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The modulation of neuronal excitability by group I metabotropic glutamate receptors (mGluRs) was studied in isolated lamprey spinal cord. At resting potential, application of the group I mGluR agonist ( R,S)-3,5-dihydroxyphenylglycine (DHPG) slightly depolarized the cells. However, at depolarized membrane potentials, this agonist induced repetitive firing. When Na+channels were blocked by TTX, DHPG induced a slight depolarization at rest that increased in amplitude as the neurons were held at more depolarized membrane potentials. In voltage-clamp conditions, DHPG application induced an inward current associated with a decrease in membrane conductance when cells were held at –40 mV. At resting membrane potential, no significant change in the current was induced by DHPG, although a decrease in membrane conductance was seen. The conductance blocked by DHPG corresponded to a leak current, since DHPG had no effect on the voltagegated current elicited by a voltage step from –60 to –40 mV, when leak currents were subtracted. The leak current blocked by DHPG is mediated by fluxes of both K+and Na+. The subtype of group I mGluR mediating the block of the leak current was characterized using specific antagonists for mGluR1 and mGluR5. The inhibition of the leak current was blocked by the mGluR1 antagonist LY 367385 but not by the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). The DHPG-induced blockage of the leak current required phospholipase C (PLC)-activation and release of Ca2+from internal stores as the effect of DHPG was suppressed by the PLC-blocker U-73122 and after depletion of intracellular Ca2+pools by thapsigargin. Our results thus show that mGluR1 activation depolarizes spinal neurons by inhibiting a leak current. This will boost membrane depolarization and result in an increase in the excitability of spinal cord neurons, which could contribute to the modulation of the activity of the spinal locomotor network.

The zebrafish (Danio rerio) has emerged as a new model system during the last three decades. The fact that the zebrafish larva is transparent enables sophisticated in vivo imaging. While being the vertebrate, the reduced complexity of its nervous system and small size make it possible to follow large-scale activity in the whole brain. Its genome is sequenced and many genetic and molecular tools have been developed that simplify the study of gene function. Since the mid 1990s, the embryonic development and neuronal function of the larval, and later, adult zebrafish have been studied using calcium imaging methods. The choice of calcium indicator depends on the desired number of cells to study and cell accessibility. Dextran indicators have been used to label cells in the developing embryo from dye injection into the one-cell stage. Dextrans have also been useful for retrograde labeling of spinal cord neurons and cells in the olfactory system. Acetoxymethyl (AM) esters permit labeling of larger areas of tissue such as the tectum, a region responsible for visual processing. Genetically encoded calcium indicators have been expressed in various tissues by the use of cell-specific promoters. These studies have contributed greatly to our understanding of basic biological principles during development and adulthood, and of the function of disease-related genes in a vertebrate system.

Alzheimer's disease (AD) is the most common neurodegenerative disease, and is clinically characterized by cognitive disturbances and the accumulation of the amyloid β (Aβ) peptides in plaques in the brain. Recent studies have shown the links between AD and the immediate-early gene Arc (activity-regulated cytoskeleton-associated protein), involved in synaptic plasticity and memory consolidation. For example, AD mouse models show a decreased expression of Arc mRNA in the brain. In additional, acute Aβ application to brain slices leads to a widespread ARC protein diffusion, unlike the normal defined localization to synapses. In this study, we investigated genetic variation in human ARC and the risk of developing AD. To this end, we genotyped 713 subjects diagnosed with AD and 841 controls without dementia. ARC was sequenced in a group of healthy individuals, and seven previously known SNPs and three novel SNPs were identified. Two of the newly found SNPs were intronic and one, +2852(G/A), was located in the 3'UTR. Three tag SNPs were selected, including the novel SNP +2852(G/A), to relate to risk of AD, Mini Mental State Examination (MMSE) scores and cerebrospinal fluid (CSF) biomarker levels of total tau (T-tau), hyperphosphorylated tau181 (P-tau(181)) and Aβ(1-42). The AA genotype of the newly found 3'-UTR SNP +2852(A/G), was associated with a decreased risk of AD (p (c) = 0.005; OR = 0.74; 95 % CI: 0.61-0.89). No associations of single SNPs or haplotypes with MMSE score or CSF biomarkers were found. Here we report a novel ARC SNP associated with a reduced risk of developing AD. To our knowledge, this is the first study associating a gene variant of ARC with any disease. The location of the SNP within the 3'UTR indicates that dendritic targeting of ARC mRNA could be involved in the molecular mechanisms underlying this protective function. However, further investigation of the importance of this SNP for ARC function, ARC processing and the pathology of AD is needed.

