A positive modulator of K Ca 2 and K Ca 3 channels, 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591), inhibits bladder afferent firing in vitro and bladder overactivity in vivo.

Calcium-activated potassium channels are attractive targets for the development of therapeutics for overactive bladder. In the current study, we addressed the role of calcium-activated potassium channels of small (SK; K(Ca)2) and intermediate (IK; K(Ca)3) conductance in bladder function pharmacologically. We identified and characterized a novel positive modulator of SK/IK channels, 4,5-dichloro-1,3-diethyl-1,3-dihydro-benzoimidazol-2-one (NS4591). In whole-cell patch-clamp experiments, NS4591 doubled IK-mediated currents at a concentration of 45 +/- 6 nM(n = 16), whereas 530 +/- 100 nM (n = 7) was required for doubling of SK3-mediated currents. In acutely dissociated bladder primary afferent neurons, the presence of SK channels was verified using apamin and 1-ethyl-2-benzimidazolinone. In these neurons, NS4591 (10 microM) inhibited the number of action potentials generated by suprathreshold depolarizing pulses. NS4591 also reduced carbachol-induced twitches in rat bladder detrusor rings in an apamin-sensitive manner. In vivo, NS4591 (30 mg/kg) inhibited bladder overactivity in rats and cats induced by capsaicin and acetic acid, respectively. In conclusion, the present study supports the involvement of calcium-activated potassium channels in bladder function and identifies NS4591 as a potent modulator of IK and SK channels that is effective in animal models of bladder overactivity.

Hypoxic/ischemic conditions induce expression of the putative pro-death gene Clca1 via activation of extrasynaptic N-methyl-D-aspartate receptors.

The stimulation of extrasynaptic N-methyl-D-aspartate (NMDA) receptors triggers cell death pathways and has been suggested to play a key role in cell degeneration and neuron loss associated with glutamate-induced excitotoxicity. In contrast, synaptic NMDA receptors promote neuronal survival. One mechanism through which extrasynaptic NMDA receptors damage neurons may involve Clca1, which encodes a putative calcium-activated chloride channel. Here we show that Clca1 expression is induced in cultured rat hippocampal neurons exposed to oxygen/glucose-free media; this induction is mediated by a signaling pathway activated by extrasynaptic NMDA receptors. Clca1 mRNA levels also increased in the gerbil hippocampus following a transient forebrain ischemia caused by bilateral carotid occlusion. Microelectrode array recordings revealed that oxygen-glucose deprivation enhances hippocampal network firing rates, which induces c-fos transcription through a signaling pathway that, in contrast to Clca1, is activated by synaptic but not extrasynaptic NMDA receptors. Thus, conditions of low oxygen/glucose lead to the activation of both extrasynaptic and synaptic NMDA receptors that regulate distinct target genes. Clca1 may be part of the genomic death program triggered by extrasynaptic NMDA receptors; it could be a marker for ischemic brain damage and a possible target for therapeutic interventions.

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

Regulation of activity-regulated cytoskeleton protein (Arc) mRNA after acute and chronic electroconvulsive stimulation in the rat.

The temporal profile of Arc gene expression after acute and chronic electroconvulsive stimulations (ECS) was studied using semi-quantitative in situ hybridisation in the rat cortex. A single ECS strongly and temporarily increased Arc mRNA levels in dentate granular cells with maximal induction seen up to 4 h after the stimulus, but returned to baseline at 24 h. A single ECS also increased expression of Arc mRNA in the CA1 and the parietal cortex, but the expression peaked within 1 h and returned to baseline levels within 2 h. Repeated or chronic ECS is a model of electroconvulsive therapy and it would be predicted that gene products involved in antidepressant effects accumulate after repeated ECS. However, repeated ECS reduced Arc gene expression in the CA1 24 h after the last stimulus. These results indicate that Arc is an immediate early gene product regulated by an acute excitatory stimulus, but not accumulated by long term repetitive ECS and therefore not a molecular biomarker for antidepressant properties. More likely, Arc is likely a molecular link to the decline in memory consolidation seen in depressive patients subjected to electroconvulsive therapy.

Homophilic NCAM interactions interfere with L1 stimulated neurite outgrowth.

