A versatile viral toolkit for functional discovery in the nervous system.

The ability to precisely control transgene expression is essential for basic research and clinical applications. Adeno-associated viruses (AAVs) are non-pathogenic and can be used to drive stable expression in virtually any tissue, cell type, or species, but their limited genomic payload results in a trade-off between the transgenes that can be incorporated and the complexity of the regulatory elements controlling their expression. Resolving these competing imperatives in complex experiments inevitably results in compromises. Here, we assemble an optimized viral toolkit (VTK) that addresses these limitations and allows for efficient combinatorial targeting of cell types. Moreover, their modular design explicitly enables further refinements. We achieve this in compact vectors by integrating structural improvements of AAV vectors with innovative molecular tools. We illustrate the potential of this approach through a systematic demonstration of their utility for targeting cell types and querying their biology using a wide array of genetically encoded tools.

Identification of the cleavage sites leading to the shed forms of human and mouse anti-aging and cognition-enhancing protein Klotho.

Klotho is an age-extending, cognition-enhancing protein found to be down-regulated in aged mammals when age-related diseases start to appear. Low levels of Klotho occur in neurodegenerative diseases, kidney disease and many cancers. Many normal and pathologic processes involve the proteolytic shedding of membrane proteins. Transmembrane (TM) Klotho contains two homologous domains, KL1 and KL2 with homology to glycosidases. After shedding by ADAM 10 and 17, a shed Klotho isoform is released into serum and urine by the kidney, and into the CSF by the choroid plexus. We previously reported that human Klotho contains two major cleavage sites. However, the exact cleavage site responsible for the cleavage between the KL1 and KL2 domains remains unknown for the human Klotho, and both sites are unknown for mouse Klotho. In this study, we aimed to identify the cleavage sites leading to the shed forms of human and mouse Klotho. Mutations in the region close to the TM domain of mouse Klotho result in the reduced shedding of the 130 kD (KL1+KL2) and 70 kD (KL1) fragments, suggesting that the cleavage site lies within the mutated region. We further identified the cleavage sites responsible for the cleavage between KL1 and KL2 of human and mouse Klotho. Moreover, mutated Klotho proteins have similar subcellular localization patterns as wild type Klotho. Finally, in an FGF23 functional assay, all Klotho mutants with a nine amino acid deletion can also function as an FGFR1 co-receptor for FGF23 signaling, however, the signaling activity was greatly reduced. The study provides new and important information on Klotho shedding, and paves the way for studies aimed to distinguish between the distinct roles of the various isoforms of Klotho.

Klotho Is Neuroprotective in the Superoxide Dismutase (SOD1G93A) Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord. ALS neuropathology is associated with increased oxidative stress, excitotoxicity, and inflammation. We and others reported that the anti-aging and cognition-enhancing protein Klotho is a neuroprotective, antioxidative, anti-inflammatory, and promyelinating protein. In mice, its absence leads to an extremely shortened life span and to multiple phenotypes resembling human aging, including motor and hippocampal neurodegeneration and cognitive impairment. In contrast, its overexpression extends life span, enhances cognition, and confers resistance against oxidative stress; it also reduces premature mortality and cognitive and behavioral abnormalities in an animal model for Alzheimer's disease (AD). These pleiotropic beneficial properties of Klotho suggest that Klotho could be a potent therapeutic target for preventing neurodegeneration in ALS. Klotho overexpression in the SOD1 mouse model of ALS resulted in delayed onset and progression of the disease and extended survival that was more prominent in females than in males. Klotho reduced the expression of neuroinflammatory markers and prevented neuronal loss with the more profound effect in the spinal cord than in the motor cortex. The effect of Klotho was accompanied by reduced expression of proinflammatory cytokines and enhanced the expression of antioxidative and promyelinating factors in the motor cortex and spinal cord of Klotho × SOD1 compared to SOD1 mice. Our study provides evidence that increased levels of Klotho alleviate ALS-associated pathology in the SOD1 mouse model and may serve as a basis for developing Klotho-based therapeutic strategies for ALS.

A novel slice preparation to study medullary oromotor and autonomic circuits in vitro.

BACKGROUND: The medulla is capable of controlling and modulating ingestive behavior and gastrointestinal function. These two functions, which are critical to maintaining homeostasis, are governed by an interconnected group of nuclei dispersed throughout the medulla. As such, in vitro experiments to study the neurophysiologic details of these connections have been limited by spatial constraints of conventional slice preparations. NEW METHOD: This study demonstrates a novel method of sectioning the medulla so that sensory, integrative, and motor nuclei that innervate the gastrointestinal tract and the oral cavity remain intact. RESULTS: Immunohistochemical staining against choline-acetyl-transferase and dopamine-ß-hydroxylase demonstrated that within a 450 µm block of tissue we are able to capture sensory, integrative and motor nuclei that are critical to oromotor and gastrointestinal function. Within slice tracing shows that axonal projections from the NST to the reticular formation and from the reticular formation to the hypoglossal motor nucleus (mXII) persist. Live-cell calcium imaging of the slice demonstrates that stimulation of either the rostral or caudal NST activates neurons throughout the NST, as well as the reticular formation and mXII. COMPARISON WITH EXISTING METHODS: This new method of sectioning captures a majority of the nuclei that are active when ingesting a meal. Tradition planes of section, i.e. coronal, horizontal or sagittal, contain only a limited portion of the substrate. CONCLUSIONS: Our results demonstrate that both anatomical and physiologic connections of oral and visceral sensory nuclei that project to integrative and motor nuclei remain intact with this new plane of section.

Prostacyclin mediates endothelial COX-2-dependent neuroprotective effects during excitotoxic brain injury.