Examining neuronal network activity in freely behaving animals is advantageous for probing the function of the vertebrate central nervous system. Here, we describe a simple, robust technique for monitoring the activity of neural circuits in unfettered, freely behaving zebrafish (Danio rerio). Zebrafish respond to unexpected tactile stimuli with short- or long-latency escape behaviors, which are mediated by distinct neural circuits. Using dipole electrodes immersed in the aquarium, we measured electric field potentials generated in muscle during short- and long-latency escapes. We found that activation of the underlying neural circuits produced unique field potential signatures that are easily recognized and can be repeatedly monitored. In conjunction with behavioral analysis, we used this technique to track changes in the pattern of circuit activation during the first week of development in animals whose trigeminal sensory neurons were unilaterally ablated. One day post-ablation, the frequency of short- and long-latency responses was significantly lower on the ablated side than on the intact side. Three days post-ablation, a significant fraction of escapes evoked by stimuli on the ablated side was improperly executed, with the animal turning towards rather than away from the stimulus. However, the overall response rate remained low. Seven days post-ablation, the frequency of escapes increased dramatically and the percentage of improperly executed escapes declined. Our results demonstrate that trigeminal ablation results in rapid reconfiguration of the escape circuitry, with reinnervation by new sensory neurons and adaptive changes in behavior. This technique is valuable for probing the activity, development, plasticity and regeneration of neural circuits under natural conditions.

The model organism zebrafish (Danio rerio) is extensively utilized in studies of developmental biology but is also being investigated in the context of a growing list of human age-related diseases. To facilitate such studies, we here present protein expression patterns of adult zebrafish organs, including blood, brain, fin, heart, intestine, liver, and skeletal muscle. Protein extracts were prepared from the different organs of two zebrafish and analyzed using liquid chromatography coupled to high-resolution tandem mass spectrometry. Zebrafish tissue was digested directly after minimal fractionation and cleaned up (the shotgun approach). Proteins were identified using Mascot software. In total, 1394 proteins were identified of which 644 were nonredundant. Of these, 373 demonstrated an organ-specific expression pattern and 57 had not been shown on protein level before. These data emphasize the need for increased research at the protein level to facilitate the selection of candidate proteins for targeted quantification and to refine systematic genetic network analysis in vertebrate development, biology, and disease.

The possibility to control at specific times and specific places the activity of biomolecules (enzymes, transcription factors, RNA, hormones, etc.) is opening up new opportunities in the study of physiological processes at the single cell level in a live organism. Most existing gene expression systems allow for tissue specific induction upon feeding the organism with exogenous inducers (e.g., tetracycline). Local genetic control has earlier been achieved by micro-injection of the relevant inducer/repressor molecule, but this is an invasive and possibly traumatic technique. In this chapter, we present the requirements for a noninvasive optical control of the activity of biomolecules and review the recent advances in this new field of research.

Metabotropic glutamate receptor subtype 1 (mGluR1) contributes importantly to the activity of the spinal locomotor network. For example, it potentiates NMDA current and inhibits leak conductance in lamprey spinal cord neurons. In this study we examined the signalling pathways underlying the mGluR1 modulation of NMDA receptors and leak channels, respectively. Our results show that mGluR1-induced potentiation of NMDA current required activation of phospholipase C (PLC) and was independent of the increase in the intracellular Ca2+ concentration because it was unaffected by the Ca2+ chelator BAPTA and by depletion of the internal Ca2+ stores with thapsigargin. We also show that the mGluR1-mediated inhibition of leak channels is mediated by activation of G-proteins. Finally, we show that blockade of protein kinase C (PKC) abolished the mGluR1-induced inhibition of leak current without affecting the potentiation of NMDA receptors. The contribution of mGluR1-mediated modulation of leak channels to the potentiation of the locomotor cycle frequency was assessed during fictive locomotion. Blockade of PKC significantly decreased the short-term potentiation of locomotor cycle frequency by mGluR1. These results show that the effects of mGluR1 activation on the two cellular targets, the NMDA receptor and leak channels, are mediated through separate signalling pathways.

Spinal networks generate the motor pattern underlying locomotion. These are subject to modulatory systems that influence their operation and thereby result in a flexible network organization. In this review, we have summarized the mechanisms by which the different metabotropic glutamate receptor subtypes fine-tune the cellular and synaptic properties and thus underlie intrinsic modulation of the activity of the locomotor network in the lamprey.