The cell adhesion molecules NCAM and L1 are considered to play key roles in neuronal development and plasticity. L1 has been shown to interact with NCAM, possibly through NCAM binding to oligomannosidic glycans present in L1. We investigated the effect of recombinant immunoglobulin (Ig) modules of NCAM involved in homophilic NCAM binding, on L1 induced neurite outgrowth from PC12-E2 cells and found a complete inhibition of L1 induced neurite outgrowth after addition of Ig-modules 1, 2 and 3 of NCAM, suggesting that the ligation state of NCAM is crucial for normal L1 signaling.

The neural cell adhesion molecule (NCAM) in development and plasticity of the nervous system.

The neural cell adhesion molecule (NCAM) is a member of the immunoglobulin superfamily and is strongly expressed in the nervous system. NCAM is found in three major forms, of which two--NCAM-140 and NCAM-180--are transmembrane proteins, while the third--NCAM-120--is attached to the membrane via a glycosylphosphatidyl inositol anchor. In addition, soluble NCAM forms exist in brain, cerebrospinal fluid, and plasma. NCAM mediates cell adhesion through homophilic as well as through heterophilic interactions. Following NCAM binding, transmembrane signalling is believed to be activated, resulting in increased intracellular calcium. By mediating cell adhesion to other cells and to the extracellular matrix and by activating intracellular signaling pathways, NCAM influences cell migration, neurite extension, and fasciculation, and possibly formation of synapses in the brain. From studies on NCAM knock-out mice, NCAM have been shown to be crucial for the formation of the olfactory bulb and the mossy fiber system in the hippocampus. In addition, NCAM is important for neuronal plasticity in the adult brain associated with learning and regeneration.

NADPH-diaphorase (NOS) is induced in pyramidal neurones of hippocampal slices.

We found NADPH-diaphorase (presumably identical with nitric oxide synthase) in pyramidal neurones of the hippocampus in slices that stayed in a chamber for 30 min or longer. In some instances parallel slices showed normal membrane properties when studied electrophysiologically. In freshly made slices the pyramidal neurones were not stained. Thus, after induction of the enzyme, the hippocampal pyramidal neurones can synthesize nitric oxide which may serve as a retrograde messenger in long-term potentiation. The enzyme may also play a role in cell loss seen in slices which stayed in a chamber for 9-22 h before fixation.

A simple procedure for quantification of neurite outgrowth based on stereological principles.

The molecular mechanisms controlling formation and remodelling of neuronal extensions are of considerable interest for the understanding of neuronal development and plasticity. Determination of neurite outgrowth in cell culture is a widely used approach to investigate these phenomena. This is generally done by a time consuming tracing of individual neurites and their branches. We have used stereological principles to determine the length of neurites. The total neuritic length per cell was estimated by counting the number of intersections between neurites and test lines of an unbiased counting frame superimposed on images of cell cultures obtained by conventional computer-assisted microscopy. The absolute length, L, of neurites per cell was subsequently estimated from the number of neurite intersections, I, per cell by means of the equation L=(pid/2)I describing the relationship between the number of neurite intersections and the vertical distance, d, between the test lines used. When measuring neurite outgrowth from PC12 cells and primary hippocampal neurons, data obtained by counting neuritic intersections correlated statistically significantly with data obtained using a conventional tracing technique. However, information was acquired more efficiently using the stereological approach. Thus, using the described set-up, the stereological procedure was approximately five times less time consuming than the conventional method based on neurite tracing. The study shows that stereological estimation of neuritic length provides a precise and efficient method for the study of neurite outgrowth in cultures of primary neurons and cell lines.

NCAM-antibodies modulate induction of long-term potentiation in rat hippocampal CA1.

The neural cell adhesion molecule (NCAM) probably plays a role in neural plasticity in the adult vertebrate brain. We here present evidence that NCAM may be involved in long-term potentiation (LTP) in the CA1-region of rat hippocampal slices. It is shown that local application of antibodies against NCAM inhibits subsequent LTP-induction. Thus NCAM may be directly involved in the initial phase of LTP-induction. These results have important implications for the possible involvement of NCAM in learning and memory.

The neural cell adhesion molecule in synaptic plasticity and ageing.