In a previous study, we found that intracerebral administration of excitotoxin (RS)-(tetrazole-5yl) glycine caused increased neural damage in the brain in an endothelial COX-2 deleted mouse line (Tie2Cre COX-2(flox/flox) ). In this study, we investigated whether prostacyclin might mediate this endothelial COX-2-dependent neuroprotection. Administration of excitotoxin into the striatum induced the production of prostacyclin (PGI2) in wild type, but not in endothelial COX-2 deleted mice. Inhibition of PGI2 synthase exacerbated brain lesions induced by the excitotoxin in wild type, but not in endothelial COX-2 deleted mice. Administration of a PGI2 agonist reduced neural damage in both wild type and endothelial COX-2 deleted mice. Increased PGI2 synthase expression was found in infiltrating neutrophils. In an ex vivo assay, PGI2 reduced the excitotoxin-induced calcium influx into neurons, suggesting a cellular mechanism for PGI2 mediated neuroprotection. These results reveal that PGI2 mediates endothelial COX-2 dependent neuroprotection.

Adrenoreceptor modulation of oromotor pathways in the rat medulla.

Regulation of feeding behavior involves the integration of multiple physiological and neurological pathways that control both nutrient-seeking and consummatory behaviors. The consummatory phase of ingestion includes stereotyped oromotor movements of the tongue and jaw that are controlled through brain stem pathways. These pathways encompass not only cranial nerve sensory and motor nuclei for processing feeding-related afferent signals and supplying the oromotor musculature but also reticular neurons for orchestrating ingestion and coordinating it with other behaviors that utilize the same musculature. Based on decerebrate studies, this circuit should be sensitive to satiety mechanisms mediated centrally by A2 noradrenergic neurons in the caudal nucleus of the solitary tract (cNST) that are potently activated during satiety. Because the first observable phase of satiety is inhibition of oromotor movements, we hypothesized that norepinephrine (NE) would act to inhibit prehypoglossal neurons in the medullary reticular formation. Using patch-clamp electrophysiology of retrogradely labeled prehypoglossal neurons and calcium imaging to test this hypothesis, we demonstrate that norepinephrine can influence both pre- and postsynaptic properties of reticular neurons through both α1- and α2-adrenoreceptors. The α1-adrenoreceptor agonist phenylephrine (PE) activated an inward current in the presence of TTX and increased the frequency of both inhibitory and excitatory miniature postsynaptic currents. The α2-adrenoreceptor agonist dexmedetomidine (DMT) inhibited cNST-evoked excitatory currents as well as spontaneous and miniature excitatory currents through presynaptic mechanisms. The diversity of adrenoreceptor modulation of these prehypoglossal neurons may reflect their role in a multifunctional circuit coordinating both ingestive and respiratory lingual function.

Live-cell imaging methods for the study of vagal afferents within the nucleus of the solitary tract.

Substantial evidence suggests that vagal afferent functions are modulated by agonists acting on afferent terminals in the solitary nucleus (NST). Actions of these agonists are implied through intracellular recordings from cultured nodose ganglion cells or second order NST neurons. While high-quality data have been obtained using these methods, techniques in which physiological measurements can be made directly on the afferent terminal fields, in situ, in the NST, would eliminate several potential interpretive problems inherent in these less direct approaches. This paper describes methods developed to directly measure changes in presynaptic cytoplasmic calcium in vagal afferents using time-lapse laser confocal microscopy on the in vitro brainstem slice. Calcium green dextran (CG) transported from the nodose ganglion clearly demonstrates vagal afferent fibers ramifying throughout the NST in the in vitro brainstem slice. CG-labeled vagal afferents can be repeatedly activated by focal electrical stimulation, by agonists acting on presynaptic ligand-gated ion channels, and by molecules that are presumed to act directly on vagal afferents based on previous physiological and immunocytochemical studies. Image and preparation stability are a challenge to the success of the experiment; however, methods described here should assist direct studies of transduction events within other afferent terminal fields in the CNS.

Alpha-1 adrenergic input to solitary nucleus neurones: calcium oscillations, excitation and gastric reflex control.

The nucleus of the solitary tract (NST) processes substantial visceral afferent input and sends divergent projections to a wide array of CNS targets. The NST is essential to the maintenance of behavioural and autonomic homeostasis and is the source, as well as the recipient, of considerable noradrenergic (NE) projections. The significance of NE projections from the NST to other CNS regions has long been appreciated, but the nature of NE action on NST neurones themselves, especially on the alpha-1 receptor subtype, is controversial. We used a combination of methodologies to establish, systematically, the effects and cellular basis of action of the alpha-1 agonist, phenylephrine (PHE), to control NST neurones responsible for vago-vagal reflex regulation of the stomach. Immunocytochemical and retrograde tracing studies verified that the area postrema, A2, A5, ventrolateral medulla and locus coeruleus regions are sources of catecholaminergic input to the NST. In vivo electrophysiological recordings showed that PHE activates physiologically identified, second-order gastric sensory NST neurones. In vivo microinjection of PHE onto NST neurones caused a significant reduction in gastric tone. Finally, in vitro calcium imaging studies revealed that PHE caused dramatic cytosolic calcium oscillations in NST neurones. These oscillations are probably the result of an interplay between agonist-induced and inositol 1,4,5-trisphosphate (IP(3))-mediated intracellular calcium release and Ca(2+)-ATPase control of intracellular calcium storage pumps. The oscillations persisted even in perfusions of zero calcium-EGTA Krebs solution suggesting that the calcium oscillation is mediated principally by intracellular calcium release-reuptake mechanisms. Cyclical activation of the NST may function to increase the responsiveness of these neurones to incoming afferent input (i.e., elevate the "gain"). An increase in gain of afferent input may cause an amplification of the response part of the reflex and help explain the powerful effects that alpha-1 agonists have in suppressing gastric motility and producing anorexia.