Neuroscience
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Extracellular adenosine triphosphate (ATP) participates in maintaining the vascular tone in the CNS, particularly in the retina, via the tonic activity of ligand gated activated P2X1 receptors. P2X1 receptors are characterized by their high affinity for ATP and their strong desensitization to concentrations of ATP that are 200-fold lower than their EC50. The mechanism behind P2X1 tonic activity remains unclear. ⋯ In the presence of extracellular Ca2+ the activity of hP2X1 receptors is greatly amplified by its coupling with Ca2+-activated Cl- channels. Future studies addressing the relationship between hP2X1 receptors and Ca2+-activated Cl- channels in vascular smooth muscle cells should provide information about additional mechanisms that regulate the vascular tone and their potential as pharmaceutical targets. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.
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Serotonin is an important neurotransmitter and neuromodulator. Disruption of the serotonergic system has been implicated in various psychiatric disorders such as schizophrenia and bipolar disorder. Most of the drugs targeting these neurotransmitter systems are classified primarily as agonists or inverse agonists/antagonists, with their described function being limited to activating the canonical signaling pathway(s), or inhibiting the pathway(s) respectively. ⋯ Using site-specific mutagenesis we have identified residues important for this functional selectivity, shown by dopamine at this receptor. Our identification of specific residues important in the functional selectivity of dopamine at 5-HT2A could have far reaching implications for the field of GPCR signaling and drug-design. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.
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The investigation on neurotransmission function during normal and pathologic development is a pivotal component needed to understand the basic mechanisms underlying neurodevelopmental pathologies. To study these diseases, many animal models have been generated which allowed to face the limited availability of human tissues and, as a consequence, most of the electrophysiology has been performed on these models of diseases. On the other hand, the technique of membrane microtransplantation in Xenopus oocytes allows the study of human functional neurotransmitter receptors thanks to the use of tissues from autopsies or surgeries, even in quantities that would not permit other kinds of functional studies. ⋯ Our findings retrace previous results and, in the light of this, further argue in favor of Prof. Miledi's technique of membrane microtransplantation that proves itself a very useful tool of investigation in the field of neurophysiology. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.
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Genetically encoded biosensors are widely used in cell biology for the non-invasive imaging of concentrations of ions or the activity of enzymes, to evaluate the distribution of small molecules, proteins and organelles, and to image protein interactions in living cells. These fluorescent molecules can be used either by transient expression in cultured cells or in entire organisms or through stable expression by producing transgenic animals characterized by genetically encoded and heritable biosensors. Using the mouse Thy1 mini-promoter, we generated a line of transgenic mice expressing a genetically encoded sensor for the simultaneous measurements of intracellular Cl- and pH. ⋯ This approach allowed us to assess cell morphology and track axonal projection, as well as to confirm E2GFP and DsRedm fluorescence colocalization. This analysis also provides a map of the brain areas suitable for non-invasive monitoring of intracellular Cl-/pH in normal and pathological conditions. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.
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A new mammalian neuromuscular preparation is introduced for physiology and microscopy of all sorts: the intrinsic muscle of the mouse ear. The great utility of this preparation is demonstrated by illustrating how it has permitted us to develop a wholly new technique for staining muscle T-tubules, the critical conductive-elements in muscle. This involves sequential immersion in dilute solutions of osmium and ferrocyanide, then tannic acid, and then uranyl acetate, all of which totally blackens the T-tubules but leaves the muscle pale, thereby revealing that the T-tubules in mouse ear-muscles become severely distorted in several pathological conditions. ⋯ These new observations strongly encourage further in-depth studies of ear-muscle architecture, in the many available mouse-models of various devastating human muscle-diseases. Finally, we demonstrate that the delicate and defined physical characteristics of this 'new' mammalian muscle are ideal for ultrastructural study, and thereby facilitate the imaging of synaptic vesicle membrane recycling in mammalian neuromuscular junctions, a topic that is critical to myasthenia gravis and related diseases, but which has, until now, completely eluded electron microscopic analysis. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.