By mediating cell adhesion and signal transduction, the neural cell adhesion molecule (NCAM) regulates neurite outgrowth, fasciculation and target recognition in the developing nervous system. In addition, a number of studies suggest an important role for the NCAM in regeneration and learning in the adult nervous system. NCAM-deficient mice are impaired in spatial learning. Moreover, by interfering with normal NCAM function by intracranial injections of NCAM-antibodies, long-term potentiation (LTP) in rat hippocampal slices and learning in rats and chicks have been inhibited. In the vertebrate nervous system, NCAM is the dominant carrier of polysialic acid (PSA), an unusual carbohydrate consisting of long homopolymers of sialic acid. The PSA-NCAM expression decreases markedly during development. However, an upregulation of polysialic acid (PSA) in restricted brain areas including the hippocampus has been observed following learning. Moreover, enzymatic removal of PSA results in impaired LTP and learning. In muscle, the PSA-NCAM expression is upregulated following denervation. This response is weakened in aging rats. The expression of NCAM and PSA have been shown to be regulated by neuronal activity suggesting that the NCAM may promote structural remodelling in an activity dependent manner associated with learning and regeneration.

Positive allosteric modulation of α4ß2 nAChR agonist induced behaviour.

Neuronal cholinergic transmission is a prerequisite for proper CNS function. Consequently, disturbance of this system is associated with a number of pathophysiological conditions such as Parkinson's disease, Alzheimer's disease, schizophrenia and ADHD. Consequently, drug discovery efforts have spurred considerable research endeavours into identifying specific compounds for this system. Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels involved in cholinergic transmission. nAChRs are homo- or heteromeric pentamers with α4ß2 receptors being the most abundant heteromer. The stoichiometry of α4ß2 receptors can be either (α4)(3)(ß2)(2) or (α4)(2)(ß2)(3) representing channels with low (LS) or high (HS) sensitivity, respectively, to endogenous ligands. In the present study we applied the partial nAChR α4ß2 LS and HS agonist NS3956 and the LS selective positive allosteric modulator NS9283 to investigate the role of α4ß2 in Parkinson and pain models. In 6-OHDA lesioned rats, NS3956 increased rotational behaviour when rats were co-treated with nomifensine. This effect was absent in the presence of mecamylamine. In contrast, co-treatment with NS3956 and NS9283 reduced rotational behaviour in the animals. In a rat formalin pain model NS3956 induced an analgesic response that was strongly potentiated by NS9283. Finally in vitro experiments were applied to determine dopamine release from striatal minces. NS3956 induced a concentration dependent release while NS9283 was unable to potentiate agonist induced release. Together these results emphasize involvement of α4ß2 nAChR in rotational and analgesic responses and confirm striatal α4ß2 receptors to be of the HS form.

Extracellular adenosine triphosphate affects neural cell adhesion molecule (NCAM)-mediated cell adhesion and neurite outgrowth.

The neural cell adhesion molecule (NCAM) plays an important role in synaptic plasticity in embryonic and adult brain. Recently, it has been demonstrated that NCAM is capable of binding and hydrolyzing extracellular ATP. The purpose of the present study was to evaluate the role of extracellular ATP in NCAM-mediated cellular adhesion and neurite outgrowth. We here show that extracellularly added adenosine triphosphate (ATP) and its structural analogues, adenosine-5'-O-(3-thiothiophosphate), beta, gamma-methylenadenosine-5'-triphosphate, beta, gamma-imidoadenosine-5-triphosphate, and UTP, in varying degrees inhibited aggregation of hippocampal neurons. Rat glial BT4Cn cells are unable to aggregate when grown on agar but acquire this capacity when transfected with NCAM. However, addition of extracellular ATP to NCAM-transfected BT4Cn cells inhibited aggregation. Furthermore, neurite outgrowth from hippocampal neurons in cultures allowing NCAM-homophilic interactions was inhibited by addition of extracellular nucleotides. These findings indicate that NCAM-mediated adhesion may be modulated by extracellular ATP. Moreover, extracellularly added ATP stimulated neurite outgrowth from hippocampal neurons under conditions non-permissive for NCAM-homophilic interactions, and neurite outgrowth stimulated by extracellular ATP could be inhibited by a synthetic peptide corresponding to the so-called cell adhesion molecule homology domain (CHD) of the fibroblast growth factor receptor (FGFR) and by FGFR antibodies binding to this domain. Antibodies against the fibronectin type-III homology modules of NCAM, in which a putative site for ATP binding and hydrolysis is located, also abolished the neurite outgrowth-promoting effect of ATP. The non-hydrolyzable analogues of ATP all strongly inhibited neurite outgrowth. Our results indicate that extracellular ATP may be involved in synaptic plasticity through a modulation of NCAM-mediated adhesion and neurite outgrowth.

