highly selective CRF-R1 antagonist CP154,526. CRF even conferred neuroprotection when its application was delayed by up to 8 h following LY294002 addition. The CRF peptides also protected cortical and hippocampal neurons against death induced by β-amyloid peptide (1-42), in a CRF-R1 dependent manner. In separate experiments, LY294002 reduced neuronal protein kinase B activity while increasing glycogen synthase kinase-3, whilst CRF (and related peptides) promoted phosphorylation of glycogen synthase kinase-3 without protein kinase B activation. Taken together, these results suggest that the neuroprotective activity of CRF may involve cAMP-dependent phosphorylation of glycogen synthase kinase-3.

Anatomical distribution of the chemorepellent semaphorin III/collapsin-1 in the adult rat and human brain: predominant expression in structures of the olfactory-hippocampal pathway and the motor system.

Alterations in neuronal connectivity of the mature central nervous system (CNS) appear to depend on a delicate balance between growth-promoting and growth-inhibiting molecules. To begin to address a potential role of the secreted chemorepulsive protein semaphorin(D)III/collapsin-1 (semaIII/coll-1) in structural plasticity during adulthood, we used high-resolution nonradioactive in situ hybridization to identify neural structures that express semaIII/coll-1 mRNA in the mature rat and human brain. SemaIII/coll-1 was expressed in distinct but anatomically and functionally linked structures of the adult nervous system. The olfactory-hippocampal pathway displayed semaIII/coll-1 expression in a continuum of neuronal structures, including mitral and tufted cells of the olfactory bulb, olfactory tubercle, and piriform cortex; and distinct nuclei of the amygdaloid complex, the superficial layers of the entorhinal cortex, and the subiculum of the hippocampal formation. In addition, prominent labeling was found in neuronal components of the motor system, particularly in cerebellar Purkinje cells and in subpopulations of cranial and spinal motoneurons. Retrograde tracing combined with in situ hybridization also revealed that the staining of semaIII/coll-1 within the entorhinal cortex was present in the stellate neurons that project via the perforant path to the molecular layer of the dentate gyrus. Like in the rat, the human brain displayed discrete expression of semaIII/coll-1. Among the structures examined, the most prominent staining was observed in the cellular islands of the superficial layers of the human entorhinal cortex. The constitutive expression of the chemorepellent semaIII/coll-1 in discrete populations of neurons in the mature rat and human CNS raises the possibility that, in addition to its function as repulsive axon guidance cue during development, semaIII/coll-1 might be involved in restricting structural changes that occur in the wiring of the intact CNS.

Ondansetron modulates GABA A current of rat central nervous system neurons

We examined the effect of ondansetron, a 5-HT 3 receptor antagonist, on the whole cell current response of freshly isolated hypothalamic and hippocampal neurons of rats to γ-aminobutyric acid (GABA). The nystatin perforated patch technique was used to minimize run-down of the GABA current. While 1–150 μM ondansetron had no effect on membrane conductance, co-application with agonist reversibly depressed the maximal end GABA current. The concentration–response relation of GABA reveals a non-competitive mechanism. However, the inhibitory effect was more potent when ondansetron was co-applied with lower concentrations of GABA: i.e., the ondansetron concentration needed to depress the current induced by 5 μM GABA to half amplitude was 7 μM compared to 28 μM for the current induced by 10 μM GABA. Analysis of the current–voltage relationship with and without ondansetron indicated that the effect of ondansetron is not voltage dependent. Current–voltage relations also showed that the effect of ondansetron was not due to activation of a GABA-independent current because the reversal potentials were the same with and without ondansetron. The present data suggest that ondansetron's suppression of GABA-activated current may be the molecular basis of ondansetron-induced seizures observed in vivo.

Permeability to various cations of the voltage-dependent sodium channel of isolated rat hippocampal pyramidal neurons

The permeability to various cations of the voltage-dependent sodium channel of isolated rat hippocampal pyramidal neurons was investigated under the voltage-clamp condition. The neurons were dispersed enzymatically and mechanically and perfused internally using a suction pipette technique. The permeability sequence, estimated from the reveral potential of the inward current was Li + > Na + > hydrazine + > formamidine + > guanidine + > methylguanidine + > monomethylamine +. Thus the ionic selectivity of the voltage-dependent sodium channel of rat central nervous system neurons is similar to that in the squid axon, myelinated frog nerve fiber and rat ventricle muscle.