Metabotropic glutamate receptors (mGluRs) act as modulators in the CNS of vertebrates, but their role in motor pattern generation in particular is primarily unknown. The intracellular signaling mechanisms of the group I mGluRs (mGluR1 and mGluR5), and their endogenous role in regulating locomotor pattern generation have been investigated in the spinal cord of the lamprey. Application of the group I mGluR agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) produced oscillations of the intracellular Ca(2+) concentration ([Ca(2+)](i)) in neurons. The oscillations were blocked by the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP) but not by the mGluR1 antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester. These [Ca(2+)](i)oscillations were abolished by a phospholipase C blocker and after depletion of internal Ca(2+) stores by thapsigargin but did not involve protein kinase C activation. Furthermore, they were dependent on Ca(2+) influx, because no [Ca(2+)](i) oscillations were produced by DHPG in a Ca(2+)-free solution or after blockade of L-type Ca(2+) channels. The mGluR5 is activated by an endogenous release of glutamate during locomotion, and a receptor blockade by MPEP caused an increase in the burst frequency. Thus, our results show that mGluR5 induces [Ca(2+)](i) oscillations and regulates the activity of locomotor networks through endogenous activation.

γ-Secretase inhibitors have been considered promising drug candidates against Alzheimer's disease (AD) due to their ability to reduce amyloid-β (Aβ) production. However, clinical trials have been halted due to lack of clinical efficacy and/or side effects. Recent in vitro studies suggest that low doses of γ-secretase inhibitors may instead increase Aβ production. Using a stem cell-derived human model of cortical neurons and low doses of the γ-secretase inhibitor DAPT, the effects on a variety of Aβ peptides were studied using mass spectrometry. One major focus was to develop a novel method for specific detection of oligomeric Aβ (oAβ), and this was used to study the effects of low-dose γ-secretase inhibitor treatment on intracellular oAβ accumulation. Low-dose treatment (2 and 20nM) with DAPT increased the secretion of several Aβ peptides, especially Aβx-42. Furthermore, using the novel method for oAβ detection, we found that 2nM DAPT treatment of cortical neurons resulted in increased oAβ accumulation. Thus, low dose-treatment with DAPT causes both increased production of long, aggregation-prone Aβ peptides and accumulation of intracellular Aβ oligomers, both believed to contribute to AD pathology.

The amyloid precursor protein (APP) is a transmembrane protein mostly recognized for its association with Alzheimer's disease. The physiological function of APP is still not completely understood much because of the redundancy between genes in the APP family. In this study we have used zebrafish to study the physiological function of the zebrafish APP homologue, appb, during development. We show that appb is expressed in post-mitotic neurons in the spinal cord. Knockdown of appb by 50–60% results in a behavioral phenotype with increased spontaneous coiling and prolonged touch-induced activity. The spinal cord motor neurons in these embryos show defective formation and axonal outgrowth patterning. Reduction in Appb also results in patterning defects and changed density of pre- and post-synapses in the neuromuscular junctions. Together, our data show that development of functional locomotion in zebrafish depends on a critical role of Appb in the patterning of motor neurons and neuromuscular junctions.

Experimental and computational techniques have been used to investigate the group I metabotropic glutamate receptor (mGluR)-mediated increase in the frequency of spinal cord network activity underlying locomotion in the lamprey. Group I mGluR activation potentiated the amplitude of NMDA-induced currents in identified motoneurons and crossed caudally projecting network interneurons. Group I mGluRs also potentiated NMDA-induced calcium responses. This effect was blocked by a group I mGluR-specific antagonist, but not by blockers of protein kinase A, C, or G. The effect of group I mGluRs activation was also tested on NMDA-induced oscillations known to occur during fictive locomotion. Activation of these receptors increased the duration of the plateau phase and decreased the duration of the hyperpolarizing phase. These effects were blocked by a group I mGluR antagonist. To determine its role in the modulation of NMDA-induced oscillations and the locomotor burst frequency, the potentiation of NMDA receptors by mGluRs was simulated using computational techniques. Simulating the interaction between these receptors reproduced the modulation of the plateau and hyperpolarized phases of NMDA-induced oscillations, and the increase in the frequency of the locomotor rhythm.Our results thus show a postsynaptic interaction between group I mGluRs and NMDA receptors in lamprey spinal cord neurons, which can account for the regulation of the locomotor network output by mGluRs.