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation.

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.

Characterization of microwell cultures of dissociated brain tissue for studies of cell-cell interactions.

Microwell cultures of dissociated tissue from prenatal rat hippocampus and cerebral cortex as well as from early postnatal cerebellum were used for quantification of neuronal aggregation, process extension, and fasciculation. It was shown that the cells in culture from these different brain regions developed differently with regard to both architecture and rate of differentiation. The effect of a polyclonal antibody against the neural cell adhesion molecule (NCAM), the excitatory amino acid receptor agonist N-methyl-D-aspartate (NMDA), and the neurotoxin acrylamide on aggregation and fiber formation was investigated. Exposure to the NCAM antibody led to formation of fewer but larger aggregates and stimulated the morphological development of the cultures. Acrylamide affected aggregate formation, leading to smaller but more numerous aggregates, and it inhibited process extension and fasciculation. Treatment with NMDA affected process formation and led to formation of more numerous but smaller aggregates. Some of these effects were strongly tissue-dependent. Thus, large differences were seen regarding the effect of the NCAM antibody on aggregation and process extension in cultures from the different brain areas. The culture systems appear to represent convenient and reliable screening tools to study the influence of putative morphoregulatory substances on cell-cell interactions during early neuronal development.

Direct evidence of nitric oxide presence within mitochondria.

Nitric oxide (NO) has been implicated in the modulation of mitochondrial respiration, membrane potential, and subsequently in apoptosis. Although the presence of a mitochondrial NO synthase (mtNOS) has been described, there is no direct evidence in vivo of the presence of NO within mitochondria. It was the aim of this study to demonstrate the in vivo production of NO within mitochondria. Using the novel fluorometric NO detection system, 4,5-diaminofluorescein diacetate (DAF-2/DA), we observed the presence of NO production in PC12 and COS-1 cells by conventional and confocal fluorescence microscopy. Part of the overall NO signal was colocalized within a subpopulation of mitochondria, labeled with the potential-dependent probe MitoTracker red. These findings demonstrate for the first time that the subcellular distribution of NO production is consistent with the presence of a mitochondrial NOS. Our results provide a new tool to directly study the modulatory role of NO in mitochondrial respiration and membrane potential, in vivo.

A synthetic peptide ligand of neural cell adhesion molecule (NCAM) IgI domain prevents NCAM internalization and disrupts passive avoidance learning.

The neural cell adhesion molecule (NCAM) mediates cell adhesion and signal transduction through trans-homophilic- and/or cis-heterophilic-binding mechanisms. Intraventricular infusions of anti-NCAM have revealed a functional requirement of NCAM for the consolidation of memory in rats and chicks in a specific interval 6-8 h after training. We have now extended these studies to a synthetic peptide ligand of NCAM (C3) with an affinity for the IgI domain and the capability of inhibiting NCAM-mediated neurite outgrowth in vitro. Intraventricular administration of a single 5 microg bolus of C3 strongly inhibited recall of a passive avoidance response in adult rats, when given during training or in the 6-8-h posttraining period. The effect of C3 on memory consolidation was similar to that obtained with anti-NCAM as the amnesia was not observed until the 48-h recall time. The unique amnesic action of C3 during training could be related to disrupted NCAM internalization following training. In the 3-4-h posttraining period NCAM 180, the synapse-associated isoform, was down-regulated in the hippocampal dentate gyrus. This effect was mediated by ubiquitination and was prevented by C3 administration during training. These findings indicate NCAM to be involved in both the acquisition and consolidation of a passive avoidance response in the rat. Moreover, the study provides the first in vivo evidence for NCAM internalization in learning and identifies a synthetic NCAM ligand capable of modulating memory processes in vivo.